{"paper_id":"16014b5f-564a-482e-acab-4ed88930165f","body_text":"Overexpression of splicing factor poly(rC)-binding protein 1 elicits cycle arrest, apoptosis induction, and p73 splicing in human cervical carcinoma cells | 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 Research article Overexpression of splicing factor poly(rC)-binding protein 1 elicits cycle arrest, apoptosis induction, and p73 splicing in human cervical carcinoma cells Hong Zhang, Yuhong Chen, Cuixia Di, Caipeng Xu, Xiaohua Chen, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.24804/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 Background Splicing factor poly(rC)-binding protein 1 (PCBP1) is a novel tumor suppressor that is downregulated in many cancers thereby regulates tumor formation and metastasis. However, to date, little information has been available on the molecular mechanisms by which PCBP1 evokes apoptosis. Results Here, we explored the molecular mechanism by which PCBP1 triggers apoptosis in human cervical cancer cells. We testified that overexpression of PCBP1 greatly repressed proliferation of HeLa cells in time-dependent manner. It also induced a significant increase in G2 / M phase arrest and apoptosis. Furthermore, it was shown that overexpression of PCBP1 caused p73 splicing, and thus efficiently downregulated the ratio of Bax / Bcl-2, the release of cytochrome c and the expression of caspase-3. Conclusion Our results revealed that PCBP1 played a vital role in cycle arrest, apoptosis induction, and p73 splicing in human cervical carcinoma cells and targeting PCBP1 may be a promising approach in cervical cancer therapy. General Cell Biology & Physiology PCBP1 p73 splicing apoptosis cancer therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Cervical cancer ranks as the fourth most frequently diagnosed cancer and the fourth leading cause of cancer death in women [ 1 ]. Therefore, research on its pathogenesis is crucial. Poly(rC) binding protein 1 (PCBP1) is a member of the RNA binding protein family and is firstly cloned from human lymphocyte cDNA library in 1994 [ 2 ]. With the deepening of research in recent years, PCBP1 has been found to be ubiquitously expressed in many tissues and plays a multifunction role in various life activities, such as intracellular transcription and post-transcriptional regulation, including alternative splicing of pre-mRNA, mRNA stability and translation [ 3 ]. In recent years, researches focus on the relationship between PCBP1 and tumors. Studies have been found that PCBP1 acts as a tumor suppressor in tumors and the expression is significantly downregulated in various tumors, including gastric cancer [ 4 ], acute myeloid leukemia [ 5 ], non-small-cell lung cancer [ 6 ], cervical cancer [ 7 ] et al . In general, PCBP1 plays a multifunctional role in tumor progress. For instance, PCBP1 has an influence on apoptosis in variety cancers [ 8 , 9 ]. It is also involved in alternative splicing which dysregulation usually leads to disease and is increasingly associated with tumorigenesis [ 10 , 11 ]. In addition, PCBP1 negatively regulated the tumor hypoxic microenvironment and inhibited autophagy to further affect the tumor formation and development [ 8 , 12 , 13 ]. Moreover, PCBP1 prevented the process of EMT to reduce cancer metastasis [ 6 ]. The above cases all turn out that PCBP1 is involved in the development of tumors as a tumor suppressor, but little information has been available on the molecular mechanisms by which PCBP1 causes cervical cancer apoptosis. In the present study, we would provide some preliminary data to illustrate the distinct functions of PCBP1 in p73 alternative splicing and the mechanisms of PCBP1 on cervical cancer cells apoptosis. The results suggested that PCBP1 may be an attractive novel target for cervical cancer therapy. Results The expression of PCBP1 and its spacial distribution To explore the biologic function of PCBP1, we initially transfected HeLa cells with pEGFP-N1 or pEGFP-N1-PCBP1, and then we verified whether the PCBP1 was successfully transfected and overexpressed in cells. We performed real-time PCR and immunofluorescence experiments. The results demonstrated that PCBP1 mRNA expression was significantly increased in cells transfected with pEGFP-N1-PCBP1 compared to mock group (Fig. 1 a) and there was no significant difference in mRNA expression in mock and vector group (Fig. 1 a). In addition, further immunofluorescence experiments results showed the PCBP1 protein expression was increased compared with mock and vector group, and the PCBP1 is distributed in both the cytoplasm and the nucleus (Fig. 1 b). Overexpression of PCBP1 and its effects on human cervical carcinoma cells viability In order to understand the effects of overexpressed PCBP1 in HeLa cells, we examined the proliferation of HeLa cells in different time points after transfection. MTS analysis proved that elevated PCBP1 significantly reduced the cell viability of HeLa cells, and the inhibition of proliferation of PCBP1 transfection. The results were significantly time-dependent (Fig. 2 a). Furthermore, the experimental results from the colony formation assay suggested that overexpression of PCBP1 in HeLa cells could significantly repress cell colony formation compared with mock and vector group (Fig. 2 b, c). PCBP1 induces cell cycle arrest and apoptosis To detect the effect of overexpressed PCBP1, after transfection, we stained the nuclei and then observed the nuclear morphology. We found the nuclear morphology changed and apoptotic bodies appear, this suggested us that apoptosis took place (Fig. 3 a). In order to further verify the mechanism of PCBP1 in inhibiting the growth of HeLa cells, we used flow cytometry to detect the cell cycle and apoptosis. The results showed that overexpressed PCBP1 would cause cell cycle arrest (Fig. 3 b), and the ratio of G2 / M cells in HeLa cells were significantly increased (Fig. 3 d). Then we detected cell apoptosis to prove that whether the decrease in viability of HeLa cells after transfected with pEGFP-N1-PCBP1 was caused by apoptosis. The results showed cells transfected with pEGFP-N1-PCBP1 for 48 h performed more apoptosis than the mock and vector group (Fig. 3 c, e). The results of the apoptosis experiment obtained by flow cytometry analysis were statistically analyzed, and the results were also verified. PCBP1 upregulates Tap73 and downregulates ΔNp73, indicating activation of apoptosis In order to demonstrate whether p73 is involved in PCBP1 induced cell cycle arrest and apoptosis in human cervical carcinoma cells, western blot and immunofluorescence were used to detect the level of Tap73 and ΔNp73 proteins. We found the level of ΔNp73 was significantly reduced in HeLa cells at 24 h after transfected with pEGFP-N1-PCBP1 compared with the mock group. On the contrary, the level of Tap73 was increased compared with the mock group (Fig. 4 a). Quantitative results showed, PCBP1 induced an increase in the Tap73 / ΔNp73 ratio (Fig. 4 b). Next, we used immunofluorescent to further verify its spatiotemporal distribution in cells (Fig. 4 c). ΔNp73 expression clearly reduced but Tap73 expression obviously upregulated. Moreover, Tap73 mainly detected in the cytoplasm, but ΔNp73 mainly detected in the nucleus. These findings suggested that p73 splicing is involved in PCBP1 induced cell cycle arrest and apoptosis in HeLa cells. PCBP1 regulates apoptosis via mitochondrial pathway In order to further verify whether PCBP1 induced apoptosis is associated with mitochondrial apoptosis pathway, we used western blot to examine several key proteins in the mitochondrial apoptosis signalling pathway. As shown in Fig. 5 , overexpression of PCBP1 upregulated the ratio of Bax / Bcl-2 to promote cell apoptosis (Fig. 5 b). In addition, there was a substantial increase in the expression of cytochrome c at 24 h after transfected with pEGFP-N1-PCBP1 (Fig. 5 d). Furthermore, decreased procaspase-3 and increased cleaved caspase-3 both indicated the occurrence of apoptosis (Fig. 5 c). Thereby confirming that overexpression of PCBP1 upregulated Bax / Bcl-2 ratio, promoted