Importin 7 Mediated Nuclear Transport of MSI2 as a Therapeutic Target in Cervical Cancer

Importin 7 Mediated Nuclear Transport of MSI2 as a Therapeutic Target in Cervical Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Importin 7 Mediated Nuclear Transport of MSI2 as a Therapeutic Target in Cervical Cancer Xu Qinyang, Wan-Zhen zhou, Tian Qiu, Juan Wang, jing cheng, Rongzhen Jiang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5254983/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cervical cancer (CC) remains a significant health threat to women worldwide, with a pressing need for novel therapeutic targets. Despite recent advances, the molecular mechanisms underlying CC progression are not fully understood. Nuclear transport proteins, key regulators of macromolecule movement between cellular compartments, have emerged as potential targets in cancer therapy. However, the specific role of Importin 7 (IPO7) in CC development and its clinical implications remain poorly characterized, indicating a significant gap in our current understanding. Here, we investigated IPO7's role in CC, leveraging clinical samples, bioinformatics analyses from TCGA and GEO databases, and experimental models. We found that IPO7 is upregulated in CC and associated with poor prognosis. IPO7 knockdown in cell lines and animal models revealed decreased cell proliferation, reduced colony formation, inhibited migration and invasion, and enhanced apoptosis. To uncover IPO7's molecular mechanisms, we performed mass spectrometry analysis, identifying MSI2, an RNA-binding protein, as a potential cargo. Further validation confirmed a direct interaction between IPO7 and MSI2, with IPO7 overexpression coupled with MSI2 knockdown abrogating oncogenic effects. Sequencing analysis of IPO7-knockdown cells indicated the MYC targets pathway and co-immunoprecipitation assays confirmed a direct interaction between MYC and MSI2, suggesting that IPO7 may facilitate the nuclear transport of MSI2 and MYC, thereby promoting cancer progression. Clinically, elevated MSI2 expression in CC patients, particularly in advanced stages, correlated with poorer outcomes. Our findings elucidate the role of IPO7 in CC, demonstrating its potential as a therapeutic target. The interaction between IPO7, MSI2, and MYC provides a novel avenue for developing targeted therapies. Importantly, our results underscore the importance of IPO7-mediated nuclear transport in CC progression, presenting a promising strategy for enhancing patient outcomes and advancing CC treatment. Health sciences/Diseases/Cancer/Gynaecological cancer/Cervical cancer Biological sciences/Cancer/Gynaecological cancer/Cervical cancer Cervical cancer nuclear transport Importin 7 MSI2 Prognosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Cervical cancer (CC) is the predominant malignancy affecting the female reproductive system. According to the Global Cancer Statistics 2020 report, CC is the fourth leading cause of cancer-related mortality in women, with an estimated 604,000 new cases and 342,000 deaths worldwide 1 . In 2020, the World Health Organization Assembly introduced a comprehensive initiative to eradicate CC, including strategies such as HPV vaccination, CC screening, and management of precancerous lesions 2 . The management of CC faces challenges in achieving satisfactory outcomes, largely due to the difficulty of implementing interventions on a large scale 3 . The development of CC associated with high-risk HPV infections is a multifaceted process involving various stages and factors, which complicates the effectiveness of vaccines in improving the prognosis of HPV-infected individuals or CC patients 4 . T Targeted therapy represents a significant advancement in treating late-stage CC patients who have undergone standard surgical procedures. Therefore, a thorough investigation into the pathogenesis of CC and the discovery of new molecular targets is essential to improve prognostic outcomes. Nuclear transport proteins, or karyopherins, are crucial in mediating the transport of signals and substances between the nucleus and cytoplasm 5 . These proteins facilitate the translocation of macromolecular proteins through the nuclear pore complex, which is essential for the transportation process 6 . The superfamily of nuclear transport proteins is comprised of α and β families, each possessing distinct structural and functional characteristics. The Karyopherin-β family, encompassing importins, exportins, and biportins, plays a vital role in mediating the transport of macromolecules, especially proteins, through the nuclear pore complex (NPC) by recognizing specific nuclear localization signals (NLSs) or nuclear export signals (NESs) 5 , 7 . This transport process relies on the GTPase RanGTP for cargo binding and dissociation 8 , 9 . Under normal conditions, karyopherins precisely transport specific cargo proteins to their designated locations, thus facilitating proper physiological functions. Conversely, dysfunction of karyopherins results in the mislocalization of nucleo-cytoplasmic elements, including proto-oncogenes, tumor suppressor genes, and cell cycle regulatory proteins, thus contributing to tumor development 5 . Targeting karyopherins to disrupt nuclear-cytoplasmic communication and material transport in cancer cells could be a promising strategy to enhance the survival rates of individuals with CC. Importin 7 (IPO7), a notable member of the karyopherin family, belongs to the β nuclear transport protein family. The sequence features two antiparallel helices, enhancing its binding affinity with associated proteins 10 , and thus making it a key component of the nuclear-cytoplasmic transport mechanism. Nonetheless, the specific biological functions of IPO7 in CC remain insufficiently understood and necessitate further investigation. This study aims to clarify the role of IPO7 in CC progression and, concurrently, to identify and validate potential IPO7 cargo proteins by isolating cytoplasmic and nuclear proteins and conducting mass spectrometry analysis. 2. Materials and methods 2.1 Clinical samples and database analysis The CC tissues microarray (TMA) containing 15 normal cervix tissues, 78 cervical intraepithelial neoplasia tissues, and 100 CC tissues from Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. All tissue specimens were evaluated by two pathologists. This research was authorized by the Research Ethics Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital(2020-YS-075), and written informed consent was obtained from all patients before this study. CC data were acquired from The Cancer Genome Atlas (TCGA) database ( https://portal.gdc.cancer.gov/ ), and normal cervix uterus data were acquired from The Cancer Genome Atlas (GTEx) ( https://www.gtexportal.org/home/ ). The gene expression profile results were downloaded from the Gene Expression Omnibus database (GSE9750, GSE7803, GSE6791, GSE63514) at the NCBI. Database between a high-IPO7 expression group and a low-IPO7 expression group based on the median of TCGA-CESC IPO7 profiles and Kaplan–Meier analysis was conducted by the above grouping method. Gene Ontology Metascape analysis was performed using Metascape software ( http://metascape.org/gp/index.html ) using default settings 11 . 2.2 Culture of cell lines and transfection Human CC Cell lines HeLa and SiHa were preserved in Shanghai Cancer Institute. All of the cells were cultured in a suggested medium (Invitrogen, USA) according to ATCC protocols. siRNA and plasmids transfection were performed using Lipofectamine 2000 (Invitrogen, USA). siRNAs targeting various genes were purchased from Asia-Vector Biotechnology (Shanghai, China), and the sequences are listed in Supplementary Files. To generate stable cell lines with IPO7 knockdown, Hela cells were infected with LV3-puro-shIPO7 (sh1) and cell clones were selected for puromycin resistance. Human IPO7 coding sequence and Ubiquitin coding sequence was amplified and cloned into the expression plasmid PcDNA3.1 + for overexpressing IPO7-FLAG and UB-HA. The entire coding sequences of wild-type MSI2 and del-type MSI2 (deleted the NLS region) were subcloned into the expression plasmid PcDNA3.1+. The detailed information of plasmids and used primers is given in supplemental file. 2.3 Quantitative real-time PCR (qRT-PCR) The total RNA was isolated from CC cell lines, quantified by NANO 2000 (Thermo Fisher Scientific, USA), subjected to reverse transcription and subsequently conducted to quantitative real-time PCR utilizing a 7500 Real-time PCR system (Applied Biosystems, USA). Relative mRNA expression was calculated by the 2−△△CT method, and was normalized by endogenous β-actin. 2.4 Western blotting analysis For Western blotting, total protein was extracted using cell lysis buffer (Beyotime, China). Otherwise, subcellular nuclear cytoplasm protein fractionation was proceeded using NE-PER Nuclear and Cytoplasm Extraction Reagents (Thermo Fisher Scientific, USA) according to instructions. Western blotting was performed as previously describe 12 . Concentrations of antibodies were used according to the manufacturer's instructions and the antibodies are listed in Supplementary File 1 (Table 4). 2.5 Co-Immunoprecipitation (Co-IP) For Co-IP, immunoglobulin G (IgG) or specific antibody was incubated with protein G-agarose beads for 30 min at room temperature, and immunoprecipitated with protein at 4°C overnight. Then the beads were washed three times with PBS and eluted by 1x protein SDS loading buffer. Extracted proteins were separated by SDS-PAGE in a 7–15% gel, transferred to a nitrocellulose membrane (Millipore, Burlington, USA), and blocked in 5% BSA. Then, the nitrocellulose membrane was exposed to primary antibodies and species-specific secondary antibodies. Antibodies are presented in Supplementary file1. 2.6 Immunohistochemistry (IHC) analysis Briefly, the primary antibodies anti-IPO7 (1:200, Abcam), anti-MSI2 (1:100, Abcam) were incubated with the slides first. Each tissue sample was scored according to the proportion of stained cells (0–5% scored “–”, 5–25% scored “+”, 25–50% scored “++”, 50–75% scored “++”, and 75–100% scored “+++”). The scoring was judged independently by two pathologists in a blinded manner. We designated the final score as a high or low expression group as follows: a score of “-” and “+” were defined low expression of IPO7 (or MSI2) and a score of “++” and “+++” were defined high expression IPO7 (or MSI2). 2.7 Immunofluorescence (IF) assay Cells were cultured on coverslips in 8-well plates, and were fixed and permeabilized with 4% paraformaldehyde and 0.05% Triton X-10. BSA (1%) was used for blocking. Then cells were probed with the anti-IPO7 antibody (1:200, Abcam) and anti-MSI2 antibody (1:50, Abcam) overnight at 4°C, followed by an Alexa Fluor 594 conjugated anti-rabbit antibody (1:200 Jackson) and Fluor 488–conjugated anti-mouse antibody (1:200, Jackson). DAPI (4′,6-diamidine-2′-phenylindole, 1:1000, Invitrogen) was used for nuclear staining. IF images were captured by confocal laser scanning microscope (Carl Zeiss). 2.8 Cell proliferation assay Cell proliferation was measured by using a Cell Counting Kit-8 (CCK-8) kit (Dojindo, Japan). The cells were cultured on 96-well plates, and OD450 was measured 1 h after addition of CCK-8 at 0, 24, 48, 72, and 96 h. 2.9 Colony formation assay The CC cells (2 × 10 3 ) were cultured on per well of a 6-well plate for 2 weeks. Then the cell culture plates were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Photographs were acquired and the cell numbers were counted. 2.10 Cell migration and invasion assay The Transwell migration assay was performed using Corning chambers (Corning, USA), featuring 8-µm pores. A total of 1 × 105 cells were resuspended in serum-free culture medium and seeded in the upper chamber, conversely complete medium containing 10% FBS was placed in the lower chamber. After 24-hour incubation, the migrated cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet, and then observed and quantified under an inverted microscope. Transwell invasion assay were performed as previously described except the chambers were coated with Matrigel (Corning, USA). 2.11 Cell apoptosis assay Target cells were cultured under serum deprivation for 24 hours to assess apoptosis. Subsequently, the cells were detached with 0.25% trypsin and resuspended for propidium iodide (PI) and Annexin V-FITC staining. The samples were processed to FACS Canto-Plus flow cytometer (BD Biosciences). 2.12 Cell cycle assay Cell cycle distribution was assessed by flow cytometry using the cell cycle/apoptosis analysis kit. The target cells were fixed in pre-cooling 75% ethanol for 12h at 4°C, and incubated in PI for 30 min at 37°C. The cells were harvested for flow cytometry analysis (BD Biosciences) and analysed with the Modfit LT 5.1 software. 2.13 Mouse xenograft model a total of 1×106 IPO7-shRNA or control shRNA Hela cells were injected subcutaneously in the axilla of BALB/c female mice (5–6 weeks of age, n = 5 per group). The tumor diameters were measured by Vernier caliper every 7 days. The tumor volume was estimated based on the equation V = 1/2 (a x b x b). After 5 weeks, the mice were killed to dissect and weight xenograft tumors. The xenograft tumor samples were fixed and used for subsequent IHC analysis. 2.14 Ubiquitination assay Cells were transfected with the HA-Ubiquitin plasmid, treated with the proteasome inhibitor MG132 (25 mM) (Sigma, M7449) for 6 h, and subsequently lysed in ubiquitination assay buffer. The cell lysis was co-immunoprecipitated with protein G-agarose beads conjugated with MSI2 antibody, and Western blotting was performed with an anti-HA antibody (Proteintech, China) to detect ubiquitinated MSI2. 2.15 Liquid Chromatography–mass spectrometry Tryptic peptides were dissolved in 0.1% formic acid (solvent A) and separated utilizing the EASY-nLC 1000 ultra-high performance liquid system. Subsequently, the peptides undergo separation by the ultra-high performance liquid system, followed by injection into the NSI ion source for ionization and analysis using Orbitrap Fusion mass spectrometry. The ion source operates at 2.2 kV and facilitates the detection and analysis of peptide precursor ions and their secondary fragments utilizing high-resolution Orbitrap. 2.16 RNA-seq Hela cells (with IPO7-shRNA and control RNA) were seeded into a 6 cm plate. Cell lysates were prepared using Trizol (TaKaRa) and then stored at − 80◦C. The following steps were performed by Newcore Biotech. Cell lysate was sequentially treated with chloroform, isopropanol, and ethanol to extract total RNA. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, Los Angeles, USA). RNA concentration was measured using Qubit® RNA Assay Kit in Qubit®2.0 Flurometer (Life Technologies, Carlsbad, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, USA). Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, Ipswich, USA) following manufacturer’s recommendations. The gene set enrichment analysis (GSEA) analysis was performed using GSEA software following the manufacturer’s introduction. 2.17 mRNA stability assay Cells (2 × 10^5 per well) were seeded into a 12-well plate. Transcriptional inhibition was achieved using Actinomycin D at a final concentration of 2 µg/mL (Leagene, Beijing, China). Total RNA was isolated at 0-, 3-, and 6-hours post-treatment using Trizol reagent. Subsequently, RNA was extracted, reverse transcribed into cDNA, and subjected to quantitative real-time PCR (qRT-PCR) as previously described. 2.16 Statistical analysis The statistical analyses were performed using SPSS Statistics software (SPSS 20, USA) and GraphPad Prism 8.0 software. The data are presented as means ± SD and were calculated using the two-tailed Student’s t-test. The relationship between IPO7/MSI2 expression level and clinical parameters was determined by the χ2 test and Fisher’s exact test. Survival analysis was conducted using the Kaplan-Meier method. The survival curves were analyzed using the log-rank test. p < 0.05 was considered significant statistically for all tests. 3. Result 3.1 IPO7 is highly expressed in CC and is associated with unfavorable prognosis This study aims to investigate the role of nuclear transport proteins in the development of CC. We analyzed the expression profiles of 27 known nuclear transport proteins in the TCGA-CESC database to evaluate their potential as prognostic indicators. Furthermore, comparative analysis of expression levels in control and tumor samples from the GEO database identified IPO7 and RanBP17 as genes with increased expression and significant prognostic value in CC. Thus, the present study concentrated on elucidating the role of IPO7 in CC progression (Supplementary Fig. 1 and Supplementary Table 1). Through the integration of TCGA and GEO databases, a substantial increase in IPO7 expression was observed in CC tissues relative to normal controls. Additionally, an escalation in IPO7 expression was noted in tandem with the advancement of cervical lesions (Supplementary Fig. 1B, Fig. 1 A, and 1 B). High IPO7 expression was associated with a poor prognosis for patients (Fig. 1 C). However, no correlation was detected between elevated IPO7 expression and patient age, BMI, histopathological type, or clinical stage when analyzing the TCGA database. Moreover, individuals with high IPO7 expression had decreased overall survival (OS) and disease-specific survival (DSS) rates (Supplementary Table 2). Additionally, we obtained clinical specimens from patients diagnosed with CC who underwent surgical treatment at our hospital over the past five years. IPO7 protein expression was evaluated via immunohistochemical staining (IHC) (Fig. 1 D). Table 1 presents the statistical analysis associated with the clinical data. Our results demonstrate an incremental rise in IPO7 protein expression in tandem with the severity and stage of cervical lesions (Figs. 1 F- 1 H). Table 1 Correlation between IPO7 expression and clinicopathological features in cervical cancer (n = 100) Expression of IPO7 total Low (%) High (%) P value age < 55 years old 72 30 (41.7%) 42 (58.3) 0.121 ≥ 55 years old 28 7 (25.0) 21 (75.0) Menopause Yes 82 32 (39.0) 50 (61.0) 0.532 No 18 5 (27.8) 13 (72.2) Pregnancy Yes 53 20 (37.7) 33 (62.3) 0.436 No 47 17 (45.9) 20 (54.1) HPV infection Yes 93 35 (40.0) 58 (60.0) 0.942 No 7 2 (47.7) 5 (52.3) Clinical Stage (FIGO) I 13 9 (69.2) 4 (30.8) 0.028* II 44 16 (36.4) 28 (63.3) III 43 12 (27.9) 31 (72.1) Tumor size T1 84 36 (42.9) 48 (57.1) 0.002* T2-4 16 1 (6.3) 15 (93.8) FIGO, International Federation of Gynecology and Obstetrics; * P < 0.05, Statistically significant 3.2 IPO7 plays an oncogenic role in CC To determine the function of IPO7, this study employed two cervical cancer (CC) cell lines, SiHa and HeLa, exhibiting high levels of IPO7 expression (Supplementary Fig. 2A-2C). Subsequently, IPO7 knockdown experiments were performed in these cell lines (Fig. 2 A). Following IPO7 knockdown, the CC cells exhibited reduced cell proliferation (Fig. 2 B, P < 0.001), decreased colony formation (Fig. 2 C and 2 D, P < 0.01), and impaired migration and invasion (Fig. 2 H- 2 I, P < 0.01, Supplementary Fig. 2D and 2E), along with increased apoptosis (Fig. 2 E- 2 G, P < 0.01). Furthermore, we established HeLa cell lines with stable IPO7 knockdown and evaluated their effect on tumorigenesis using subcutaneous xenograft models in nude mice. The group with IPO7 shRNA exhibited markedly slower tumor growth and lower tumor burden compared to the control shRNA group (Fig. 3 A- 3 D). IHC analysis revealed decreased expression of the proliferation marker Ki67 in the IPO7-shRNA xenografts (Fig. 3 E and 3 F). This result indicated that IPO7 promoted the proliferation of HeLa cells. In summary, cellular and in vivo experiments collectively support the oncogenic function of IPO7 in CC cells. 3.3 MSI2 is a potential target protein transported by IPO7 in CC As a nuclear transport protein, IPO7 is responsible for the import of cargo proteins into the cell nucleus. Thus, we investigated the molecular mechanism of IPO7 in CC by focusing on nuclear-cytoplasmic transport. We manipulated IPO7 expression and isolated nuclear-cytoplasmic fractions from both IPO7 knockdown and control groups. Subsequently, mass spectrometry analysis identified differentially expressed proteins. The analysis identified 74 proteins that were downregulated in the cell nucleus following IPO7 knockdown (fold change > 2 and P < 0.05). These proteins are considered potential IPO7 cargo proteins (Fig. 4 A). The enrichment analysis indicated that these proteins are involved in processes such as protein localization, Rho GTP cycling, and translation regulation (Fig. 4 B). Additionally, we performed enrichment analysis using Metascape ( http://metascape.org/ ) on the top 200 genes most correlated with IPO7 expression in the TCGA- CESC database. This analysis showed that IPO7 is predominantly linked to mRNA processes involving transport and regulation (Fig. 4 C). Among the candidates, the RNA-binding protein Musashi-2 (MSI2) attracted our attention. MSI2 is a member of the Musashi family, which includes MSI1 and MSI2, and possesses two RNA-recognition motifs (RRM1 and RRM2) at the N-terminus 13 . MSI2 can bind to the 3'-end of target RNAs and modulate the stability and translation of mRNA of proteins implicated in tumorigenesis, including SMAD3, PTEN and others 14 . MSI2 is considered a potential IPO7 cargo protein in this study. We utilized the NLS-mapper website ( https://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi ) to predict the NLS of MSI2, identifying a highly conserved sequence (Fig. 4 D) with a score of 5, indicating its potential for nucleocytoplasmic shuttling. Furthermore, MSI2 shows increased expression in CC (Fig. 4 E) and is positively correlated with IPO7 expression (Fig. 4 F, P < 0.001, R = 0.38). Further analysis showed decreased MSI2 protein levels after IPO7 knockdown (Fig. 4 G), while qPCR analysis showed no significant change in MSI2 mRNA levels following knockdown (Fig. 4 H). After isolating nuclear and cytoplasmic fractions, we performed individual analyses of MSI2 expression. The findings showed a reduction in MSI2 expression in both the nucleus and cytoplasm subsequent to IPO7 knockdown (Fig. 4 I). The reduction in MSI2 expression could be attributed to hindered nuclear entry and subsequent degradation of the protein. This hypothesis is supported by the identification of seven ubiquitination sites on the MSI2 protein, as predicted using the SMART website ( http://smart.embl-heidelberg.de/ ) (Fig. 5 D). To further investigate, UB-HA was introduced into both normal and IPO7 knockdown CC cells, followed by immunoprecipitation (IP) experiments to evaluate the ubiquitination level of MSI2. The results indicated elevated MSI2 ubiquitination upon IPO7 knockdown, implying that this could be a potential cause for the decreased cytoplasmic levels of MSI2 (Fig. 4 J). 3.4 MSI2 directly interacts with IPO7, which exerts oncogenic effects by transporting MSI2 In this study, we examined the interaction between IPO7 and MSI2. Initially, Co-IP experiments and IF assays were conducted to confirm a direct interaction (Fig. 5 A- 5 C). Subsequently, we used the SMART website for predicting the structure of the MSI2 protein and generated a corresponding model diagram (Fig. 5 D). This model indicated that the nuclear localization sequence (NLS) is predominantly located in the RRM1 region of MSI2. Then, we deleted the NLS fragment from the MSI2 DNA sequence to observe its effects on binding to IPO7 in cell cultures. A significant reduction in the binding affinity between MSI2 (del-NLS) and IPO7 was observed, particularly in HeLa cells where almost no binding was detected (Fig. 5 E and 5 F). The results demonstrate the importance of the NLS region in mediating the interaction between IPO7 and MSI2, suggesting that IPO7 likely facilitates the nuclear transport of MSI2. To investigate whether the oncogenic effects of IPO7 are mediated through the transportation of MSI2, we conducted experiments with the overexpression of IPO7 and simultaneous knockdown of MSI2 in two cell lines (SiHa and HeLa). Firstly, we assessed the efficacy of MSI2 knockdown and selected the most potent shRNA sequence for further experiments (Supplementary Fig. 3). MSI2 knockdown did not influence IPO7 mRNA expression levels, and IPO7 overexpression had no impact on MSI2 mRNA levels in both cell lines (Fig. 6 A- 6 D). Nevertheless, the overexpression of IPO7 and knockdown of MSI2 simultaneously resulted in a notable decrease in both cell proliferation capacity and colony formation ability (Fig. 6 E- 6 I, P < 0.01). These results suggest that IPO7 exerts its oncogenic effects through the transportation of MSI2 into the nucleus. 3.5 MSI2 acts as an oncogene by directly interacting with c-MYC and is co-transported into the nucleus by IPO7. To further investigate how MSI2 promotes cancer progression, we RNA-sequenced IPO7-knockdown HeLa cells and performed a GSEA analysis (Fig. 7 A), which showed that the knockdown primarily led to the enrichment of MYC TARGET V1 and V2 (Fig. 7 B and 7 C). The MYC gene, a well-known oncogene, plays a pivotal role in the formation and progression of many tumors. Although our mass spectrometry analysis failed to detect changes in c-MYC expression following IPO7 knockdown, we identified a direct interaction between the MYC family and IPO7 using the BioGRID database (Supplementary Fig. 4A). Analysis of the TCGA cervical cancer database revealed a positive correlation between MYC and IPO7 expression levels (Supplementary Fig. 4B), and IPO7 expression levels was also positively correlated with MYC target pathways (Supplementary Fig. 4C). After confirming that IPO7 can bind to MSI2 and translocate it into the nucleus, we investigated whether MSI2 also interacts directly with c-MYC. Co-IP experiments validated a direct interaction between MSI2 and c-MYC in Hela and SiHa cells (Fig. 7 D and 7 E), leading us to suggest that IPO7 may synergistically transport the MSI2 and c-MYC complex into the nucleus, thereby promoting oncogenic effects. Furthermore, we found that MSI2 knockdown reduced the expression of c-MYC at both the mRNA and protein levels (Fig. 7 F- 7 I). Given that MSI2 is an RNA-binding protein, we examined whether it influences the stability of MYC mRNA. Following treatment of MSI2-knockdown cells with actinomycin D for 0, 3, and 6 hours, qPCR analysis showed that MSI2 knockdown significantly decreased the mRNA stability of c-MYC compared to the control group (Figs. 7 J and 7 K). Therefore, we suggest that IPO7 may systematically transport the MSI2-c-MYC complex into the nucleus, where MSI2 plays a crucial role in maintaining the stability of MYC, potentially facilitating the nuclear import of c-MYC to exert oncogenic activities. 3.6 Elevated MSI2 and IPO7 expression correlate with a worse prognosis in CC patients To investigate the oncogenic effect of IPO7 via MSI2 transport, we conducted a comprehensive examination of MSI2 expression in CC. Analysis using the GEO database (GSE6791, GSE63514) revealed a significant upregulation of MSI2 expression in CC (Fig. 8 A and 8 B, *P < 0.05). IHC staining of clinical samples (Fig. 8 C) showed a considerable elevation in MSI2 expression in CC compared to normal cervical tissues (Fig. 8 D), especially in advanced-stage cases (Fig. 8 E). The CCK-8 assay demonstrated a reduction in the proliferation capacity of both CC cell lines following MSI2 knockdown (Fig. 8 F and 8 G). Moreover, after MSI2 knockdown, the majority of cells exhibited arrest in the S phase (Fig. 8 H and 8 I, **P < 0.01). Survival analysis performed on CC patients from TCGA indicated that high MSI2 expression alone did not impact the prognosis (Fig. 8 J). However, CC patients with high MSI2 expression alongside high IPO7 expression experienced the poorest prognosis (Fig. 8 K and 8 L, P = 0.00069). Our results strongly suggest that IPO7 may serve as a promising target for CC treatment. In conclusion, our research demonstrates a correlation between elevated IPO7 levels and unfavorable outcomes in patients with CC. IPO7 contributes to the progression of CC by promoting cellular proliferation, migration, and invasion, and inhibiting apoptosis. This oncogenic effect is primarily due to the transportation of MSI2 and c-MYC into the nucleus by IPO7, which that recognizes and binds to the nuclear localization signal (NLS) region of MSI2. Thus, in the presence of Ran-GTPase, the MSI2-c-MYC complex is released from IPO7 and exhibits its oncogenic characteristics within the nucleus. The oncogenic effects of IPO7 occur through the presence of MSI2, as illustrated in Fig. 9 . If the cytoplasmic transport of MSI2 is hindered, it is subject to degradation via ubiquitination 4. Discussion Appropriate subcellular localization is crucial for the functioning of biological macromolecules, such as proteins and RNAs. Nuclear transport is a fundamental cellular process that regulates the localization of numerous macromolecules within the nucleus or cytoplasm 5 . Dysfunctional nuclear transport leads to changes in the physiological levels and spatiotemporal localization of tumor suppressors, proto-oncogenes, and other macromolecules, thereby affecting tumorigenesis and drug sensitivity in cancer cells 15 . Our study investigates the role of karyopherins, specifically IPO7, in the development of CC. By analyzing expression profiles of 27 karyopherins in the TCGA-CESC database and comparing them to control samples in the GEO database, we discovered IPO7 to be significantly upregulated in CC, exhibiting a significant prognostic