cytochrome c release and activated caspase-3 to induce apoptosis. Discussion Cervical cancer is one of the most diseases threatening women's health, but our understanding of its pathogenesis is still not deep enough. It is imminent to further study its pathogenesis [ 14 ]. PCBP1 is an evolutionarily conserved RNA-binding protein that regulates transcription, translation, and alternative splicing of genes [ 3 , 15 , 16 ]. Increasing evidence revealed that PCBP1 is significantly downregulated in gastric cancer [ 4 ], acute myeloid leukemia [ 5 ], non-small-cell lung cancer [ 6 ], and cervical cancer [ 7 ] et al . and is involved in tumor metastasis. Subsequently, scientists pointed out that PCBP1 might act as a tumor suppressor in various tumors [ 17 , 18 ]. For instance, it has been proved that PCBP1 is involved in cell apoptosis. Zhang et al . reported that overexpression of PCBP1 decreased the Bcl-2 expression and caused cancer cells apoptosis [ 8 ]. Shi et al . confirmed that PCBP1 increased p27 expression via stabilizing its mRNA to further facilitate cell apoptosis [ 19 ]. Ishii et al . reported PCBP1 activated apoptosis through interacted with more severely oxidized RNA [ 20 ]. Interestingly, PCBP1 has gained more attention due to its multiple functions in tumor progression, but the real mechanism is relatively unexplored. Therefore, it is worthy to further study the relationship between PCBP1 and cervical cancer. Here, we transfected PCBP1 into HeLa cells and testified that overexpression of PCBP1 greatly repressed proliferation of HeLa cells in time-dependent manner (Fig. 2 ). It also induced G2 / M phase arrest and significant rise of apoptotic cells (Fig. 3 ). Overall, these results indicated that elevated PCBP1 is an efficient way to inhibit tumor cell progression, and this will provide a reference for further understanding of the pathogenesis of cervical cancer. RNA splicing is the key to the pathology of numerous diseases, and experiments have shown that dysregulation of splicing isoforms were increasingly associated with tumor proliferation, metastasis and apoptosis [ 21 , 22 ]. Additionally, PCBP1 is related to alternative splicing. In pancreatic cancer, upregulated PCBP1 reduced tumor metastasis by interacting with integrin β1 to regulate its alternative splicing [ 11 ]. Moreover, overexpression of PCBP1 inhibited the tumor invasion and metastasis in HepG2 cells via regulating exon inclusion of CD44 [ 10 ]. In our study, we transfected PCBP1 into HeLa cells and indicated that overexpressed PCBP1 obviously enhanced the expression of Tap73 and decreased ΔNp73 expression (Fig. 4 ). Tap73 and ΔNp73 are two variants of p73, which is a structural homolog of p53 and acts as a tumor suppressor. This gene often encodes two opposing variants: the transcriptionally active TAp73 and the dominant-negative ΔNp73 [ 23 ]. ΔNp73 overexpressed in a variety of cancers and it is correlated with poor prognosis [ 24 , 25 ]. Additionally, ΔNp73 can interact with wild-type p53 or Tap73 to efficiently counteract wild-type p53 and TAp73 mediated apoptosis, and growth suppression [ 26 ]. Therefore, ΔNp73 has become a novel tumor-specific molecular target for cancer because of its anti-apoptotic functions. TAp73 contains the NH2-terminal domain and plays a similar role to p53 as a tumor suppressor [ 27 , 28 ]. TAp73 is relevant to DNA damage and upregulates proapoptotic Bcl-2 family members and causes apoptosis via the mitochondrial pathway [ 29 ]. Recently, some studies elucidated that the ratio between Tap73 and ∆Np73 might contribute to tumorigenesis and resistance to chemotherapy and determined the fate of the cell [ 30 ]. Our results showed overexpressed PCBP1 upregulated the ratio of Tap73 / ΔNp73 and caused HeLa cell apoptosis. These data are evidenced by our finding that that PCBP1 is involved in p73 gene splicing and it will induce cell apoptosis via upregulation of Tap73 / ΔNp73 ratio in human cervical cancer (Fig. 4 ). This may have a certain inspiration for cancer treatment. Taken together, our data indicated that PCBP1 is an important gene and a tumor suppressor in cervical cancer. To further study the mechanism of PCBP1 induced apoptosis, we examined the expression of some related proteins after transfection of PCBP1. Indeed, our data revealed that overexpressed PCBP1 significantly decreased anti-apoptosis Bcl-2 expression and increased the Bax / Bcl-2 ratio (Fig. 5 ). It has been confirmed that Bax / Bcl-2 ratio regulated cytochrome c release from mitochondria [ 31 ]. In addition, we further detected the expression level of cytochrome c, which can activate the caspase-3 and downstream cell death pathway. We discovered that procaspase-3 expression levels were significantly reduced, while cytochrome c levels were elevated. In light of our previous work, Tap73 / ΔNp73 ratio also plays an important role in regulating apoptosis via mitochondrial pathway [ 32 , 33 ]. PCBP1 may initiate a mitochondria-mediated apoptotic pathway by inducing p73 alternative splicing. As expected from the above results, we believe that PCBP1 plays a pivotal role in arresting cell cycle, inducing apoptosis, regulating p73 splicing in human cervical carcinoma cells, and it induced splice regulation of p73 may be another downstream signaling pathway independent of p53. In conclusion, our results suggested that PCBP1 could be used as a potential candidate for cervical cancer therapy and it has broad prospects as a molecular therapeutic target for cervical cancer. However, this also requires the use of tumor-bearing animal models and clinical trials to further study the effects of PCBP1. Materials And Methods Cell culture and transfection The human cervical carcinoma HeLa cells were obtained from the First Hospital of Lanzhou University and cultured in Dulbecco's modified Eagle's medium (DMEM, Minghai Biochem, Lanzhou, China) supplemented with 10% fetal bovine serum (Minghai Biochem, Lanzhou, China) at a culture temperature of 37℃, 5% CO 2 in incubator (Thermo, USA). DNA transfection was carried out using Exfect2000 transfection reagent (Vazyme, Nanjing, China) as a mediator according to the manufacturer's instruction. The pEGFP-N1-PCBP1 and non-targeting negative control pEGFP-N1 was purchased from Invitrogen (Invitrogen Life Technologies, CA, USA). To transfect HeLa cells with plasmid vector, cells were plated into either 60 mm dish or a 100 mm dish and allowed to adhere for 24 h. Exfect2000 transfection reagent was utilized for the transfection. After pEGFP-N1 or pEGFP-N1-PCBP1 transfection, cells were cultured for 5 h and then the medium was replaced with fresh medium supplemented with 10% fetal bovine serum. Cells were harvested 24–48 h after transfection. Cell survival detection Cell viability was investigated using the methyl tetrazolium salt (MTS) assay. Cells were plated into 96-well plates (Promega, Beijing, China) and incubated 24 h. Then used transfection reagent to transfect pEGFP-N1 or pEGFP-N1-PCBP1 and cultured 24 h and 48 h to detect viability. 20 µl / well of MTS solution was added to each 100 µl of DMEM medium, and incubated for 60 min at a 37℃ constant temperature incubator. Then the absorbance was detected with multifunction microplate reader (Tecan Infinite M200, Swiss) at 490 nm. The survival rate of cells in each well was shown as a percentage of control. Quantitative RT-PCR analysis and agarose gel electrophoresis Total RNA was extracted from cells with TRIzol reagent (Takara Biotech Co., Ltd.) and the complementary DNA (cDNA) was synthesized by using Transcriptor First Strand cDNA Synthesis System kit, real-time PCR analysis of PCBP1 and the reference gene β-Actin was treated by using a SYBR Green reaction kit (TIANGEN, China) in real-time PCR instrument (Thermo, USA), according to instruction. All experiments were carried out in triplicate and analyzed using the comparative threshold cycle (2 −ΔΔCT ) method. Products were run in 1% agarose gel and the band intensity was scanned. The results were normalized by β-Actin levels. Colony formation assay The cells were harvested for 24 h after transfection, and seeded into 60 mm dishes. After incubated at 37 °C for 10–14 days, the culture was terminated when macroscopic clones appeared in the dishes. The clone was fixed with paraformaldehyde and stained with crystal violet. Count clones containing more than 50 cells, calculated clone formation rate and collected images. Each experiment was performed in triplicate. Cell cycle assay After pEGFP-N1 or pEGFP-N1-PCBP1 transfection and cultured for 24 h, then collected the cells, fixed with pre-cooled 70% ethanol in PBS, and overnight at 4℃. Then, the supernatant was centrifuged at 800 rpm for 4 min. 100 µl of propidium iodide (PI, Sigma, USA) was added after washing three times with PBS, and the cells were incubated for 30 min in the dark. The samples were collected with a minimum of 20,000 cells and analyzed with a flow cytometer FlowSight (Amnis, Seattle, WA, USA). The results were analyzed with FlowJo 7.6 software and each experiment was repeated at least three times. Cell apoptosis assay We used 4', 6-diamidino-2-phenylindole (DAPI) (Vector, Laboratories, USA) staining to observe nuclear morphology after pEGFP-N1 or pEGFP-N1-PCBP1 transfection. HeLa cells were expressed PCBP1 after transfection and cultured for 48 h. First, cells were collected and washed twice with PBS and fixed with the 4% paraformaldehyde for 20 min. And then permeabilized with 0.5% Triton X-100 / PBS for 15 min on ice and finally treated with DAPI. Finally fluorescence microscope was used to observe after the tablet was sealed with anti-fluorescence quench sealing solution. In order to determine the apoptosis rate more accurately, Annexin V-FITC / PI double staining flow cytometry was used to detect apoptosis. Cells were collected and washed twice with PBS. Next, 75 µl of binding buffer was added to the cell suspension, and then 5 µl of annexin V and 5 µl of PI (Annexin V-FITC Apoptosis Detection Kit I, BD, USA) were also added to the cell suspension. The samples were detected with a flow cytometer FlowSight (Amnis, Seattle, WA, USA) after incubated in the dark for 15 min at RT, and immediately analyzed on IDEAS Application v6.0. Western blot analysis After pEGFP-N1 or pEGFP-N1-PCBP1 transfection and cultured for 24 h and 48 h, cells were collected. Whole proteins were lysed from the cells using RIPA lysis buffer (Solarbio, China) added PMSF. Western blot analysis was performed according to standard procedures. Proteins were fractionated by 10% SDS-PAGE and transferred to a methanol activated PVDF membrane (GE Healthcare, Beijing, China). Antibodies against Tap73, ΔNp73 (Imgenex, San Diego, USA), β-Actin (Bioss, Beijing, China) and Bax, Bcl-2, Cytochrome c, procaspase-3, cleaved caspase-3 (Santa Cruz, CA, USA) were used according to the instruction. HRP-linked anti-mouse or anti-rabbit IgG antibodies (Bioss, Beijing, China) were used as secondary antibodies. The results were normalized by β-Actin levels. Immunofluorescence Cells were transfected with pEGFP-N1 or pEGFP-N1-PCBP1 and harvested at 48 h. First, cells were washed in 0.01 M PBS (pH 7.4), fixed with paraformaldehyde, permeabilized with 0.5% Triton X-100 / PBS for 15 min on ice, and blocked with 5% BSA for 60 min at RT. Incubated with antibodies against PCBP1 (Santa Cruz, CA, USA) at 4℃ overnight, then added fluorescent secondary antibody and incubated at RT for 1 h in the dark. Finally, 0. 01 M PBS was used to wash three times for 5 min each wash and DAPI was added. After added glycerol, the samples were detected with a confocal laser microscope (LSM, Carl Zeiss AG, Germany). Statistical analysis Data are presented as means ± SD. Statistical analysis were showed on the means of the data obtained from at least three independent experiments. Student's t-tests program in Microsoft Excel was used to compare the differences between the mock group, vector group and the PCBP1 group. P < 0.05 was considered significant. Availability Of Data And Materials All data generated or analysed during this study are included in this published article. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials All data generated or analysed during this study are included in this published article. Competing interests The authors report no conflict of interest in this work. Acknowledgments The authors thank anonymous reviewers who helped in improving the article by their valuable comments. Funding We thank the grants of the national Key R&D project of the Chinese Ministry of Science and Technology (2018YFE0205100), the Key Program of the National Natural Science Foundation of China (U1632270), the National Natural Science Foundation of China (11675234, 11875061), and the Natural Science Foundation of Gansu (17JR5RA310) for financial support. Author information Hong Zhang and Cuixia Di are senior authors who contributed equally. Affiliations Bio-Medical Research Center, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, 730000, China Yuhong Chen, Cuixia Di, Caipeng Xu, Xiaohua Chen, Xuetian Zhang, Junfang Yan, Fang Wang, Hongyan Li & Hong Zhang Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou, 730000, China Yuhong Chen, Cuixia Di, Caipeng Xu, Xiaohua Chen, Xuetian Zhang, Junfang Yan, Fang Wang, Hongyan Li & Hong Zhang College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 100039, China Yuhong Chen, Cuixia Di, Caipeng Xu, Xiaohua Chen, Xuetian Zhang, Junfang Yan, Fang Wang, Hongyan Li & Hong Zhang School of Nuclear Science and Technology, University of Chinese Academy of Sciences, Beijing 100039, China Yuhong Chen, Cuixia Di, Caipeng Xu, Xiaohua Chen, Xuetian Zhang, Junfang Yan, Fang Wang, Hongyan Li & Hong Zhang Laboratory of Precision Medicine and Translational Medicine, Suzhou Hospital Affiliated to Nanjing Medical University, Suzhou Science and Technology Town Hospital, Suzhou, 215153, China Tuanjie Che Key Laboratory of Functional Genomic and Molecular Diagnosis of Gansu Province, Lanzhou, 730030, China Tuanjie Che Department of Biotherapy Center, Gansu Provincial Hospital, Lanzhou, China Guoying Miao Medical College of Soochow University, Soochow University, Suzhou, 215000, China Hongying Yang Contributions YC, CD and HZ conceived the manuscript. YC, CD, CX and HC consulted the literature and wrote the initial draft of the manuscript. TC, GM, XZ, JY, FW, HL and HY participated in writing the manuscript. All authors reviewed the manuscript. Corresponding author Correspondence to Hong Zhang. References 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. Ca-a Cancer Journal for Clinicians. 2018;68(6):394–424. 2. Aasheim HC, Loukianova T, Deggerdal A, Smeland EB. Tissue specific expression and cDNA structure of a human transcript encoding a nucleic acid binding [oligo(dC)] protein related to the pre-mRNA binding protein K. Nucleic acids research. 1994;22(6):959–64. 3. Chaudhury A, Chander P, Howe PH. Heterogeneous nuclear ribonucleoproteins (hnRNPs) in cellular processes: Focus on hnRNP E1's multifunctional regulatory roles. RNA. 2010;16(8):1449–62. 4. Ji FJ, Wu YY, An Z, Liu XS, Jiang JN, Chen FF, et al. Expression of both poly r(C) binding protein 1 (PCBP1) and miRNA-3978 is suppressed in peritoneal gastric cancer metastasis. Sci Rep. 2017;7(1):15488. 5. Zhou M, Tong X. Downregulated Poly-C binding protein-1 is a novel predictor associated with poor prognosis in Acute Myeloid Leukemia. Diagnostic pathology. 2015;10:147–7. 6. Liu Y, Gai L, Liu J, Cui Y, Zhang Y, Feng J. Expression of poly(C)-binding protein 1 (PCBP1) in NSCLC as a negative regulator of EMT and its clinical value. Int J Clin Exp Pathol. 2015;8(6):7165–72. 7. Pillai MR, Chacko P, Kesari LA, Jayaprakash PG, Jayaram HN, Antony AC. Expression of folate receptors and heterogeneous nuclear ribonucleoprotein E1 in women with human papillomavirus mediated transformation of cervical tissue to cancer. J Clin Pathol. 2003;56(8):569–74. 8. Zhang W, Shi H, Zhang M, Liu B, Mao S, Li L, et al. Poly C binding protein 1 represses autophagy through downregulation of LC3B to promote tumor cell apoptosis in starvation. Int J Biochem Cell Biol. 2016;73:127–36. 9. Ishii T, Hayakawa H, Igawa T, Sekiguchi T, Sekiguchi M. Specific binding of PCBP1 to heavily oxidized RNA to induce cell death. Proc Natl Acad Sci U S A. 2018;115(26):6715–20. 