correlation. IPO7 demonstrates elevated expression levels, as confirmed by histochemical analysis of our clinical samples. Consequently, we proceeded to conduct subsequent studies into IPO7's role in CC and its potential cargo proteins. Recent studies 16 have found that increased IPO7 expression in breast cancer is linked to poor prognosis, mainly by aiding USP22-AR nuclear transport. NUAK1, an AMPK-related kinase, plays a key role in tumorigenesis, relying on Importin β family transporters like KPNB1, IPO7, and IPO9 for nuclear transport 17 . Moreover, An NTS-derived myristoylated phosphomimetic peptide specifically blocked ERK1/2's interaction with IPO7, hindering ERK1/2's nuclear translocation 18 . These results, along with our study, demonstrates that IPO7 significantly contributes to tumorigenesis and tumor progression by transporting various cargo proteins. Knockdown of IPO7 significantly reduced cell proliferation, colony formation, migration, and invasion, and increased apoptosis in Hela and Siha cells. Furthermore, xenograft model experiments showed that IPO7 knockdown delayed tumor growth and decreased tumor burden, reinforcing its oncogenic role. Subsequently, we aimed to elucidate the mechanisms underlying IPO7's tumor-promoting effects, focusing on the cargo proteins that IPO7 transports. Cells efficiently regulate protein synthesis and degradation 19 . Non-functional proteins are quickly degraded and removed. Therefore, we knocked down IPO7 expression and isolated nuclear and cytoplasmic proteins for mass spectrometry analysis. We selected proteins with reduced nuclear expression after IPO7 knockdown for further study. We systematically screened 74 potential cargo proteins and performed enrichment analysis. These proteins are predominantly involved in the regulation of mRNA and the nuclear-cytoplasmic transport process. Notably, elevated IPO7 expression primarily facilitates protein localization. Our identification of MSI2, a member of the Musashi protein family, as a potential IPO7 cargo protein was based on its function as an RNA-binding protein that regulates mRNA stability and translation in key oncogenic pathways 13 . The NLS-mapper website 20 facilitated the prediction of IPO7's probable NLS sequence and location. Notably, the conservation of the NLS sequence suggests its crucial role in IPO7 binding and transportation. Direct interactions between IPO7 and MSI2 were confirmed using Co-IP and IF assays. Intriguingly, deleting the NLS region in MSI2 significantly reduced its binding to IPO7, especially in HeLa cells. Moreover, proliferation and colony formation assays further supported IPO7's role in tumor promotion via MSI2. The Musashi protein family, including MSI1 and MSI2, is a widespread and highly conserved group of RNA-binding proteins 21 . MSI proteins contain two RNA recognition motifs (RRMs) for interacting with target RNA. Notably, the first RRM exhibits a greater affinity towards RNA compared to the second RRM 22 , 23 . MSI proteins contain two RNA recognition motifs (RRMs) for interacting with target RNA 24 . MSI2 is widely expressed in various tumors, including leukemia, gastric, colorectal, and triple-negative breast cancers 25 , 26 , 27 , 28 . How does IPO7-mediated nuclear transport of MSI2 enhance its oncogenic effects? After RNA-sequencing HeLa cells with IPO7 knockdown, GSEA analysis revealed significant enrichment in the "MYC targets" pathway. Research shows that c-MYC is vital in CC, with MYC genes often being HPV integration targets 29 . Microhomology aids HPV integration near MYC, boosting c-Myc expression 30 . Microbial overgrowth, such as Prevotella, may also enhance persistent HPV-related cervical lesions by influencing c-Myc expression 31 . Can IPO7 facilitate MYC's nuclear transport? Using the BioGRID database, interactions between IPO7 and MYC were identified. Co-IP assays further confirmed that MSI2 directly binds to c-MYC, suggesting that IPO7 is capable of transporting both MSI2 and c-MYC into the nucleus. Additionally, reduced c-MYC expression at both the protein and RNA levels was observed following MSI2 knockdown, suggesting that MYC is targeted for degradation if it is not transported to the nucleus. Meanwhile, as an RNA-binding protein, MSI2 stabilizes MYC mRNA. Upon IPO7 knockdown, MSI2 fails to be transported into the nucleus, leading to its ubiquitination and reduced expression. Concurrently, MSI2 knockdown impedes MYC nuclear transport, thereby curtailing its oncogenic potential, and reduces MYC mRNA stability, resulting in decreased MYC expression. Additionally, we found that overexpression of MSI2 promotes CC cell proliferation. MSI2 has been identified as a target for small molecule inhibitors such as largazole and Aza-9, which have effectively reduced MSI2 levels and suppressed the proliferation of non-small cell lung cancer (NSCLC), chronic myeloid leukemia, and colon cancer cells 32 , 33 . A thorough analysis of the TCGA database showed that patients with high MSI2 and IPO7 expression had the worst prognosis. Thus, targeting the IPO7-MSI2 axis could be a more effective treatment strategy for CC. In this study, we observed that downregulating IPO7 led to decreased MSI2 protein expression in both the nucleus and cytoplasm. However, IPO7 knockdown did not affect MSI2 mRNA levels, implying a post-translational mechanism. Knockdown-induced IPO7 reduction impeded MSI2 nuclear transport, causing cytoplasmic degradation and loss of function. Notably, ubiquitination is a key mechanism for cellular protein degradation. Using the SMART database 34 , we identified seven ubiquitination sites on MSI2. This evidence indicates that IPO7 is crucial for nuclear transport of MSI2, underscoring the complex precision of cellular protein regulation. Ubiquitination of MSI2 is commonly observed in tumors. For instance, MSI2's interaction with LncRNA LINC00942 inhibits ubiquitination, thereby enhancing the stability and expression of target mRNAs, including c-MYC 26 . Intriguingly, our research revealed that MSI2 enhances c-MYC mRNA stability, and c-MYC can bind directly to MSI2. Both are transported to the nucleus by IPO7 to perform their functions. In conclusion, our study identifies IPO7 as a pivotal factor in the progression of CC. Its oncogenic role is mediated by nuclear transport of MSI2 and c-MYC. Targeting the IPO7-MSI2 interaction may lead to new therapeutic strategies for CC. Abbreviations CC cervical cancer DSS disease-specific survival GEO the Gene Expression Omnibus IHC Immunohistochemistry IP immunoprecipitation IPO7 Importin 7 MSI2 Musashi 2 NESs Nuclear Export Signals NLSs Nuclear Localization Signals OS overall survival PI propidium iodide RRM RNA-recognition motifs TCGA The Cancer Genome Atlas TMA tissues microarray Declarations Grant support: This work was supported by grants from the National Natural Science Foundation of China (82002730) The authors declare no conflict of interest. Ethics approval and consent to participate All of patients provided written informed consent (ethic approval number: 2020-YS-075), and all the experiments, including animal work (ethic approval number: 2020 − 0204) were approved by the Research Ethics Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital and complied with the Declaration of Helsinki. Consent for publication All authors have consented for publication Funding This research was supported by the National Natural Science Foundation of China (No: 82002730). Authors' contributions Q.Y.X., R.Z. and Y.B designed and supervised this study. W.Z.Z, J.C., and L.M. performed the data analysis, statistical analysis and finished the manuscript writing. Q.Y.X., and T.Q. performed the whole experiments. Z.H.A., Y.C.T and R.Z.J. provided data collection and technical support. W.Z.Z, T.Q., and J.W. contributed equally to this work. All authors participated in revising, reviewing, and approving the final submission. Acknowledgements We are grateful to Professor Zhigang Zhang and Lipeng Hu for their mentoring and assistance on this study. We are grateful to the Asia-Vector Biotechnology for assistance with our experiments. Availability of data and material The datasets utilized and/or examined in this research are accessible from the corresponding author upon request. The database of Cervical cancer data produced in this study can be accessed through the Gene Expression Omnibus (GEO) with accession numbers GSE9750, GSE7803, GSE6791, GSE63514, as well as the TCGA-CESC libraries. References Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: a cancer journal for clinicians 2021, 71(3): 209–249. WHO. Global strategy to accelerate the elimination of cervical cancer as a public health problem. 2020. Ji L, Chen M, Yao L. Strategies to eliminate cervical cancer in China. Frontiers in oncology 2023, 13. Markowitz LE, Unger ER. Human Papillomavirus Vaccination. The New England journal of medicine 2023, 388(19): 1790–1798. Yang Y, Guo L, Chen L, Gong B, Jia D, Sun Q. Nuclear transport proteins: structure, function, and disease relevance. Signal Transduction and Targeted Therapy 2023, 8(1). Strambio-De-Castillia C, Niepel M, Rout MP. The nuclear pore complex: bridging nuclear transport and gene regulation. Nature Reviews Molecular Cell Biology 2010, 11(7): 490–501. Wing CE, Fung HYJ, Chook YM. Karyopherin-mediated nucleocytoplasmic transport. Nature Reviews Molecular Cell Biology 2022, 23(5): 307–328. Koepp DM, Silver PA. A GTPase controlling nuclear trafficking: running the right way or walking RANdomly? Cell 1996, 87(1): 1–4. Kalita J, Kapinos LE, Lim RYH. On the asymmetric partitioning of nucleocytoplasmic transport - recent insights and open questions. Journal of cell science 2021, 134(7). Soniat M, Chook YM. Nuclear localization signals for four distinct karyopherin-β nuclear import systems. The Biochemical journal 2015, 468(3): 353–362. Zhou Y, Zhou B, Pache L, Chang M, Khodabakhshi AH, Tanaseichuk O, et al. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nature communications 2019, 10(1): 1523. Xu Q, Ge Q, Zhou Y, Yang B, Yang Q, Jiang S, et al. MELK promotes Endometrial carcinoma progression via activating mTOR signaling pathway. EBioMedicine 2020, 51: 102609–102609. Kudinov AE, Karanicolas J, Golemis EA, Boumber Y. Musashi RNA-Binding Proteins as Cancer Drivers and Novel Therapeutic Targets. Clinical cancer research: an official journal of the American Association for Cancer Research 2017, 23(9): 2143–2153. Sun J, Sheng W, Ma Y, Dong M. Potential Role of Musashi-2 RNA-Binding Protein in Cancer EMT. Onco Targets Ther 2021, 14: 1969–1980. Çağatay T, Chook YM. Karyopherins in cancer. Current opinion in cell biology 2018, 52: 30–42. Cai GX, Kong WY, Liu Y, Zhong SY, Liu Q, Deng YF, et al. Nuclear transport maintenance of USP22-AR by Importin-7 promotes breast cancer progression. Cell Death Discov 2023, 9(1): 211. Palma M, Riffo EN, Suganuma T, Washburn MP, Workman JL, Pincheira R, et al. Identification of a nuclear localization signal and importin beta members mediating NUAK1 nuclear import inhibited by oxidative stress. Journal of cellular biochemistry 2019, 120(9): 16088–16107. Plotnikov A, Flores K, Maik-Rachline G, Zehorai E, Kapri-Pardes E, Berti DA, et al. The nuclear translocation of ERK1/2 as an anticancer target. Nature communications 2015, 6: 6685. Zhu J, Thompson CB. Metabolic regulation of cell growth and proliferation. Nat Rev Mol Cell Biol 2019, 20(7): 436–450. Kosugi S, Hasebe M, Tomita M, Yanagawa H. Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs. Proceedings of the National Academy of Sciences of the United States of America 2009, 106(25): 10171–10176. Siddall NA, McLaughlin EA, Marriner NL, Hime GR. The RNA-binding protein Musashi is required intrinsically to maintain stem cell identity. Proceedings of the National Academy of Sciences of the United States of America 2006, 103(22): 8402–8407. Kurihara Y, Nagata T, Imai T, Hiwatashi A, Horiuchi M, Sakakibara S, et al. Structural properties and RNA-binding activities of two RNA recognition motifs of a mouse neural RNA-binding protein, mouse-Musashi-1. Gene 1997, 186(1): 21–27. Miyanoiri Y, Kobayashi H, Imai T, Watanabe M, Nagata T, Uesugi S, et al. Origin of higher affinity to RNA of the N-terminal RNA-binding domain than that of the C-terminal one of a mouse neural protein, musashi1, as revealed by comparison of their structures, modes of interaction, surface electrostatic potentials, and backbone dynamics. J Biol Chem 2003, 278(42): 41309–41315. Sakakibara S, Nakamura Y, Satoh H, Okano H. Rna-binding protein Musashi2: developmentally regulated expression in neural precursor cells and subpopulations of neurons in mammalian CNS. The Journal of neuroscience: the official journal of the Society for Neuroscience 2001, 21(20): 8091–8107. Zhang J, Duan Y, Wu P, Chang Y, Wang Y, Hu T, et al. Clonal evolution dissection reveals that a high MSI2 level promotes chemoresistance in T-cell acute lymphoblastic leukemia. Blood 2024, 143(4): 320–335. Zhu Y, Zhou B, Hu X, Ying S, Zhou Q, Xu W, et al. LncRNA LINC00942 promotes chemoresistance in gastric cancer by suppressing MSI2 degradation to enhance c-Myc mRNA stability. Clinical and translational medicine 2022, 12(1): e703. Zhang X, Su K, Liu Y, Zhu D, Pan Y, Ke X, et al. Small Molecule Palmatine Targeting Musashi-2 in Colorectal Cancer. Front Pharmacol 2021, 12: 793449. Li M, Li AQ, Zhou SL, Lv H, Wei P, Yang WT. RNA-binding protein MSI2 isoforms expression and regulation in progression of triple-negative breast cancer. Journal of experimental & clinical cancer research: CR 2020, 39(1): 92. Wang X, Jia W, Wang M, Liu J, Zhou X, Liang Z, et al. Human papillomavirus integration perspective in small cell cervical carcinoma. Nature communications 2022, 13(1): 5968. Hu Z, Zhu D, Wang W, Li W, Jia W, Zeng X, et al. Genome-wide profiling of HPV integration in cervical cancer identifies clustered genomic hot spots and a potential microhomology-mediated integration mechanism. Nature genetics 2015, 47(2): 158–163. Dong B, Huang Y, Cai H, Chen Y, Li Y, Zou H, et al. Prevotella as the hub of the cervicovaginal microbiota affects the occurrence of persistent human papillomavirus infection and cervical lesions in women of childbearing age via host NF-κB/C-myc. J Med Virol 2022, 94(11): 5519–5534. Wang M, Sun XY, Zhou YC, Zhang KJ, Lu YZ, Liu J, et al. Suppression of Musashi–2 by the small compound largazole exerts inhibitory effects on malignant cells. International journal of oncology 2020, 56(5): 1274–1283. Lan L, Liu J, Xing M, Smith AR, Wang J, Wu X, et al. Identification and Validation of an Aspergillus nidulans Secondary Metabolite Derivative as an Inhibitor of the Musashi-RNA Interaction. Cancers (Basel) 2020, 12(8). Letunic I, Khedkar S, Bork P. SMART: recent updates, new developments and status in 2020. Nucleic acids research 2021, 49(D1): D458-d460. Additional Declarations (Not answered) Supplementary Files SupplementaryFigure1.tif SupplementaryFigure2.tif SupplementaryFigure3.tif SupplementaryFigure4.tif SupplementaryFigurelegend.docx SupplementaryFile1.docx Supplementarytable1.docx Supplementarytable2.docx WBXXXXXXXXXXXX.