10. Zhang T, Huang XH, Dong L, Hu D, Ge C, Zhan YQ, et al. PCBP-1 regulates alternative splicing of the CD44 gene and inhibits invasion in human hepatoma cell line HepG2 cells. Mol Cancer. 2010;9:72. 11. Jiang P, Li Z, Tian F, Li X, Yang J. Fyn/heterogeneous nuclear ribonucleoprotein E1 signaling regulates pancreatic cancer metastasis by affecting the alternative splicing of integrin beta1. Int J Oncol. 2017;51(1):169–83. 12. Nandal A, Ruiz JC, Subramanian P, Ghimire-Rijal S, Sinnamon RA, Stemmler TL, et al. Activation of the HIF prolyl hydroxylase by the iron chaperones PCBP1 and PCBP2. Cell Metab. 2011;14(5):647–57. 13. Wang H, Vardy LA, Tan CP, Loo JM, Guo K, Li J, et al. PCBP1 suppresses the translation of metastasis-associated PRL-3 phosphatase. Cancer Cell. 2010;18(1):52–62. 14. Shafabakhsh R, Reiter RJ, Mirzaei H, Teymoordash SN, Asemi Z. Melatonin: A new inhibitor agent for cervical cancer treatment. J Cell Physiol. 2019;234(12):21670–82. 15. Dejgaard K, Leffers H. Characterisation of the nucleic-acid-binding activity of KH domains. Different properties of different domains. Eur J Biochem. 1996;241(2):425–31. 16. Chkheidze AN, Liebhaber SA. A novel set of nuclear localization signals determine distributions of the alphaCP RNA-binding proteins. Mol Cell Biol. 2003;23(23):8405–15. 17. Lu J, Gao FH. Role and molecular mechanism of heterogeneous nuclear ribonucleoprotein K in tumor development and progression. Biomed Rep. 2016;4(6):657–63. 18. Barboro P, Ferrari N, Balbi C. Emerging roles of heterogeneous nuclear ribonucleoprotein K (hnRNP K) in cancer progression. Cancer Lett. 2014;352(2):152–9. 19. Shi HS, Li H, Yuan RH, Guan W, Zhang XM, Zhang SY, et al. PCBP1 depletion promotes tumorigenesis through attenuation of p27(Kip1) mRNA stability and translation. Journal of Experimental Clinical Cancer Research. 2018;37:18. 20. Ishii T, Hayakawa H, Igawa T, Sekiguchi T, Sekiguchi M. Specific binding of PCBP1 to heavily oxidized RNA to induce cell death. Proc Natl Acad Sci USA. 2018;115(26):6715–20. 21. Guo JH, Jia R. Splicing factor poly(rC)-binding protein 1 is a novel and distinctive tumor suppressor. J Cell Physiol. 2019;234(1):33–41. 22. Di CX, Syafrizayanti QJ, Zhang YH, Chen YP, Wang XT, Zhang, et al. Function, clinical application, and strategies of Pre-mRNA splicing in cancer. Cell Death Differ. 2019;26(7):1181–94. 23. Melino G, De Laurenzi V, Vousden KH. p73: Friend or foe in tumorigenesis. Nat Rev Cancer. 2002;2(8):605–15. 24. Prieto-Nieto MI, Pastor D, Rodriguez-Cobos J, Perez JP, Mendez C, Palacios E, et al. Delta Np73 status in peritoneal and ovarian dissemination of appendicular adenocarcinoids (goblet cells). Clinical Translational Oncology. 2019;21(10):1432–9. 25. Gomez LC, Sottile ML, Guerrero-Gimenez ME, Zoppino FCM, Redondo AL, Gago FE, et al. TP73 DNA methylation and upregulation of Delta Np73 are associated with an adverse prognosis in breast cancer. J Clin Pathol. 2018;71(1):52–8. 26. Zaika AI, Slade N, Erster SH, Sansome C, Joseph TW, Pearl M, et al. Delta Np73, a dominant-negative inhibitor of wild-type p53 and TAp73, is up-regulated in human tumors. J Exp Med. 2002;196(6):765–80. 27. Rodriguez N, Pelaez A, Barderas R, Dominguez G. Clinical implications of the deregulated TP73 isoforms expression in cancer. Clinical Translational Oncology. 2018;20(7):827–36. 28. Tomasini R, Tsuchihara K, Wilhelm M, Fujitani M, Rufini A, Cheung CC, et al. TAp73 knockout shows genomic instability with infertility and tumor suppressor functions. Genes Dev. 2008;22(19):2677–91. 29. Muller M, Schilling T, Sayan AE, Kairat A, Lorenz K, Schulze-Bergkamen H, et al. TAp73/Delta Np73 influences apoptotic response, chemosensitivity and prognosis in hepatocellular carcinoma. Cell Death Differ. 2005;12(12):1564–77. 30. Lucena-Araujo AR, Kim HT, Thome C, Jacomo RH, Melo RA, Bittencourt R, et al. High DeltaNp73/TAp73 ratio is associated with poor prognosis in acute promyelocytic leukemia. Blood. 2015;126(20):2302–6. 31. Raisova M, Hossini AM, Eberle J, Riebeling C, Wieder T, Sturm I, et al. The Bax/Bcl-2 ratio determines the susceptibility of human melanoma cells to CD95/Fas-mediated apoptosis. Journal of Investigative Dermatology. 2001;117(2):333–40. 32. Di CX, Han L, Zhang H, Xu S, Mao AH, Sun C, et al. Diallyl disulfide attenuated carbon ion irradiation-induced apoptosis in mouse testis through changing the ratio of Tap73/DeltaNp73 via mitochondrial pathway. Sci Rep. 2015;5:16020. 33. Zhang Q, Di C, Yan J, Wang F, Qu T, Wang Y, et al. Inhibition of SF3b1 by pladienolide B evokes cycle arrest, apoptosis induction and p73 splicing in human cervical carcinoma cells. Artificial Cells Nanomedicine Biotechnology. 2019;47(1):1273–80. Declarations Declarations. 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-15362\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research article\",\"associatedPublications\":[],\"authors\":[{\"id\":374059,\"identity\":\"eaa292bb-9f88-42fc-8c2b-4f0d3c24d8e5\",\"order_by\":1,\"name\":\"Hong Zhang\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYDACCSBmbLDhIVlLGulaDpOgQ35287OHX3ecl9Gd3fzw4w8GO3kG9rMH8GphnHPM3Fj2zG0eszvHjKV5GJING3jyEvBqYZZIMJOWbANquZHDIA3kJzBI8Bjg1cImkf4NqOUcSAvzzx8M9YS18EjkmEl+bDsA0sImwcNwmLAWCYmcMmnGtmSQX8yseQyOG7bx5ODXIj8jfZvkzzY7e7PbzY9v/qioludnP4NfCwgwg+MRFEEMQMVsBNUDAeMPuJZRMApGwSgYBVgAACVKO8RMtttCAAAAAElFTkSuQmCC\",\"orcid\":\"\",\"institution\":\"Insititute of Mordern Physics, Chinese Academy of Sciences\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Hong\",\"middleName\":\"\",\"lastName\":\"Zhang\",\"suffix\":\"\"},{\"id\":374060,\"identity\":\"4594d420-1a59-4a8a-ae95-268b92951778\",\"order_by\":2,\"name\":\"Yuhong Chen\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Institute of Modern Physics,Chinese Academy of Sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yuhong\",\"middleName\":\"\",\"lastName\":\"Chen\",\"suffix\":\"\"},{\"id\":374061,\"identity\":\"b53e34c1-f0bc-4169-9f41-d2aabff491d1\",\"order_by\":3,\"name\":\"Cuixia Di\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Institute of Modern Physics,Chinese Academy of Science\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Cuixia\",\"middleName\":\"\",\"lastName\":\"Di\",\"suffix\":\"\"},{\"id\":374062,\"identity\":\"744da980-b015-4c5b-9ea5-cba29cfcbc56\",\"order_by\":4,\"name\":\"Caipeng Xu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"institute of modern physics,chinese academy of sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Caipeng\",\"middleName\":\"\",\"lastName\":\"Xu\",\"suffix\":\"\"},{\"id\":374063,\"identity\":\"78080675-332e-47e4-9e94-4c379d241dbf\",\"order_by\":5,\"name\":\"Xiaohua Chen\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"institute of modern phycics, chinese academy of sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xiaohua\",\"middleName\":\"\",\"lastName\":\"Chen\",\"suffix\":\"\"},{\"id\":374064,\"identity\":\"e1d9c0b6-aa90-4245-945b-dceb09e48bda\",\"order_by\":6,\"name\":\"Tuanjie Che\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"suzhou science and technology town hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Tuanjie\",\"middleName\":\"\",\"lastName\":\"Che\",\"suffix\":\"\"},{\"id\":374065,\"identity\":\"195d8baf-2c70-47fd-95c3-2c4e9f401f66\",\"order_by\":7,\"name\":\"Guoying Miao\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Gansu Provincial Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Guoying\",\"middleName\":\"\",\"lastName\":\"Miao\",\"suffix\":\"\"},{\"id\":374066,\"identity\":\"9015e6e8-38d4-4cf7-a575-148c77604adb\",\"order_by\":8,\"name\":\"Xuetian Zhang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Institute of modern physics, Chinese academy of sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xuetian\",\"middleName\":\"\",\"lastName\":\"Zhang\",\"suffix\":\"\"},{\"id\":374067,\"identity\":\"d62eb542-cf6a-40de-9261-36dcf3553a13\",\"order_by\":9,\"name\":\"Junfang Yan\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Institute of modern physics,Chinese academy of sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Junfang\",\"middleName\":\"\",\"lastName\":\"Yan\",\"suffix\":\"\"},{\"id\":374068,\"identity\":\"563ae200-8017-4177-b72f-b3a179537f1c\",\"order_by\":10,\"name\":\"Fang Wang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Institute of modern physics, Chinese academy of sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Fang\",\"middleName\":\"\",\"lastName\":\"Wang\",\"suffix\":\"\"},{\"id\":374069,\"identity\":\"3a3fa75e-5ab1-402a-887a-ad6760c4eabb\",\"order_by\":11,\"name\":\"Hongyan Li\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Institute of modern phycics,Chinese academy of sciences\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Hongyan\",\"middleName\":\"\",\"lastName\":\"Li\",\"suffix\":\"\"},{\"id\":374070,\"identity\":\"d34f611f-10b5-4240-8c10-87d04f07375d\",\"order_by\":12,\"name\":\"Hongying Yang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Soochow University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Hongying\",\"middleName\":\"\",\"lastName\":\"Yang\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2020-02-26 13:37:29\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.2.24804/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.2.24804/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":568645,\"identity\":\"186ba080-3f99-4a5f-a732-580aa8112769\",\"added_by\":\"auto\",\"created_at\":\"2020-02-28 15:41:49\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":242098,\"visible\":true,\"origin\":\"\",\"legend\":\"The expression of PCBP1 mRNA and protein and its spacial distribution. a Expression of PCBP1 mRNA was determined in HeLa cells, the data was normalized to β-Actin expression. b PCBP1 protein is distributed in both the cytoplasm and the nucleus. All experiments were repeated at least three times. The data are expressed as the mean ± SD. *** P \\u003c 0.001 (vs. mock group).\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/39b3635a-0faa-4725-b325-f5a57a96e177/v1/1.png\"},{\"id\":568646,\"identity\":\"8da2239f-f320-4530-b3aa-fbef91c2e0bf\",\"added_by\":\"auto\",\"created_at\":\"2020-02-28 15:41:49\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":382992,\"visible\":true,\"origin\":\"\",\"legend\":\"Overexpression of PCBP1 significantly inhibited human cervical carcinoma HeLa cells viability and colony formation. a The cell viability transfected with pEGFP-N1-PCBP1 was detected by MTS assay. b Quantification of the colony counts. c Representative images of the colony formation potential of HeLa cells treated with transfection. All experiments were repeated at least three times. The data are performed as the mean ± SD. *** P \\u003c 0.001 (vs. mock group).\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/39b3635a-0faa-4725-b325-f5a57a96e177/v1/2.png\"},{\"id\":568647,\"identity\":\"c352195b-3fc9-45b3-aca7-f0d131a16b62\",\"added_by\":\"auto\",\"created_at\":\"2020-02-28 15:41:49\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":206023,\"visible\":true,\"origin\":\"\",\"legend\":\"Overexpression of PCBP1 induced cell cycle arrest and apoptosis in human cervical carcinoma HeLa cells. a Nuclear morphology was observed after transfection in human cervical carcinoma HeLa cells. b Flow cytometry analysis of cell cycle arrest in HeLa cells transfected with PCBP1. c Apoptotic effect of overexpression of PCBP1 in HeLa cells. d Percentages of G2 / M phase of cell population was shown. e Total percentages of apoptotic and dead rate of cell population was calculated. All experiments were repeated at least three times. The data are expressed as the mean ± SD, *** P \\u003c 0.001 (vs. mock group). ** P \\u003c 0.01 (vs. mock group).\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/39b3635a-0faa-4725-b325-f5a57a96e177/v1/3.png\"},{\"id\":568648,\"identity\":\"1f5e8fe3-9e21-4614-842a-98a607fef70d\",\"added_by\":\"auto\",\"created_at\":\"2020-02-28 15:41:50\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":466500,\"visible\":true,\"origin\":\"\",\"legend\":\"Overexpression of PCBP1 increased the protein ratio of Tap73/ΔNp73 in human cervical carcinoma HeLa cells. a Western blot results. b Quantitative analysis of Tap73 and ΔNp73 proteins in HeLa cells by western blot analysis. c The expression and localization of Tap73 and ΔNp73 proteins in HeLa cells detected by fluorescent microscopy. All experiments were repeated at least three times. The data are expressed as the mean ± SD. *** P \\u003c 0.001 (vs. control group). ** P \\u003c 0.01 (vs. mock group).\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/39b3635a-0faa-4725-b325-f5a57a96e177/v1/4.png\"},{\"id\":568649,\"identity\":\"d7e15bc6-c9cd-4226-af19-d0d4b138a9da\",\"added_by\":\"auto\",\"created_at\":\"2020-02-28 15:41:50\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":124424,\"visible\":true,\"origin\":\"\",\"legend\":\"Overexpression of PCBP1 induced apoptosis through mitochondrial pathway in human cervical carcinoma HeLa cells. a Western blot results. b Quantitative analysis of Bax / Bcl-2 ratio in HeLa cells. c Quantitative analysis of procaspase-3 expression in HeLa cells. d Quantitative analysis of cytochrome c expression in HeLa cells. All experiments were repeated at least three times. The data are expressed as the mean ± SD. *** P \\u003c 0.001 (vs. mock group). ** P \\u003c 0.01 (vs. mock group).\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/39b3635a-0faa-4725-b325-f5a57a96e177/v1/5.png\"},{\"id\":13491099,\"identity\":\"fa97eb09-fdae-47f4-9e23-5af0aab6f0c0\",\"added_by\":\"auto\",\"created_at\":\"2021-09-16 22:26:14\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1863914,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-15362/v1/33eabfd0-4b85-4d4a-b596-a0432e1a4deb.pdf\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Overexpression of splicing factor poly(rC)-binding protein 1 elicits cycle arrest, apoptosis induction, and p73 splicing in human cervical carcinoma cells\",\"fulltext\":[{\"header\":\"Background\",\"content\":\" \\u003cp\\u003eCervical cancer ranks as the fourth most frequently diagnosed cancer and the fourth leading cause of cancer death in women [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. Therefore, research on its pathogenesis is crucial. Poly(rC) binding protein 1 (PCBP1) is a member of the RNA binding protein family and is firstly cloned from human lymphocyte cDNA library in 1994 [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. With the deepening of research in recent years, PCBP1 has been found to be ubiquitously expressed in many tissues and plays a multifunction role in various life activities, such as intracellular transcription and post-transcriptional regulation, including alternative splicing of pre-mRNA, mRNA stability and translation [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. In recent years, researches focus on the relationship between PCBP1 and tumors. Studies have been found that PCBP1 acts as a tumor suppressor in tumors and the expression is significantly downregulated in various tumors, including gastric cancer [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e], acute myeloid leukemia [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e], non-small-cell lung cancer [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e], cervical cancer [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e] \\u003cspan type=\\\"Italic\\\" class=\\\"Italic\\\" name=\\\"Emphasis\\\"\\u003eet al\\u003c/span\\u003e. In general, PCBP1 plays a multifunctional role in tumor progress. For instance, PCBP1 has an influence on apoptosis in variety cancers [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. It is also involved in alternative splicing which dysregulation usually leads to disease and is increasingly associated with tumorigenesis [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. In addition, PCBP1 negatively regulated the tumor hypoxic microenvironment and inhibited autophagy to further affect the tumor formation and development [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. Moreover, PCBP1 prevented the process of EMT to reduce cancer metastasis [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e]. The above cases all turn out that PCBP1 is involved in the development of tumors as a tumor suppressor, but little information has been available on the molecular mechanisms by which PCBP1 causes cervical cancer apoptosis. In the present study, we would provide some preliminary data to illustrate the distinct functions of PCBP1 in p73 alternative splicing and the mechanisms of PCBP1 on cervical cancer cells apoptosis. The results suggested that PCBP1 may be an attractive novel target for cervical cancer therapy.