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5254983","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":369872180,"identity":"f62a867b-1243-4665-8e48-569b4538f01e","order_by":0,"name":"Xu 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11:00:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5254983/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5254983/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":67620705,"identity":"df949ea3-f08e-4a7e-8f1b-1e3592d2ef46","added_by":"auto","created_at":"2024-10-28 07:06:00","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2680036,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIPO7 is highly expressed in cervical cancer and is associated with unfavorable prognosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. mRNA expression levels of IPO7 in CC (n=21) and normal cervical tissues (n=10) in the GSE7803 database (\u003cem\u003et\u003c/em\u003e-test). B. mRNA expression levels of IPO7 from normal cervical tissues(n=24), Squamous Intraepithelial Lesion (SIL, n=76), and CC(n=28) lesions in the GSE63514 database (\u003cem\u003et\u003c/em\u003e-test). C. Kaplan-Meier analysis of overall survival based on IPO7 expression in CC patients, using TCGA database. D. Representative immunohistochemical images showing IPO7 expression from Normal, LSIL, HSIL, and CC tissues according to samples in TMA. Scale bar, 50 μm. E. The proportion of IPO7 expression was assessed by blinded IHC analyses in normal tissues (n = 15), LSIL (n = 43), HSIL(n=35) and CC (n = 100) (chi-square test). F-H. Relationship between IPO7 expression level and FIGO stages (F), TNM stages (G), and tumor tissue types (H) of CC samples in TMA (Kruskal-Walli’s test). * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5254983/v1/a18b0301d610f18da58d5ca5.jpg"},{"id":67620717,"identity":"c41ef759-649e-43ef-98b6-7ec1a5317852","added_by":"auto","created_at":"2024-10-28 07:06:00","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2049235,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of IPO7 inhibited the growth and metastasis of CC cells and promoted apoptosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Knockdown efficiency of IPO7 was detected by qRT-PCR assay. B. The cell proliferation capability after IPO7 knockdown in Hela and SiHa cells was evaluated by CCK-8 assay. Values are means ± SD, n = 3. *** p \u0026lt; 0.001. C. The colony formation assay was performed in Hela and SiHa cells to assess the cell proliferation capability, and the representative images of the colony formation assay are shown. D. Quantitative analysis of the colonies from panels (C). E. Detection of Apoptosis by Flow Cytometry in Hela and SiHa cells after IPO7 knockdown. F. Quantitative analysis of cell apoptosis rates from panels (E). G. Western blotting was used to detected the expression of Bcl-2, caspase 3/cleaved-caspase3, caspase7/ cleaved caspase7 and IPO7 in Hela and SiHa cells after IPO7 knockdown. H and I. The Transwell assay was performed to assess the effect of IPO7 knockdown on the migration and invasion of Hela and SiHa cells. Quantitative analysis from panels (H and I). * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5254983/v1/1c4e4fdaaddb88c9260cb100.jpg"},{"id":67622304,"identity":"3a1d40da-f8c8-401d-ba64-9a923e1733eb","added_by":"auto","created_at":"2024-10-28 07:14:00","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2845770,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of IPO7 suppresses tumor proliferation\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e in vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA and B. Hela cells were infected with either the lentivirus expressing IPO7-knockdown shRNA or control shRNA, and the cell clones were selected for puromycin resistance. IPO7-shRNA or control shRNA Hela cells were injected into BALB/c female mice which were randomly divided into two groups (n = 5). Mice were sacrificed at 5 weeks after injection. Tumors were excised from the mice and weighed. C. The growth curve of subcutaneous xenograft tumors. D. Statistical analysis of tumor weight measured in mice after 5 weeks. E. Representative images of Ki67 staining in xenograft tumors from IPO7-shRNA and control shRNA mice. F. statistical analysis from panels(E). * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5254983/v1/e89d169eefe2530693adf82c.jpg"},{"id":67620718,"identity":"6572b1d0-c3bc-423d-bcee-986f42a9f7e5","added_by":"auto","created_at":"2024-10-28 07:06:00","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2420206,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMSI2 is a potential target protein transported by IPO7 in CC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. After knockdown of IPO7, nuclear cytoplasm was isolated and mass spectrometry was utilized to identify alterations in nucleoproteins (by Figdraw); B. The top 200 genes most closely related to IPO7 were enriched using Metascape (http://metascape.org); C. 74 differential proteins were identified through mass spectrometry (|FC|\u0026gt; 1.5; P \u0026lt; 0.01), and GO and KEGG enrichment analyses were conducted; D. The NLS-Mapper website (http://NLS-mapper.iab.keio.ac.jp) was used to predict NLS sequences of MSI2 in various species, with different cutoff scores indicating diverse NLS localizations. A score of 5 suggests that the NLS is localized in both the cytoplasm and nucleus; E. A comparative analysis was conducted on the expression of MSI2 in 306 tumor cases and 13 normal control cases sourced from the TCGA-CESC and GTEx databases (**p\u0026lt;0.01); F. Correlation analysis of MSI2 and IPO7 mRNA expression in TCGA-CESC; G and H. Following the knockdown of IPO7, the expression of MSI2 was assessed at both the protein level (G) and mRNA level (H) in Siha and Hela cells (N.S. no significance, **P\u0026lt; 0.01). I. Western blotting was used to identify the expression of MSI2 in the cytoplasm and nucleus following the knockdown of IPO7 expression. J. The ubiquitination of MSI2 in Hela and SiHa cells transfected with HA-Ub expression constructs was demonstrated through anti-MSI2 immunoprecipitation and anti-HA Western blot analysis.\u003c/p\u003e","description":"","filename":"Figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5254983/v1/ffd270ceec8bb67fb217e71e.jpg"},{"id":67620708,"identity":"eb8b40b2-0d7a-4d0f-928e-efa5e60a8e3a","added_by":"auto","created_at":"2024-10-28 07:06:00","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2533859,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMSI2 directly interacts with IPO7 and deletion of MSI2-NLS affects its binding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA and B. Co-IP was performed in Hela and SiHa cell lines to detect whether there is a direct interaction between IPO7 and MSI2; C. Immunofluorescence assays were conducted to verify the colocalization of IPO7 and MSI2 (scale bar 10um); D. Based on the amino acid sequence information of MSI2 provided by the uniprot website (https://www.uniprot.org/), the protein structure model of MSI2 was generated in the SMART website (\u003ca href=\"http://smart.embl.de/\"\u003ehttp://smart.embl.de/\u003c/a\u003e) and the potential post-translational modification targets were predicted. E and F. Exogenous plasmids labeled with IPO7-Flag, MSI2-HA or MSI2-Del-HA (NLS-del) were transfected into Siha and Hela cells, and the direct interaction between IPO7 and MSI2 was verified by Co-IP.\u003c/p\u003e","description":"","filename":"Figure5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5254983/v1/057a9eaa1cc7f5aef8ee7079.jpg"},{"id":67622306,"identity":"b9153b50-5673-4e1f-8bdf-4f02f11f34e2","added_by":"auto","created_at":"2024-10-28 07:14:00","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1732874,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe oncogenic role of IPO7 is played by mediating MSI2 nuclear transport.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn Hela and SiHa cells, carcinogenic effects were evaluated by transfecting exogenous plasmids to either overexpress IPO7-Flag (Flag-IPO7), knock down MSI2 (MSI2-si2), or simultaneously overexpress IPO7 and knock down MSI2 (FLAG-IPO7 + MSI2-si2). A and B represent the mRNA relative expression of IPO7 in the two cell lines; C and D represent the mRNA relative expression of MSI2 in the two cell lines (N.S. no significant difference, *** p \u0026lt; 0.001, t-test). E and F. The cell proliferation capability of different groups (NC, FLAG-IPO7, MSI2-si2, FLAG-IPO7 + MSI2-si2) in Hela and SiHa cells was evaluated by CCK-8 assay. (Values are means ± SD, n = 3, ** compared with NC, ## compared with FLAG-IPO7, **p \u0026lt; 0.01 ## p \u0026lt; 0.01). G-I. The colony formation assay was performed in Hela and SiHa cells to assess the cell proliferation capability of different groups (NC, FLAG-IPO7, MSI2-si2, FLAG-IPO7 + MSI2-si2), and the representative images of the colony formation assay are shown. H and I. Quantitative analysis of the colonies from panels (G) (Values are means ± SD, n = 3, ** compared with NC, ## compared with FLAG-IPO7, **p \u0026lt; 0.01 ## p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Figure6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5254983/v1/fa7d574fb1c51f7d0e873587.jpg"},{"id":67620714,"identity":"bc3aea4b-205e-47c7-9e23-baf859b3a11a","added_by":"auto","created_at":"2024-10-28 07:06:00","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1967807,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMSI2 acts as an oncogene by directly interacting with c-MYC and stabilizes c-MYC mRNA. \u003c/strong\u003eA. IPO7 knockdown in Hela cells was performed by second-generation sequencing, and the sequencing results were analyzed by GSEA, as shown in this figure; B. GSEA was performed on the Hela control group and IPO7 knockdown group (Hallmark dataset); C. Enrichment analysis is mainly concentrated on the MYC target; D and E. Co-IP was performed in Hela and SiHa cell lines to detect whether there is a direct interaction between c-MYC and MSI2; F and G. Western blot detection of c-MYC protein expression in SiHa (F) and Hela (G) cells after MSI2 knockdown; H and I. q-PCR detection of mRNA expression of c-MYC in SiHa (H) and Hela (I) cells after MSI2 knockdown. J and K. mRNA level alteration of c-MYC in NC group and MSI2-knockdown group (MSI2-si1 and MSI2-si2) after treatment with actinomycin D for 0,3 or 6 h in SiHa and Hela cells. ∗P \u0026lt; 0.05, ∗∗P \u0026lt; 0.01, ∗∗∗P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5254983/v1/4fa8d41483ddca93582cc85b.jpg"},{"id":67622724,"identity":"4d7ebd97-e042-4e3a-b486-eef3e39f604d","added_by":"auto","created_at":"2024-10-28 07:22:00","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2224846,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElevated MSI2 and IPO7 expression correlate with a worse prognosis in CC patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA and B. MSI2 expression in Cervical Cancer (CC) tissues, CIN, and normal controls was examined using the GEO database (GSE6791, GSE63514) (*p \u0026lt; 0.05, t-test); C. Representative IHC images displaying MSI2 expression in Normal and CC tissues from Tissue Microarray (TMA). Scale bar: 200 μm and 50 μm; D. The proportion of IPO7 expression was assessed by blinded IHC analyses in normal tissues (n = 15) and CC (n = 100) (***p\u0026lt;0.001, Fisher’s exact probability method); E. Examination of the relationship between MSI2 expression levels and FIGO stages in CC samples from TMA; F and G. Cell proliferation capability subsequent to MSI2 knockdown in Hela and SiHa cells was assessed via CCK-8 assay (Values as means ± SD, n = 3, **p \u0026lt; 0.01); H and I. Flow cytometry was utilized to identify cell cycle changes after MSI2 knockdown (Values as means ± SD, n = 3, **p \u0026lt; 0.01); J. Kaplan-Meier analysis of overall survival based on MSI2 expression in CC patients using the TCGA database; K. The gene expression of IPO7, survival time, and status in high MSI2 expression CC patients (grouped by median of MSI2 expression) according to TCGA dataset. The scatterplot illustrates gene expression from low to high, with different colors indicating distinct groups divided based on the quartile of IPO7 expression; L. Kaplan-Meier survival examination of overall survival linked to IPO7 expression in high MSI2 expression patients from TCGA dataset. Comparison among various groups (groups as per K) was conducted using the log-rank test.\u003c/p\u003e","description":"","filename":"Figure8.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5254983/v1/91037e534b098c3474e3adbe.jpg"},{"id":67622310,"identity":"2bffa9c9-ed3c-4310-9aba-69cfc6b4a62f","added_by":"auto","created_at":"2024-10-28 07:14:00","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":831482,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic summary of IPO7 promoting CC progression by transporting MSI2 to the nucleus (by Figdraw).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure9.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5254983/v1/22fe799ede8e5ba46f0eb852.jpg"},{"id":71664814,"identity":"aa14a8e2-5d3a-4c04-a1c3-04889c2c1a6d","added_by":"auto","created_at":"2024-12-17 14:09:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20258092,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5254983/v1/037b4f23-01f5-4450-9f7d-cc7cfd1ec85d.pdf"},{"id":67622303,"identity":"64479542-1f13-423b-9370-80ad469847ab","added_by":"auto","created_at":"2024-10-28 07:14:00","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1992956,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-5254983/v1/c41726ab13d117b84d96edd1.tif"},{"id":67620720,"identity":"6ab3d18c-72d9-4cdc-b036-e4b1bdbd2c55","added_by":"auto","created_at":"2024-10-28 07:06:01","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":35225332,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-5254983/v1/e0a9bb7232e6da649e8c482d.tif"},{"id":67620704,"identity":"e5d33e75-e7b3-40ec-b017-b8735915d875","added_by":"auto","created_at":"2024-10-28 07:06:00","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":416152,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-5254983/v1/b938e99294c7bb5aa7def73c.tif"},{"id":67620715,"identity":"d205f38c-dbcc-46df-bfa7-5875df346cba","added_by":"auto","created_at":"2024-10-28 07:06:00","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":11324564,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-5254983/v1/cba498fcfa7abd8b2e88389d.tif"},{"id":67622723,"identity":"7af235b8-0a0f-4578-a5b7-71a8a2fd3f0e","added_by":"auto","created_at":"2024-10-28 07:22:00","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":16168,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigurelegend.docx","url":"https://assets-eu.researchsquare.com/files/rs-5254983/v1/2edb3bc101cf218210b2e9c3.docx"},{"id":67620712,"identity":"71bfed08-b016-4ba1-80c9-c09907b15e56","added_by":"auto","created_at":"2024-10-28 07:06:00","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":23501,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5254983/v1/f6146d440ccb60ea89d5f0da.docx"},{"id":67622309,"identity":"7308e2d4-f519-4903-a0f6-6b6d1c14995b","added_by":"auto","created_at":"2024-10-28 07:14:00","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":22989,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5254983/v1/04412eb16e08f7b4dac0ba2b.docx"},{"id":67622305,"identity":"741d1af8-a06c-4116-bc80-77405b306f62","added_by":"auto","created_at":"2024-10-28 07:14:00","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":20320,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5254983/v1/563e9b750e50aa24b088211c.docx"},{"id":67620721,"identity":"f000f9e8-1715-47a9-8024-1e78e13482c6","added_by":"auto","created_at":"2024-10-28 07:06:01","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":1306272,"visible":true,"origin":"","legend":"","description":"","filename":"WBXXXXXXXXXXXX.