\\u003c/p\\u003e \"},{\"header\":\"Results\",\"content\":\" \\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eThe expression of PCBP1 and its spacial distribution\\u003c/h2\\u003e \\u003cp\\u003eTo explore the biologic function of PCBP1, we initially transfected HeLa cells with pEGFP-N1 or pEGFP-N1-PCBP1, and then we verified whether the PCBP1 was successfully transfected and overexpressed in cells. We performed real-time PCR and immunofluorescence experiments. The results demonstrated that PCBP1 mRNA expression was significantly increased in cells transfected with pEGFP-N1-PCBP1 compared to mock group (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ea) and there was no significant difference in mRNA expression in mock and vector group (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ea). In addition, further immunofluorescence experiments results showed the PCBP1 protein expression was increased compared with mock and vector group, and the PCBP1 is distributed in both the cytoplasm and the nucleus (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eb).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003eOverexpression of PCBP1 and its effects on human cervical carcinoma cells viability\\u003c/h2\\u003e \\u003cp\\u003eIn order to understand the effects of overexpressed PCBP1 in HeLa cells, we examined the proliferation of HeLa cells in different time points after transfection. MTS analysis proved that elevated PCBP1 significantly reduced the cell viability of HeLa cells, and the inhibition of proliferation of PCBP1 transfection. The results were significantly time-dependent (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea). Furthermore, the experimental results from the colony formation assay suggested that overexpression of PCBP1 in HeLa cells could significantly repress cell colony formation compared with mock and vector group (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eb, c).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePCBP1 induces cell cycle arrest and apoptosis\\u003c/h2\\u003e \\u003cp\\u003eTo detect the effect of overexpressed PCBP1, after transfection, we stained the nuclei and then observed the nuclear morphology. We found the nuclear morphology changed and apoptotic bodies appear, this suggested us that apoptosis took place (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn order to further verify the mechanism of PCBP1 in inhibiting the growth of HeLa cells, we used flow cytometry to detect the cell cycle and apoptosis. The results showed that overexpressed PCBP1 would cause cell cycle arrest (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eb), and the ratio of G2 / M cells in HeLa cells were significantly increased (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ed). Then we detected cell apoptosis to prove that whether the decrease in viability of HeLa cells after transfected with pEGFP-N1-PCBP1 was caused by apoptosis. The results showed cells transfected with pEGFP-N1-PCBP1 for 48\\u0026nbsp;h performed more apoptosis than the mock and vector group (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ec, e). The results of the apoptosis experiment obtained by flow cytometry analysis were statistically analyzed, and the results were also verified.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePCBP1 upregulates Tap73 and downregulates ΔNp73, indicating activation of apoptosis\\u003c/h2\\u003e \\u003cp\\u003eIn order to demonstrate whether p73 is involved in PCBP1 induced cell cycle arrest and apoptosis in human cervical carcinoma cells, western blot and immunofluorescence were used to detect the level of Tap73 and ΔNp73 proteins. We found the level of ΔNp73 was significantly reduced in HeLa cells at 24\\u0026nbsp;h after transfected with pEGFP-N1-PCBP1 compared with the mock group. On the contrary, the level of Tap73 was increased compared with the mock group (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea). Quantitative results showed, PCBP1 induced an increase in the Tap73 / ΔNp73 ratio (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eb). Next, we used immunofluorescent to further verify its spatiotemporal distribution in cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ec). ΔNp73 expression clearly reduced but Tap73 expression obviously upregulated. Moreover, Tap73 mainly detected in the cytoplasm, but ΔNp73 mainly detected in the nucleus. These findings suggested that p73 splicing is involved in PCBP1 induced cell cycle arrest and apoptosis in HeLa cells.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePCBP1 regulates apoptosis via mitochondrial pathway\\u003c/h2\\u003e \\u003cp\\u003eIn order to further verify whether PCBP1 induced apoptosis is associated with mitochondrial apoptosis pathway, we used western blot to examine several key proteins in the mitochondrial apoptosis signalling pathway. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e, overexpression of PCBP1 upregulated the ratio of Bax / Bcl-2 to promote cell apoptosis (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eb). In addition, there was a substantial increase in the expression of cytochrome c at 24\\u0026nbsp;h after transfected with pEGFP-N1-PCBP1 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ed). Furthermore, decreased procaspase-3 and increased cleaved caspase-3 both indicated the occurrence of apoptosis (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ec). Thereby confirming that overexpression of PCBP1 upregulated Bax / Bcl-2 ratio, promoted cytochrome c release and activated caspase-3 to induce apoptosis.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \"},{\"header\":\"Discussion\",\"content\":\" \\u003cp\\u003eCervical cancer is one of the most diseases threatening women's health, but our understanding of its pathogenesis is still not deep enough. It is imminent to further study its pathogenesis [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. PCBP1 is an evolutionarily conserved RNA-binding protein that regulates transcription, translation, and alternative splicing of genes [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. Increasing evidence revealed that PCBP1 is significantly downregulated in gastric cancer [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e], acute myeloid leukemia [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e], non-small-cell lung cancer [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e], and cervical cancer [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e] \\u003cspan type=\\\"Italic\\\" class=\\\"Italic\\\" name=\\\"Emphasis\\\"\\u003eet al\\u003c/span\\u003e. and is involved in tumor metastasis. Subsequently, scientists pointed out that PCBP1 might act as a tumor suppressor in various tumors [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]. For instance, it has been proved that PCBP1 is involved in cell apoptosis. Zhang \\u003cspan type=\\\"Italic\\\" class=\\\"Italic\\\" name=\\\"Emphasis\\\"\\u003eet al\\u003c/span\\u003e. reported that overexpression of PCBP1 decreased the Bcl-2 expression and caused cancer cells apoptosis [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. Shi \\u003cspan type=\\\"Italic\\\" class=\\\"Italic\\\" name=\\\"Emphasis\\\"\\u003eet al\\u003c/span\\u003e. confirmed that PCBP1 increased p27 expression via stabilizing its mRNA to further facilitate cell apoptosis [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]. Ishii \\u003cspan type=\\\"Italic\\\" class=\\\"Italic\\\" name=\\\"Emphasis\\\"\\u003eet al\\u003c/span\\u003e. reported PCBP1 activated apoptosis through interacted with more severely oxidized RNA [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. Interestingly, PCBP1 has gained more attention due to its multiple functions in tumor progression, but the real mechanism is relatively unexplored. Therefore, it is worthy to further study the relationship between PCBP1 and cervical cancer. Here, we transfected PCBP1 into HeLa cells and testified that overexpression of PCBP1 greatly repressed proliferation of HeLa cells in time-dependent manner (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). It also induced G2 / M phase arrest and significant rise of apoptotic cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). Overall, these results indicated that elevated PCBP1 is an efficient way to inhibit tumor cell progression, and this will provide a reference for further understanding of the pathogenesis of cervical cancer.