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5254983/v1/6643a2c744f4abd8fc38dc80.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"Importin 7 Mediated Nuclear Transport of MSI2 as a Therapeutic Target in Cervical Cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCervical cancer (CC) is the predominant malignancy affecting the female reproductive system. According to the Global Cancer Statistics 2020 report, CC is the fourth leading cause of cancer-related mortality in women, with an estimated 604,000 new cases and 342,000 deaths worldwide\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In 2020, the World Health Organization Assembly introduced a comprehensive initiative to eradicate CC, including strategies such as HPV vaccination, CC screening, and management of precancerous lesions\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The management of CC faces challenges in achieving satisfactory outcomes, largely due to the difficulty of implementing interventions on a large scale\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The development of CC associated with high-risk HPV infections is a multifaceted process involving various stages and factors, which complicates the effectiveness of vaccines in improving the prognosis of HPV-infected individuals or CC patients\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. T Targeted therapy represents a significant advancement in treating late-stage CC patients who have undergone standard surgical procedures. Therefore, a thorough investigation into the pathogenesis of CC and the discovery of new molecular targets is essential to improve prognostic outcomes.\u003c/p\u003e \u003cp\u003eNuclear transport proteins, or karyopherins, are crucial in mediating the transport of signals and substances between the nucleus and cytoplasm\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. These proteins facilitate the translocation of macromolecular proteins through the nuclear pore complex, which is essential for the transportation process\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The superfamily of nuclear transport proteins is comprised of α and β families, each possessing distinct structural and functional characteristics.\u003c/p\u003e \u003cp\u003eThe Karyopherin-β family, encompassing importins, exportins, and biportins, plays a vital role in mediating the transport of macromolecules, especially proteins, through the nuclear pore complex (NPC) by recognizing specific nuclear localization signals (NLSs) or nuclear export signals (NESs)\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. This transport process relies on the GTPase RanGTP for cargo binding and dissociation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Under normal conditions, karyopherins precisely transport specific cargo proteins to their designated locations, thus facilitating proper physiological functions. Conversely, dysfunction of karyopherins results in the mislocalization of nucleo-cytoplasmic elements, including proto-oncogenes, tumor suppressor genes, and cell cycle regulatory proteins, thus contributing to tumor development\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Targeting karyopherins to disrupt nuclear-cytoplasmic communication and material transport in cancer cells could be a promising strategy to enhance the survival rates of individuals with CC.\u003c/p\u003e \u003cp\u003eImportin 7 (IPO7), a notable member of the karyopherin family, belongs to the β nuclear transport protein family. The sequence features two antiparallel helices, enhancing its binding affinity with associated proteins\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, and thus making it a key component of the nuclear-cytoplasmic transport mechanism. Nonetheless, the specific biological functions of IPO7 in CC remain insufficiently understood and necessitate further investigation. This study aims to clarify the role of IPO7 in CC progression and, concurrently, to identify and validate potential IPO7 cargo proteins by isolating cytoplasmic and nuclear proteins and conducting mass spectrometry analysis.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Clinical samples and database analysis\u003c/h2\u003e \u003cp\u003eThe CC tissues microarray (TMA) containing 15 normal cervix tissues, 78 cervical intraepithelial neoplasia tissues, and 100 CC tissues from Shanghai Jiao Tong University Affiliated Sixth People\u0026rsquo;s Hospital. All tissue specimens were evaluated by two pathologists. This research was authorized by the Research Ethics Committee of Shanghai Jiao Tong University Affiliated Sixth People\u0026rsquo;s Hospital(2020-YS-075), and written informed consent was obtained from all patients before this study. CC data were acquired from The Cancer Genome Atlas (TCGA) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://portal.gdc.cancer.gov/\u003c/span\u003e\u003cspan address=\"https://portal.gdc.cancer.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and normal cervix uterus data were acquired from The Cancer Genome Atlas (GTEx) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.gtexportal.org/home/\u003c/span\u003e\u003cspan address=\"https://www.gtexportal.org/home/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The gene expression profile results were downloaded from the Gene Expression Omnibus database (GSE9750, GSE7803, GSE6791, GSE63514) at the NCBI. Database between a high-IPO7 expression group and a low-IPO7 expression group based on the median of TCGA-CESC IPO7 profiles and Kaplan\u0026ndash;Meier analysis was conducted by the above grouping method. Gene Ontology Metascape analysis was performed using Metascape software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://metascape.org/gp/index.html\u003c/span\u003e\u003cspan address=\"http://metascape.org/gp/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ) using default settings\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Culture of cell lines and transfection\u003c/h2\u003e \u003cp\u003eHuman CC Cell lines HeLa and SiHa were preserved in Shanghai Cancer Institute. All of the cells were cultured in a suggested medium (Invitrogen, USA) according to ATCC protocols. siRNA and plasmids transfection were performed using Lipofectamine 2000 (Invitrogen, USA). siRNAs targeting various genes were purchased from Asia-Vector Biotechnology (Shanghai, China), and the sequences are listed in Supplementary Files. To generate stable cell lines with IPO7 knockdown, Hela cells were infected with LV3-puro-shIPO7 (sh1) and cell clones were selected for puromycin resistance.\u003c/p\u003e \u003cp\u003eHuman IPO7 coding sequence and Ubiquitin coding sequence was amplified and cloned into the expression plasmid PcDNA3.1\u0026thinsp;+\u0026thinsp;for overexpressing IPO7-FLAG and UB-HA. The entire coding sequences of wild-type MSI2 and del-type MSI2 (deleted the NLS region) were subcloned into the expression plasmid PcDNA3.1+. The detailed information of plasmids and used primers is given in supplemental file.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Quantitative real-time PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eThe total RNA was isolated from CC cell lines, quantified by NANO 2000 (Thermo Fisher Scientific, USA), subjected to reverse transcription and subsequently conducted to quantitative real-time PCR utilizing a 7500 Real-time PCR system (Applied Biosystems, USA). Relative mRNA expression was calculated by the 2\u0026minus;△△CT method, and was normalized by endogenous β-actin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Western blotting analysis\u003c/h2\u003e \u003cp\u003eFor Western blotting, total protein was extracted using cell lysis buffer (Beyotime, China). Otherwise, subcellular nuclear cytoplasm protein fractionation was proceeded using NE-PER Nuclear and Cytoplasm Extraction Reagents (Thermo Fisher Scientific, USA) according to instructions. Western blotting was performed as previously describe\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Concentrations of antibodies were used according to the manufacturer's instructions and the antibodies are listed in Supplementary File 1 (Table\u0026nbsp;4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Co-Immunoprecipitation (Co-IP)\u003c/h2\u003e \u003cp\u003eFor Co-IP, immunoglobulin G (IgG) or specific antibody was incubated with protein G-agarose beads for 30 min at room temperature, and immunoprecipitated with protein at 4\u0026deg;C overnight. Then the beads were washed three times with PBS and eluted by 1x protein SDS loading buffer. Extracted proteins were separated by SDS-PAGE in a 7\u0026ndash;15% gel, transferred to a nitrocellulose membrane (Millipore, Burlington, USA), and blocked in 5% BSA. Then, the nitrocellulose membrane was exposed to primary antibodies and species-specific secondary antibodies. Antibodies are presented in Supplementary file1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Immunohistochemistry (IHC) analysis\u003c/h2\u003e \u003cp\u003eBriefly, the primary antibodies anti-IPO7 (1:200, Abcam), anti-MSI2 (1:100, Abcam) were incubated with the slides first. Each tissue sample was scored according to the proportion of stained cells (0\u0026ndash;5% scored \u0026ldquo;\u0026ndash;\u0026rdquo;, 5\u0026ndash;25% scored \u0026ldquo;+\u0026rdquo;, 25\u0026ndash;50% scored \u0026ldquo;++\u0026rdquo;, 50\u0026ndash;75% scored \u0026ldquo;++\u0026rdquo;, and 75\u0026ndash;100% scored \u0026ldquo;+++\u0026rdquo;). The scoring was judged independently by two pathologists in a blinded manner. We designated the final score as a high or low expression group as follows: a score of \u0026ldquo;-\u0026rdquo; and \u0026ldquo;+\u0026rdquo; were defined low expression of IPO7 (or MSI2) and a score of \u0026ldquo;++\u0026rdquo; and \u0026ldquo;+++\u0026rdquo; were defined high expression IPO7 (or MSI2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Immunofluorescence (IF) assay\u003c/h2\u003e \u003cp\u003eCells were cultured on coverslips in 8-well plates, and were fixed and permeabilized with 4% paraformaldehyde and 0.05% Triton X-10. BSA (1%) was used for blocking. Then cells were probed with the anti-IPO7 antibody (1:200, Abcam) and anti-MSI2 antibody (1:50, Abcam) overnight at 4\u0026deg;C, followed by an Alexa Fluor 594 conjugated anti-rabbit antibody (1:200 Jackson) and Fluor 488\u0026ndash;conjugated anti-mouse antibody (1:200, Jackson). DAPI (4\u0026prime;,6-diamidine-2\u0026prime;-phenylindole, 1:1000, Invitrogen) was used for nuclear staining. IF images were captured by confocal laser scanning microscope (Carl Zeiss).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Cell proliferation assay\u003c/h2\u003e \u003cp\u003eCell proliferation was measured by using a Cell Counting Kit-8 (CCK-8) kit (Dojindo, Japan). The cells were cultured on 96-well plates, and OD450 was measured 1 h after addition of CCK-8 at 0, 24, 48, 72, and 96 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Colony formation assay\u003c/h2\u003e \u003cp\u003eThe CC cells (2 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e) were cultured on per well of a 6-well plate for 2 weeks. Then the cell culture plates were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Photographs were acquired and the cell numbers were counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Cell migration and invasion assay\u003c/h2\u003e \u003cp\u003eThe Transwell migration assay was performed using Corning chambers (Corning, USA), featuring 8-\u0026micro;m pores. A total of 1 \u0026times; 105 cells were resuspended in serum-free culture medium and seeded in the upper chamber, conversely complete medium containing 10% FBS was placed in the lower chamber. After 24-hour incubation, the migrated cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet, and then observed and quantified under an inverted microscope. Transwell invasion assay were performed as previously described except the chambers were coated with Matrigel (Corning, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Cell apoptosis assay\u003c/h2\u003e \u003cp\u003eTarget cells were cultured under serum deprivation for 24 hours to assess apoptosis. Subsequently, the cells were detached with 0.25% trypsin and resuspended for propidium iodide (PI) and Annexin V-FITC staining. The samples were processed to FACS Canto-Plus flow cytometer (BD Biosciences).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Cell cycle assay\u003c/h2\u003e \u003cp\u003eCell cycle distribution was assessed by flow cytometry using the cell cycle/apoptosis analysis kit. The target cells were fixed in pre-cooling 75% ethanol for 12h at 4\u0026deg;C, and incubated in PI for 30 min at 37\u0026deg;C. The cells were harvested for flow cytometry analysis (BD Biosciences) and analysed with the Modfit LT 5.1 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Mouse xenograft model\u003c/h2\u003e \u003cp\u003ea total of 1\u0026times;106 IPO7-shRNA or control shRNA Hela cells were injected subcutaneously in the axilla of BALB/c female mice (5\u0026ndash;6 weeks of age, n\u0026thinsp;=\u0026thinsp;5 per group). The tumor diameters were measured by Vernier caliper every 7 days. The tumor volume was estimated based on the equation V\u0026thinsp;=\u0026thinsp;1/2 (a x b x b). After 5 weeks, the mice were killed to dissect and weight xenograft tumors. The xenograft tumor samples were fixed and used for subsequent IHC analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Ubiquitination assay\u003c/h2\u003e \u003cp\u003eCells were transfected with the HA-Ubiquitin plasmid, treated with the proteasome inhibitor MG132 (25 mM) (Sigma, M7449) for 6 h, and subsequently lysed in ubiquitination assay buffer. The cell lysis was co-immunoprecipitated with protein G-agarose beads conjugated with MSI2 antibody, and Western blotting was performed with an anti-HA antibody (Proteintech, China) to detect ubiquitinated MSI2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Liquid Chromatography\u0026ndash;mass spectrometry\u003c/h2\u003e \u003cp\u003eTryptic peptides were dissolved in 0.1% formic acid (solvent A) and separated utilizing the EASY-nLC 1000 ultra-high performance liquid system. Subsequently, the peptides undergo separation by the ultra-high performance liquid system, followed by injection into the NSI ion source for ionization and analysis using Orbitrap Fusion mass spectrometry. The ion source operates at 2.2 kV and facilitates the detection and analysis of peptide precursor ions and their secondary fragments utilizing high-resolution Orbitrap.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16 RNA-seq\u003c/h2\u003e \u003cp\u003eHela cells (with IPO7-shRNA and control RNA) were seeded into a 6 cm plate. Cell lysates were prepared using Trizol (TaKaRa) and then stored at \u0026minus;\u0026thinsp;80◦C. The following steps were performed by Newcore Biotech. Cell lysate was sequentially treated with chloroform, isopropanol, and ethanol to extract total RNA. RNA purity was checked using the NanoPhotometer\u0026reg; spectrophotometer (IMPLEN, Los Angeles, USA). RNA concentration was measured using Qubit\u0026reg; RNA Assay Kit in Qubit\u0026reg;2.0 Flurometer (Life Technologies, Carlsbad, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, USA). Sequencing libraries were generated using NEBNext\u0026reg; UltraTM RNA Library Prep Kit for Illumina\u0026reg; (NEB, Ipswich, USA) following manufacturer\u0026rsquo;s recommendations. The gene set enrichment analysis (GSEA) analysis was performed using GSEA software following the manufacturer\u0026rsquo;s introduction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.17 mRNA stability assay\u003c/h2\u003e \u003cp\u003eCells (2 \u0026times; 10^5 per well) were seeded into a 12-well plate. Transcriptional inhibition was achieved using Actinomycin D at a final concentration of 2 \u0026micro;g/mL (Leagene, Beijing, China). Total RNA was isolated at 0-, 3-, and 6-hours post-treatment using Trizol reagent. Subsequently, RNA was extracted, reverse transcribed into cDNA, and subjected to quantitative real-time PCR (qRT-PCR) as previously described.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.16 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical analyses were performed using SPSS Statistics software (SPSS 20, USA) and GraphPad Prism 8.0 software. The data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and were calculated using the two-tailed Student\u0026rsquo;s t-test. The relationship between IPO7/MSI2 expression level and clinical parameters was determined by the χ2 test and Fisher\u0026rsquo;s exact test. Survival analysis was conducted using the Kaplan-Meier method. The survival curves were analyzed using the log-rank test. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant statistically for all tests.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Result","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.1 IPO7 is highly expressed in CC and is associated with unfavorable prognosis\u003c/h2\u003e \u003cp\u003eThis study aims to investigate the role of nuclear transport proteins in the development of CC. We analyzed the expression profiles of 27 known nuclear transport proteins in the TCGA-CESC database to evaluate their potential as prognostic indicators. Furthermore, comparative analysis of expression levels in control and tumor samples from the GEO database identified IPO7 and RanBP17 as genes with increased expression and significant prognostic value in CC. Thus, the present study concentrated on elucidating the role of IPO7 in CC progression (Supplementary Fig.\u0026nbsp;1 and Supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eThrough the integration of TCGA and GEO databases, a substantial increase in IPO7 expression was observed in CC tissues relative to normal controls. Additionally, an escalation in IPO7 expression was noted in tandem with the advancement of cervical lesions (Supplementary Fig.\u0026nbsp;1B, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). High IPO7 expression was associated with a poor prognosis for patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). However, no correlation was detected between elevated IPO7 expression and patient age, BMI, histopathological type, or clinical stage when analyzing the TCGA database. Moreover, individuals with high IPO7 expression had decreased overall survival (OS) and disease-specific survival (DSS) rates (Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, we obtained clinical specimens from patients diagnosed with CC who underwent surgical treatment at our hospital over the past five years. IPO7 protein expression was evaluated via immunohistochemical staining (IHC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the statistical analysis associated with the clinical data. Our results demonstrate an incremental rise in IPO7 protein expression in tandem with the severity and stage of cervical lesions (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation between IPO7 expression and clinicopathological features in cervical cancer (n\u0026thinsp;=\u0026thinsp;100)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eExpression of IPO7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003etotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eLow (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eHigh (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eP value\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eage\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;55 years old\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 (41.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42 (58.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.121\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e\u0026ge;\u0026thinsp;55 years old\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 (25.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21 (75.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMenopause\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eYes\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32 (39.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50 (61.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.532\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNo\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (27.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13 (72.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePregnancy\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eYes\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20 (37.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33 (62.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.436\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNo\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17 (45.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20 (54.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHPV infection\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eYes\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35 (40.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58 (60.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.942\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNo\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (47.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 (52.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eClinical Stage (FIGO)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9 (69.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 (30.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.028*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eII\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16 (36.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28 (63.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIII\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12 (27.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31 (72.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTumor size\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36 (42.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48 (57.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.002*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT2-4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (6.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15 (93.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eFIGO, International Federation of Gynecology and Obstetrics; * P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Statistically significant\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.2 IPO7 plays an oncogenic role in CC\u003c/h2\u003e \u003cp\u003eTo determine the function of IPO7, this study employed two cervical cancer (CC) cell lines, SiHa and HeLa, exhibiting high levels of IPO7 expression (Supplementary Fig.\u0026nbsp;2A-2C). Subsequently, IPO7 knockdown experiments were performed in these cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Following IPO7 knockdown, the CC cells exhibited reduced cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), decreased colony formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and impaired migration and invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Supplementary Fig.\u0026nbsp;2D and 2E), along with increased apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, we established HeLa cell lines with stable IPO7 knockdown and evaluated their effect on tumorigenesis using subcutaneous xenograft models in nude mice. The group with IPO7 shRNA exhibited markedly slower tumor growth and lower tumor burden compared to the control shRNA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). IHC analysis revealed decreased expression of the proliferation marker Ki67 in the IPO7-shRNA xenografts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). This result indicated that IPO7 promoted the proliferation of HeLa cells. In summary, cellular and in vivo experiments collectively support the oncogenic function of IPO7 in CC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.3 MSI2 is a potential target protein transported by IPO7 in CC\u003c/h2\u003e \u003cp\u003eAs a nuclear transport protein, IPO7 is responsible for the import of cargo proteins into the cell nucleus. Thus, we investigated the molecular mechanism of IPO7 in CC by focusing on nuclear-cytoplasmic transport. We manipulated IPO7 expression and isolated nuclear-cytoplasmic fractions from both IPO7 knockdown and control groups. Subsequently, mass spectrometry analysis identified differentially expressed proteins. The analysis identified 74 proteins that were downregulated in the cell nucleus following IPO7 knockdown (fold change\u0026thinsp;\u0026gt;\u0026thinsp;2 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These proteins are considered potential IPO7 cargo proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The enrichment analysis indicated that these proteins are involved in processes such as protein localization, Rho GTP cycling, and translation regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Additionally, we performed enrichment analysis using Metascape (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://metascape.org/\u003c/span\u003e\u003cspan address=\"http://metascape.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) on the top 200 genes most correlated with IPO7 expression in the TCGA- CESC database. This analysis showed that IPO7 is predominantly linked to mRNA processes involving transport and regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong the candidates, the RNA-binding protein Musashi-2 (MSI2) attracted our attention. MSI2 is a member of the Musashi family, which includes MSI1 and MSI2, and possesses two RNA-recognition motifs (RRM1 and RRM2) at the N-terminus\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. MSI2 can bind to the 3'-end of target RNAs and modulate the stability and translation of mRNA of proteins implicated in tumorigenesis, including SMAD3, PTEN and others\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. MSI2 is considered a potential IPO7 cargo protein in this study.\u003c/p\u003e \u003cp\u003eWe utilized the NLS-mapper website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi\u003c/span\u003e\u003cspan address=\"https://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ) to predict the NLS of MSI2, identifying a highly conserved sequence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) with a score of 5, indicating its potential for nucleocytoplasmic shuttling. Furthermore, MSI2 shows increased expression in CC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) and is positively correlated with IPO7 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, R\u0026thinsp;=\u0026thinsp;0.38). Further analysis showed decreased MSI2 protein levels after IPO7 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), while qPCR analysis showed no significant change in MSI2 mRNA levels following knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). After isolating nuclear and cytoplasmic fractions, we performed individual analyses of MSI2 expression. The findings showed a reduction in MSI2 expression in both the nucleus and cytoplasm subsequent to IPO7 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). The reduction in MSI2 expression could be attributed to hindered nuclear entry and subsequent degradation of the protein. This hypothesis is supported by the identification of seven ubiquitination sites on the MSI2 protein, as predicted using the SMART website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://smart.embl-heidelberg.de/\u003c/span\u003e\u003cspan address=\"http://smart.embl-heidelberg.