\\u003c/p\\u003e \\u003cp\\u003eRNA splicing is the key to the pathology of numerous diseases, and experiments have shown that dysregulation of splicing isoforms were increasingly associated with tumor proliferation, metastasis and apoptosis [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. Additionally, PCBP1 is related to alternative splicing. In pancreatic cancer, upregulated PCBP1 reduced tumor metastasis by interacting with integrin β1 to regulate its alternative splicing [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. Moreover, overexpression of PCBP1 inhibited the tumor invasion and metastasis in HepG2 cells via regulating exon inclusion of CD44 [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. In our study, we transfected PCBP1 into HeLa cells and indicated that overexpressed PCBP1 obviously enhanced the expression of Tap73 and decreased ΔNp73 expression (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). Tap73 and ΔNp73 are two variants of p73, which is a structural homolog of p53 and acts as a tumor suppressor. This gene often encodes two opposing variants: the transcriptionally active TAp73 and the dominant-negative ΔNp73 [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e]. ΔNp73 overexpressed in a variety of cancers and it is correlated with poor prognosis [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. Additionally, ΔNp73 can interact with wild-type p53 or Tap73 to efficiently counteract wild-type p53 and TAp73 mediated apoptosis, and growth suppression [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. Therefore, ΔNp73 has become a novel tumor-specific molecular target for cancer because of its anti-apoptotic functions. TAp73 contains the NH2-terminal domain and plays a similar role to p53 as a tumor suppressor [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e]. TAp73 is relevant to DNA damage and upregulates proapoptotic Bcl-2 family members and causes apoptosis via the mitochondrial pathway [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. Recently, some studies elucidated that the ratio between Tap73 and ∆Np73 might contribute to tumorigenesis and resistance to chemotherapy and determined the fate of the cell [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e]. Our results showed overexpressed PCBP1 upregulated the ratio of Tap73 / ΔNp73 and caused HeLa cell apoptosis. These data are evidenced by our finding that that PCBP1 is involved in p73 gene splicing and it will induce cell apoptosis via upregulation of Tap73 / ΔNp73 ratio in human cervical cancer (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). This may have a certain inspiration for cancer treatment. Taken together, our data indicated that PCBP1 is an important gene and a tumor suppressor in cervical cancer.\\u003c/p\\u003e \\u003cp\\u003eTo further study the mechanism of PCBP1 induced apoptosis, we examined the expression of some related proteins after transfection of PCBP1. Indeed, our data revealed that overexpressed PCBP1 significantly decreased anti-apoptosis Bcl-2 expression and increased the Bax / Bcl-2 ratio (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). It has been confirmed that Bax / Bcl-2 ratio regulated cytochrome c release from mitochondria [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. In addition, we further detected the expression level of cytochrome c, which can activate the caspase-3 and downstream cell death pathway. We discovered that procaspase-3 expression levels were significantly reduced, while cytochrome c levels were elevated. In light of our previous work, Tap73 / ΔNp73 ratio also plays an important role in regulating apoptosis via mitochondrial pathway [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e]. PCBP1 may initiate a mitochondria-mediated apoptotic pathway by inducing p73 alternative splicing. As expected from the above results, we believe that PCBP1 plays a pivotal role in arresting cell cycle, inducing apoptosis, regulating p73 splicing in human cervical carcinoma cells, and it induced splice regulation of p73 may be another downstream signaling pathway independent of p53. In conclusion, our results suggested that PCBP1 could be used as a potential candidate for cervical cancer therapy and it has broad prospects as a molecular therapeutic target for cervical cancer. However, this also requires the use of tumor-bearing animal models and clinical trials to further study the effects of PCBP1.\\u003c/p\\u003e \"},{\"header\":\"Materials And Methods\",\"content\":\" \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCell culture and transfection\\u003c/h2\\u003e \\u003cp\\u003eThe human cervical carcinoma HeLa cells were obtained from the First Hospital of Lanzhou University and cultured in Dulbecco's modified Eagle's medium (DMEM, Minghai Biochem, Lanzhou, China) supplemented with 10% fetal bovine serum (Minghai Biochem, Lanzhou, China) at a culture temperature of 37℃, 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e in incubator (Thermo, USA). DNA transfection was carried out using Exfect2000 transfection reagent (Vazyme, Nanjing, China) as a mediator according to the manufacturer's instruction. The pEGFP-N1-PCBP1 and non-targeting negative control pEGFP-N1 was purchased from Invitrogen (Invitrogen Life Technologies, CA, USA). To transfect HeLa cells with plasmid vector, cells were plated into either 60\\u0026nbsp;mm dish or a 100\\u0026nbsp;mm dish and allowed to adhere for 24\\u0026nbsp;h. Exfect2000 transfection reagent was utilized for the transfection. After pEGFP-N1 or pEGFP-N1-PCBP1 transfection, cells were cultured for 5\\u0026nbsp;h and then the medium was replaced with fresh medium supplemented with 10% fetal bovine serum. Cells were harvested 24\\u0026ndash;48\\u0026nbsp;h after transfection.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCell survival detection\\u003c/h2\\u003e \\u003cp\\u003eCell viability was investigated using the methyl tetrazolium salt (MTS) assay. Cells were plated into 96-well plates (Promega, Beijing, China) and incubated 24\\u0026nbsp;h. Then used transfection reagent to transfect pEGFP-N1 or pEGFP-N1-PCBP1 and cultured 24\\u0026nbsp;h and 48\\u0026nbsp;h to detect viability. 20\\u0026nbsp;\\u0026micro;l / well of MTS solution was added to each 100\\u0026nbsp;\\u0026micro;l of DMEM medium, and incubated for 60\\u0026nbsp;min at a 37℃ constant temperature incubator. Then the absorbance was detected with multifunction microplate reader (Tecan Infinite M200, Swiss) at 490\\u0026nbsp;nm. The survival rate of cells in each well was shown as a percentage of control.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eQuantitative RT-PCR analysis and agarose gel electrophoresis\\u003c/h2\\u003e \\u003cp\\u003eTotal RNA was extracted from cells with TRIzol reagent (Takara Biotech Co., Ltd.) and the complementary DNA (cDNA) was synthesized by using Transcriptor First Strand cDNA Synthesis System kit, real-time PCR analysis of PCBP1 and the reference gene β-Actin was treated by using a SYBR Green reaction kit (TIANGEN, China) in real-time PCR instrument (Thermo, USA), according to instruction. All experiments were carried out in triplicate and analyzed using the comparative threshold cycle (2\\u003csup\\u003e\\u0026minus;ΔΔCT\\u003c/sup\\u003e) method. Products were run in 1% agarose gel and the band intensity was scanned. The results were normalized by β-Actin levels.