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). To further investigate, UB-HA was introduced into both normal and IPO7 knockdown CC cells, followed by immunoprecipitation (IP) experiments to evaluate the ubiquitination level of MSI2. The results indicated elevated MSI2 ubiquitination upon IPO7 knockdown, implying that this could be a potential cause for the decreased cytoplasmic levels of MSI2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.4 MSI2 directly interacts with IPO7, which exerts oncogenic effects by transporting MSI2\u003c/h2\u003e \u003cp\u003eIn this study, we examined the interaction between IPO7 and MSI2. Initially, Co-IP experiments and IF assays were conducted to confirm a direct interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Subsequently, we used the SMART website for predicting the structure of the MSI2 protein and generated a corresponding model diagram (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). This model indicated that the nuclear localization sequence (NLS) is predominantly located in the RRM1 region of MSI2. Then, we deleted the NLS fragment from the MSI2 DNA sequence to observe its effects on binding to IPO7 in cell cultures. A significant reduction in the binding affinity between MSI2 (del-NLS) and IPO7 was observed, particularly in HeLa cells where almost no binding was detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). The results demonstrate the importance of the NLS region in mediating the interaction between IPO7 and MSI2, suggesting that IPO7 likely facilitates the nuclear transport of MSI2.\u003c/p\u003e \u003cp\u003eTo investigate whether the oncogenic effects of IPO7 are mediated through the transportation of MSI2, we conducted experiments with the overexpression of IPO7 and simultaneous knockdown of MSI2 in two cell lines (SiHa and HeLa). Firstly, we assessed the efficacy of MSI2 knockdown and selected the most potent shRNA sequence for further experiments (Supplementary Fig.\u0026nbsp;3). MSI2 knockdown did not influence IPO7 mRNA expression levels, and IPO7 overexpression had no impact on MSI2 mRNA levels in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Nevertheless, the overexpression of IPO7 and knockdown of MSI2 simultaneously resulted in a notable decrease in both cell proliferation capacity and colony formation ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These results suggest that IPO7 exerts its oncogenic effects through the transportation of MSI2 into the nucleus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.5 MSI2 acts as an oncogene by directly interacting with c-MYC and is co-transported into the nucleus by IPO7.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further investigate how MSI2 promotes cancer progression, we RNA-sequenced IPO7-knockdown HeLa cells and performed a GSEA analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), which showed that the knockdown primarily led to the enrichment of MYC TARGET V1 and V2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). The MYC gene, a well-known oncogene, plays a pivotal role in the formation and progression of many tumors. Although our mass spectrometry analysis failed to detect changes in c-MYC expression following IPO7 knockdown, we identified a direct interaction between the MYC family and IPO7 using the BioGRID database (Supplementary Fig.\u0026nbsp;4A). Analysis of the TCGA cervical cancer database revealed a positive correlation between MYC and IPO7 expression levels (Supplementary Fig.\u0026nbsp;4B), and IPO7 expression levels was also positively correlated with MYC target pathways (Supplementary Fig.\u0026nbsp;4C). After confirming that IPO7 can bind to MSI2 and translocate it into the nucleus, we investigated whether MSI2 also interacts directly with c-MYC. Co-IP experiments validated a direct interaction between MSI2 and c-MYC in Hela and SiHa cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE), leading us to suggest that IPO7 may synergistically transport the MSI2 and c-MYC complex into the nucleus, thereby promoting oncogenic effects. Furthermore, we found that MSI2 knockdown reduced the expression of c-MYC at both the mRNA and protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF-\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI). Given that MSI2 is an RNA-binding protein, we examined whether it influences the stability of MYC mRNA. Following treatment of MSI2-knockdown cells with actinomycin D for 0, 3, and 6 hours, qPCR analysis showed that MSI2 knockdown significantly decreased the mRNA stability of c-MYC compared to the control group (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK). Therefore, we suggest that IPO7 may systematically transport the MSI2-c-MYC complex into the nucleus, where MSI2 plays a crucial role in maintaining the stability of MYC, potentially facilitating the nuclear import of c-MYC to exert oncogenic activities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Elevated MSI2 and IPO7 expression correlate with a worse prognosis in CC patients\u003c/h2\u003e \u003cp\u003eTo investigate the oncogenic effect of IPO7 via MSI2 transport, we conducted a comprehensive examination of MSI2 expression in CC. Analysis using the GEO database (GSE6791, GSE63514) revealed a significant upregulation of MSI2 expression in CC (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). IHC staining of clinical samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC) showed a considerable elevation in MSI2 expression in CC compared to normal cervical tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD), especially in advanced-stage cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). The CCK-8 assay demonstrated a reduction in the proliferation capacity of both CC cell lines following MSI2 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG). Moreover, after MSI2 knockdown, the majority of cells exhibited arrest in the S phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Survival analysis performed on CC patients from TCGA indicated that high MSI2 expression alone did not impact the prognosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eJ). However, CC patients with high MSI2 expression alongside high IPO7 expression experienced the poorest prognosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eK and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eL, P\u0026thinsp;=\u0026thinsp;0.00069). Our results strongly suggest that IPO7 may serve as a promising target for CC treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn conclusion, our research demonstrates a correlation between elevated IPO7 levels and unfavorable outcomes in patients with CC. IPO7 contributes to the progression of CC by promoting cellular proliferation, migration, and invasion, and inhibiting apoptosis. This oncogenic effect is primarily due to the transportation of MSI2 and c-MYC into the nucleus by IPO7, which that recognizes and binds to the nuclear localization signal (NLS) region of MSI2. Thus, in the presence of Ran-GTPase, the MSI2-c-MYC complex is released from IPO7 and exhibits its oncogenic characteristics within the nucleus. The oncogenic effects of IPO7 occur through the presence of MSI2, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. If the cytoplasmic transport of MSI2 is hindered, it is subject to degradation via ubiquitination\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAppropriate subcellular localization is crucial for the functioning of biological macromolecules, such as proteins and RNAs. Nuclear transport is a fundamental cellular process that regulates the localization of numerous macromolecules within the nucleus or cytoplasm\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Dysfunctional nuclear transport leads to changes in the physiological levels and spatiotemporal localization of tumor suppressors, proto-oncogenes, and other macromolecules, thereby affecting tumorigenesis and drug sensitivity in cancer cells\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Our study investigates the role of karyopherins, specifically IPO7, in the development of CC. By analyzing expression profiles of 27 karyopherins in the TCGA-CESC database and comparing them to control samples in the GEO database, we discovered IPO7 to be significantly upregulated in CC, exhibiting a significant prognostic correlation. IPO7 demonstrates elevated expression levels, as confirmed by histochemical analysis of our clinical samples. Consequently, we proceeded to conduct subsequent studies into IPO7's role in CC and its potential cargo proteins.\u003c/p\u003e \u003cp\u003eRecent studies\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e have found that increased IPO7 expression in breast cancer is linked to poor prognosis, mainly by aiding USP22-AR nuclear transport. NUAK1, an AMPK-related kinase, plays a key role in tumorigenesis, relying on Importin β family transporters like KPNB1, IPO7, and IPO9 for nuclear transport\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Moreover, An NTS-derived myristoylated phosphomimetic peptide specifically blocked ERK1/2's interaction with IPO7, hindering ERK1/2's nuclear translocation\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. These results, along with our study, demonstrates that IPO7 significantly contributes to tumorigenesis and tumor progression by transporting various cargo proteins. Knockdown of IPO7 significantly reduced cell proliferation, colony formation, migration, and invasion, and increased apoptosis in Hela and Siha cells. Furthermore, xenograft model experiments showed that IPO7 knockdown delayed tumor growth and decreased tumor burden, reinforcing its oncogenic role.\u003c/p\u003e \u003cp\u003eSubsequently, we aimed to elucidate the mechanisms underlying IPO7's tumor-promoting effects, focusing on the cargo proteins that IPO7 transports. Cells efficiently regulate protein synthesis and degradation\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Non-functional proteins are quickly degraded and removed. Therefore, we knocked down IPO7 expression and isolated nuclear and cytoplasmic proteins for mass spectrometry analysis. We selected proteins with reduced nuclear expression after IPO7 knockdown for further study. We systematically screened 74 potential cargo proteins and performed enrichment analysis. These proteins are predominantly involved in the regulation of mRNA and the nuclear-cytoplasmic transport process. Notably, elevated IPO7 expression primarily facilitates protein localization. Our identification of MSI2, a member of the Musashi protein family, as a potential IPO7 cargo protein was based on its function as an RNA-binding protein that regulates mRNA stability and translation in key oncogenic pathways\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The NLS-mapper website\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e facilitated the prediction of IPO7's probable NLS sequence and location. Notably, the conservation of the NLS sequence suggests its crucial role in IPO7 binding and transportation. Direct interactions between IPO7 and MSI2 were confirmed using Co-IP and IF assays. Intriguingly, deleting the NLS region in MSI2 significantly reduced its binding to IPO7, especially in HeLa cells. Moreover, proliferation and colony formation assays further supported IPO7's role in tumor promotion via MSI2.\u003c/p\u003e \u003cp\u003eThe Musashi protein family, including MSI1 and MSI2, is a widespread and highly conserved group of RNA-binding proteins\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. MSI proteins contain two RNA recognition motifs (RRMs) for interacting with target RNA. Notably, the first RRM exhibits a greater affinity towards RNA compared to the second RRM\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. MSI proteins contain two RNA recognition motifs (RRMs) for interacting with target RNA\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. MSI2 is widely expressed in various tumors, including leukemia, gastric, colorectal, and triple-negative breast cancers\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHow does IPO7-mediated nuclear transport of MSI2 enhance its oncogenic effects? After RNA-sequencing HeLa cells with IPO7 knockdown, GSEA analysis revealed significant enrichment in the \"MYC targets\" pathway. Research shows that c-MYC is vital in CC, with MYC genes often being HPV integration targets\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Microhomology aids HPV integration near MYC, boosting c-Myc expression\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Microbial overgrowth, such as Prevotella, may also enhance persistent HPV-related cervical lesions by influencing c-Myc expression\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Can IPO7 facilitate MYC's nuclear transport? Using the BioGRID database, interactions between IPO7 and MYC were identified. Co-IP assays further confirmed that MSI2 directly binds to c-MYC, suggesting that IPO7 is capable of transporting both MSI2 and c-MYC into the nucleus. Additionally, reduced c-MYC expression at both the protein and RNA levels was observed following MSI2 knockdown, suggesting that MYC is targeted for degradation if it is not transported to the nucleus. Meanwhile, as an RNA-binding protein, MSI2 stabilizes MYC mRNA. Upon IPO7 knockdown, MSI2 fails to be transported into the nucleus, leading to its ubiquitination and reduced expression. Concurrently, MSI2 knockdown impedes MYC nuclear transport, thereby curtailing its oncogenic potential, and reduces MYC mRNA stability, resulting in decreased MYC expression.\u003c/p\u003e \u003cp\u003eAdditionally, we found that overexpression of MSI2 promotes CC cell proliferation. MSI2 has been identified as a target for small molecule inhibitors such as largazole and Aza-9, which have effectively reduced MSI2 levels and suppressed the proliferation of non-small cell lung cancer (NSCLC), chronic myeloid leukemia, and colon cancer cells\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. A thorough analysis of the TCGA database showed that patients with high MSI2 and IPO7 expression had the worst prognosis. Thus, targeting the IPO7-MSI2 axis could be a more effective treatment strategy for CC.\u003c/p\u003e \u003cp\u003eIn this study, we observed that downregulating IPO7 led to decreased MSI2 protein expression in both the nucleus and cytoplasm. However, IPO7 knockdown did not affect MSI2 mRNA levels, implying a post-translational mechanism. Knockdown-induced IPO7 reduction impeded MSI2 nuclear transport, causing cytoplasmic degradation and loss of function. Notably, ubiquitination is a key mechanism for cellular protein degradation. Using the SMART database\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, we identified seven ubiquitination sites on MSI2. This evidence indicates that IPO7 is crucial for nuclear transport of MSI2, underscoring the complex precision of cellular protein regulation. Ubiquitination of MSI2 is commonly observed in tumors. For instance, MSI2's interaction with LncRNA LINC00942 inhibits ubiquitination, thereby enhancing the stability and expression of target mRNAs, including c-MYC\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Intriguingly, our research revealed that MSI2 enhances c-MYC mRNA stability, and c-MYC can bind directly to MSI2. Both are transported to the nucleus by IPO7 to perform their functions.\u003c/p\u003e \u003cp\u003eIn conclusion, our study identifies IPO7 as a pivotal factor in the progression of CC. Its oncogenic role is mediated by nuclear transport of MSI2 and c-MYC. Targeting the IPO7-MSI2 interaction may lead to new therapeutic strategies for CC.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCC cervical cancer\u003c/p\u003e\u003cp\u003eDSS disease-specific survival\u003c/p\u003e\u003cp\u003eGEO the Gene Expression Omnibus\u003c/p\u003e\u003cp\u003eIHC Immunohistochemistry\u003c/p\u003e\u003cp\u003eIP immunoprecipitation\u003c/p\u003e\u003cp\u003eIPO7 Importin 7\u003c/p\u003e\u003cp\u003eMSI2 Musashi 2\u003c/p\u003e\u003cp\u003eNESs Nuclear Export Signals\u003c/p\u003e\u003cp\u003eNLSs Nuclear Localization Signals\u003c/p\u003e\u003cp\u003eOS overall survival\u003c/p\u003e\u003cp\u003ePI propidium iodide\u003c/p\u003e\u003cp\u003eRRM RNA-recognition motifs\u003c/p\u003e\u003cp\u003eTCGA The Cancer Genome Atlas\u003c/p\u003e\u003cp\u003eTMA tissues microarray\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eGrant support: This work was supported by grants from the National Natural Science Foundation of China (82002730)\u003c/p\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eAll of patients provided written informed consent (ethic approval number: 2020-YS-075), and all the experiments, including animal work (ethic approval number: 2020 − 0204) were approved by the Research Ethics Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital and complied with the Declaration of Helsinki.\u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eAll authors have consented for publication\u003c/p\u003e \u003cp\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by the National Natural Science Foundation of China (No: 82002730).\u003c/p\u003e\u003ch2\u003eAuthors' contributions\u003c/h2\u003e \u003cp\u003eQ.Y.X., R.Z. and Y.B designed and supervised this study. W.Z.Z, J.C., and L.M. performed the data analysis, statistical analysis and finished the manuscript writing. Q.Y.X., and T.Q. performed the whole experiments. Z.H.A., Y.C.T and R.Z.J. provided data collection and technical support. W.Z.Z, T.Q., and J.W. contributed equally to this work. All authors participated in revising, reviewing, and approving the final submission.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe are grateful to Professor Zhigang Zhang and Lipeng Hu for their mentoring and assistance on this study. We are grateful to the Asia-Vector Biotechnology for assistance with our experiments.\u003c/p\u003e\u003ch2\u003eAvailability of data and material\u003c/h2\u003e \u003cp\u003eThe datasets utilized and/or examined in this research are accessible from the corresponding author upon request. The database of Cervical cancer data produced in this study can be accessed through the Gene Expression Omnibus (GEO) with accession numbers GSE9750, GSE7803, GSE6791, GSE63514, as well as the TCGA-CESC libraries.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, \u003cem\u003eet al.\u003c/em\u003e Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: a cancer journal for clinicians 2021, 71(3): 209\u0026ndash;249.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWHO. Global strategy to accelerate the elimination of cervical cancer as a public health problem. 2020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJi L, Chen M, Yao L. Strategies to eliminate cervical cancer in China. Frontiers in oncology 2023, 13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarkowitz LE, Unger ER. Human Papillomavirus Vaccination. The New England journal of medicine 2023, 388(19): 1790\u0026ndash;1798.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Guo L, Chen L, Gong B, Jia D, Sun Q. Nuclear transport proteins: structure, function, and disease relevance. Signal Transduction and Targeted Therapy 2023, 8(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStrambio-De-Castillia C, Niepel M, Rout MP. The nuclear pore complex: bridging nuclear transport and gene regulation. Nature Reviews Molecular Cell Biology 2010, 11(7): 490\u0026ndash;501.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWing CE, Fung HYJ, Chook YM. Karyopherin-mediated nucleocytoplasmic transport. Nature Reviews Molecular Cell Biology 2022, 23(5): 307\u0026ndash;328.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoepp DM, Silver PA. A GTPase controlling nuclear trafficking: running the right way or walking RANdomly? Cell 1996, 87(1): 1\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalita J, Kapinos LE, Lim RYH. On the asymmetric partitioning of nucleocytoplasmic transport - recent insights and open questions. Journal of cell science 2021, 134(7).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoniat M, Chook YM. Nuclear localization signals for four distinct karyopherin-β nuclear import systems. The Biochemical journal 2015, 468(3): 353\u0026ndash;362.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou Y, Zhou B, Pache L, Chang M, Khodabakhshi AH, Tanaseichuk O, \u003cem\u003eet al.\u003c/em\u003e Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nature communications 2019, 10(1): 1523.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu Q, Ge Q, Zhou Y, Yang B, Yang Q, Jiang S, \u003cem\u003eet al.\u003c/em\u003e MELK promotes Endometrial carcinoma progression via activating mTOR signaling pathway. EBioMedicine 2020, 51: 102609\u0026ndash;102609.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKudinov AE, Karanicolas J, Golemis EA, Boumber Y. Musashi RNA-Binding Proteins as Cancer Drivers and Novel Therapeutic Targets. Clinical cancer research: an official journal of the American Association for Cancer Research 2017, 23(9): 2143\u0026ndash;2153.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun J, Sheng W, Ma Y, Dong M. Potential Role of Musashi-2 RNA-Binding Protein in Cancer EMT. Onco Targets Ther 2021, 14: 1969\u0026ndash;1980.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u0026Ccedil;ağatay T, Chook YM. Karyopherins in cancer. Current opinion in cell biology 2018, 52: 30\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai GX, Kong WY, Liu Y, Zhong SY, Liu Q, Deng YF, \u003cem\u003eet al.\u003c/em\u003e Nuclear transport maintenance of USP22-AR by Importin-7 promotes breast cancer progression. Cell Death Discov 2023, 9(1): 211.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePalma M, Riffo EN, Suganuma T, Washburn MP, Workman JL, Pincheira R, \u003cem\u003eet al.\u003c/em\u003e Identification of a nuclear localization signal and importin beta members mediating NUAK1 nuclear import inhibited by oxidative stress. Journal of cellular biochemistry 2019, 120(9): 16088\u0026ndash;16107.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePlotnikov A, Flores K, Maik-Rachline G, Zehorai E, Kapri-Pardes E, Berti DA, \u003cem\u003eet al.\u003c/em\u003e The nuclear translocation of ERK1/2 as an anticancer target. Nature communications 2015, 6: 6685.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu J, Thompson CB. Metabolic regulation of cell growth and proliferation. Nat Rev Mol Cell Biol 2019, 20(7): 436\u0026ndash;450.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKosugi S, Hasebe M, Tomita M, Yanagawa H. Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs. Proceedings of the National Academy of Sciences of the United States of America 2009, 106(25): 10171\u0026ndash;10176.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiddall NA, McLaughlin EA, Marriner NL, Hime GR. The RNA-binding protein Musashi is required intrinsically to maintain stem cell identity. Proceedings of the National Academy of Sciences of the United States of America 2006, 103(22): 8402\u0026ndash;8407.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKurihara Y, Nagata T, Imai T, Hiwatashi A, Horiuchi M, Sakakibara S, \u003cem\u003eet al.\u003c/em\u003e Structural properties and RNA-binding activities of two RNA recognition motifs of a mouse neural RNA-binding protein, mouse-Musashi-1. Gene 1997, 186(1): 21\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiyanoiri Y, Kobayashi H, Imai T, Watanabe M, Nagata T, Uesugi S, \u003cem\u003eet al.\u003c/em\u003e Origin of higher affinity to RNA of the N-terminal RNA-binding domain than that of the C-terminal one of a mouse neural protein, musashi1, as revealed by comparison of their structures, modes of interaction, surface electrostatic potentials, and backbone dynamics. J Biol Chem 2003, 278(42): 41309\u0026ndash;41315.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSakakibara S, Nakamura Y, Satoh H, Okano H. Rna-binding protein Musashi2: developmentally regulated expression in neural precursor cells and subpopulations of neurons in mammalian CNS. The Journal of neuroscience: the official journal of the Society for Neuroscience 2001, 21(20): 8091\u0026ndash;8107.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J, Duan Y, Wu P, Chang Y, Wang Y, Hu T, \u003cem\u003eet al.\u003c/em\u003e Clonal evolution dissection reveals that a high MSI2 level promotes chemoresistance in T-cell acute lymphoblastic leukemia. Blood 2024, 143(4): 320\u0026ndash;335.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu Y, Zhou B, Hu X, Ying S, Zhou Q, Xu W, \u003cem\u003eet al.\u003c/em\u003e LncRNA LINC00942 promotes chemoresistance in gastric cancer by suppressing MSI2 degradation to enhance c-Myc mRNA stability. Clinical and translational medicine 2022, 12(1): e703.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Su K, Liu Y, Zhu D, Pan Y, Ke X, \u003cem\u003eet al.\u003c/em\u003e Small Molecule Palmatine Targeting Musashi-2 in Colorectal Cancer. Front Pharmacol 2021, 12: 793449.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi M, Li AQ, Zhou SL, Lv H, Wei P, Yang WT. RNA-binding protein MSI2 isoforms expression and regulation in progression of triple-negative breast cancer. Journal of experimental \u0026amp; clinical cancer research: CR 2020, 39(1): 92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Jia W, Wang M, Liu J, Zhou X, Liang Z, \u003cem\u003eet al.\u003c/em\u003e Human papillomavirus integration perspective in small cell cervical carcinoma. Nature communications 2022, 13(1): 5968.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu Z, Zhu D, Wang W, Li W, Jia W, Zeng X, \u003cem\u003eet al.\u003c/em\u003e Genome-wide profiling of HPV integration in cervical cancer identifies clustered genomic hot spots and a potential microhomology-mediated integration mechanism. Nature genetics 2015, 47(2): 158\u0026ndash;163.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDong B, Huang Y, Cai H, Chen Y, Li Y, Zou H, \u003cem\u003eet al.\u003c/em\u003e Prevotella as the hub of the cervicovaginal microbiota affects the occurrence of persistent human papillomavirus infection and cervical lesions in women of childbearing age via host NF-κB/C-myc. J Med Virol 2022, 94(11): 5519\u0026ndash;5534.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang M, Sun XY, Zhou YC, Zhang KJ, Lu YZ, Liu J, \u003cem\u003eet al.\u003c/em\u003e Suppression of Musashi\u0026ndash;2 by the small compound largazole exerts inhibitory effects on malignant cells. International journal of oncology 2020, 56(5): 1274\u0026ndash;1283.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLan L, Liu J, Xing M, Smith AR, Wang J, Wu X, \u003cem\u003eet al.\u003c/em\u003e Identification and Validation of an Aspergillus nidulans Secondary Metabolite Derivative as an Inhibitor of the Musashi-RNA Interaction. Cancers (Basel) 2020, 12(8).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLetunic I, Khedkar S, Bork P. SMART: recent updates, new developments and status in 2020. Nucleic acids research 2021, 49(D1): D458-d460.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cervical cancer, nuclear transport, Importin 7, MSI2, Prognosis ","lastPublishedDoi":"10.21203/rs.3.rs-5254983/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5254983/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCervical cancer (CC) remains a significant health threat to women worldwide, with a pressing need for novel therapeutic targets. Despite recent advances, the molecular mechanisms underlying CC progression are not fully understood. Nuclear transport proteins, key regulators of macromolecule movement between cellular compartments, have emerged as potential targets in cancer therapy. However, the specific role of Importin 7 (IPO7) in CC development and its clinical implications remain poorly characterized, indicating a significant gap in our current understanding.\u003c/p\u003e \u003cp\u003eHere, we investigated IPO7's role in CC, leveraging clinical samples, bioinformatics analyses from TCGA and GEO databases, and experimental models. We found that IPO7 is upregulated in CC and associated with poor prognosis. IPO7 knockdown in cell lines and animal models revealed decreased cell proliferation, reduced colony formation, inhibited migration and invasion, and enhanced apoptosis. To uncover IPO7's molecular mechanisms, we performed mass spectrometry analysis, identifying MSI2, an RNA-binding protein, as a potential cargo. Further validation confirmed a direct interaction between IPO7 and MSI2, with IPO7 overexpression coupled with MSI2 knockdown abrogating oncogenic effects. Sequencing analysis of IPO7-knockdown cells indicated the MYC targets pathway and co-immunoprecipitation assays confirmed a direct interaction between MYC and MSI2, suggesting that IPO7 may facilitate the nuclear transport of MSI2 and MYC, thereby promoting cancer progression. Clinically, elevated MSI2 expression in CC patients, particularly in advanced stages, correlated with poorer outcomes.\u003c/p\u003e \u003cp\u003eOur findings elucidate the role of IPO7 in CC, demonstrating its potential as a therapeutic target. The interaction between IPO7, MSI2, and MYC provides a novel avenue for developing targeted therapies. Importantly, our results underscore the importance of IPO7-mediated nuclear transport in CC progression, presenting a promising strategy for enhancing patient outcomes and advancing CC treatment.\u003c/p\u003e","manuscriptTitle":"Importin 7 Mediated Nuclear Transport of MSI2 as a Therapeutic Target in Cervical Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-28 07:05:55","doi":"10.21203/rs.3.rs-5254983/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5325bf74-1fca-42f7-985e-8cfe052268e7","owner":[],"postedDate":"October 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":39362058,"name":"Health sciences/Diseases/Cancer/Gynaecological cancer/Cervical cancer"},{"id":39362059,"name":"Biological sciences/Cancer/Gynaecological cancer/Cervical cancer"}],"tags":[],"updatedAt":"2024-12-17T14:01:28+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-28 07:05:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5254983","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5254983","identity":"rs-5254983","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}