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eColony formation assay\\u003c/h2\\u003e \\u003cp\\u003eThe cells were harvested for 24\\u0026nbsp;h after transfection, and seeded into 60\\u0026nbsp;mm dishes. After incubated at 37\\u0026nbsp;\\u0026deg;C for 10\\u0026ndash;14 days, the culture was terminated when macroscopic clones appeared in the dishes. The clone was fixed with paraformaldehyde and stained with crystal violet. Count clones containing more than 50 cells, calculated clone formation rate and collected images. Each experiment was performed in triplicate.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCell cycle assay\\u003c/h2\\u003e \\u003cp\\u003eAfter pEGFP-N1 or pEGFP-N1-PCBP1 transfection and cultured for 24\\u0026nbsp;h, then collected the cells, fixed with pre-cooled 70% ethanol in PBS, and overnight at 4℃. Then, the supernatant was centrifuged at 800\\u0026nbsp;rpm for 4\\u0026nbsp;min. 100\\u0026nbsp;\\u0026micro;l of propidium iodide (PI, Sigma, USA) was added after washing three times with PBS, and the cells were incubated for 30\\u0026nbsp;min in the dark. The samples were collected with a minimum of 20,000 cells and analyzed with a flow cytometer FlowSight (Amnis, Seattle, WA, USA). The results were analyzed with FlowJo 7.6 software and each experiment was repeated at least three times.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCell apoptosis assay\\u003c/h2\\u003e \\u003cp\\u003eWe used 4', 6-diamidino-2-phenylindole (DAPI) (Vector, Laboratories, USA) staining to observe nuclear morphology after pEGFP-N1 or pEGFP-N1-PCBP1 transfection. HeLa cells were expressed PCBP1 after transfection and cultured for 48\\u0026nbsp;h. First, cells were collected and washed twice with PBS and fixed with the 4% paraformaldehyde for 20\\u0026nbsp;min. And then permeabilized with 0.5% Triton X-100 / PBS for 15\\u0026nbsp;min on ice and finally treated with DAPI. Finally fluorescence microscope was used to observe after the tablet was sealed with anti-fluorescence quench sealing solution.\\u003c/p\\u003e \\u003cp\\u003eIn order to determine the apoptosis rate more accurately, Annexin V-FITC / PI double staining flow cytometry was used to detect apoptosis. Cells were collected and washed twice with PBS. Next, 75\\u0026nbsp;\\u0026micro;l of binding buffer was added to the cell suspension, and then 5\\u0026nbsp;\\u0026micro;l of annexin V and 5\\u0026nbsp;\\u0026micro;l of PI (Annexin V-FITC Apoptosis Detection Kit I, BD, USA) were also added to the cell suspension. The samples were detected with a flow cytometer FlowSight (Amnis, Seattle, WA, USA) after incubated in the dark for 15\\u0026nbsp;min at RT, and immediately analyzed on IDEAS Application v6.0.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eWestern blot analysis\\u003c/h2\\u003e \\u003cp\\u003eAfter pEGFP-N1 or pEGFP-N1-PCBP1 transfection and cultured for 24\\u0026nbsp;h and 48\\u0026nbsp;h, cells were collected. Whole proteins were lysed from the cells using RIPA lysis buffer (Solarbio, China) added PMSF. Western blot analysis was performed according to standard procedures. Proteins were fractionated by 10% SDS-PAGE and transferred to a methanol activated PVDF membrane (GE Healthcare, Beijing, China). Antibodies against Tap73, ΔNp73 (Imgenex, San Diego, USA), β-Actin (Bioss, Beijing, China) and Bax, Bcl-2, Cytochrome c, procaspase-3, cleaved caspase-3 (Santa Cruz, CA, USA) were used according to the instruction. HRP-linked anti-mouse or anti-rabbit IgG antibodies (Bioss, Beijing, China) were used as secondary antibodies. The results were normalized by β-Actin levels.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eImmunofluorescence\\u003c/h2\\u003e \\u003cp\\u003eCells were transfected with pEGFP-N1 or pEGFP-N1-PCBP1 and harvested at 48\\u0026nbsp;h. First, cells were washed in 0.01\\u0026nbsp;M PBS (pH 7.4), fixed with paraformaldehyde, permeabilized with 0.5% Triton X-100 / PBS for 15\\u0026nbsp;min on ice, and blocked with 5% BSA for 60\\u0026nbsp;min at RT. Incubated with antibodies against PCBP1 (Santa Cruz, CA, USA) at 4℃ overnight, then added fluorescent secondary antibody and incubated at RT for 1\\u0026nbsp;h in the dark. Finally, 0. 01\\u0026nbsp;M PBS was used to wash three times for 5\\u0026nbsp;min each wash and DAPI was added. After added glycerol, the samples were detected with a confocal laser microscope (LSM, Carl Zeiss AG, Germany).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical analysis\\u003c/h2\\u003e \\u003cp\\u003eData are presented as means\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SD. Statistical analysis were showed on the means of the data obtained from at least three independent experiments. Student's t-tests program in Microsoft Excel was used to compare the differences between the mock group, vector group and the PCBP1 group. \\u003cspan type=\\\"Italic\\\" class=\\\"Italic\\\" name=\\\"Emphasis\\\"\\u003eP\\u003c/span\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 was considered significant.\\u003c/p\\u003e \\u003c/div\\u003e \"},{\"header\":\"Availability Of Data And Materials\",\"content\":\" \\u003cp\\u003eAll data generated or analysed during this study are included in this published article.\\u003c/p\\u003e \"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll data generated or analysed during this study are included in this published article.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors report no conflict of interest in this work.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors thank anonymous reviewers who helped in improving the article by their valuable comments.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe thank the\\u0026nbsp;grants\\u0026nbsp;of\\u0026nbsp;the\\u0026nbsp;national Key\\u0026nbsp;R\\u0026amp;D\\u0026nbsp;project\\u0026nbsp;of\\u0026nbsp;the Chinese Ministry of Science and\\u0026nbsp;Technology\\u0026nbsp;(2018YFE0205100), the Key Program of the National Natural Science Foundation of China (U1632270), the National Natural Science Foundation of China (11675234, 11875061), and the Natural Science Foundation of Gansu (17JR5RA310) for financial support.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor information\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eHong\\u0026nbsp;Zhang and Cuixia\\u0026nbsp;Di are senior authors who contributed equally.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAffiliations\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eBio-Medical Research Center, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, 730000, China \\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eYuhong\\u0026nbsp;Chen,\\u0026nbsp;Cuixia\\u0026nbsp;Di,\\u0026nbsp;Caipeng\\u0026nbsp;Xu, Xiaohua Chen, Xuetian\\u0026nbsp;Zhang,\\u0026nbsp;Junfang\\u0026nbsp;Yan, Fang\\u0026nbsp;Wang, Hongyan Li \\u0026amp; Hong Zhang\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eKey Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou, 730000, China\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eYuhong\\u0026nbsp;Chen,\\u0026nbsp;Cuixia\\u0026nbsp;Di,\\u0026nbsp;Caipeng\\u0026nbsp;Xu, Xiaohua Chen, Xuetian\\u0026nbsp;Zhang,\\u0026nbsp;Junfang\\u0026nbsp;Yan, Fang\\u0026nbsp;Wang, Hongyan Li \\u0026amp; Hong Zhang\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eCollege of Life Sciences, University of Chinese Academy of Sciences, Beijing, 100039, China\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eYuhong\\u0026nbsp;Chen,\\u0026nbsp;Cuixia\\u0026nbsp;Di,\\u0026nbsp;Caipeng\\u0026nbsp;Xu, Xiaohua Chen, Xuetian\\u0026nbsp;Zhang,\\u0026nbsp;Junfang\\u0026nbsp;Yan, Fang\\u0026nbsp;Wang, Hongyan Li \\u0026amp; Hong Zhang\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eSchool of Nuclear Science and Technology, University of Chinese Academy of Sciences, Beijing 100039, China\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eYuhong\\u0026nbsp;Chen,\\u0026nbsp;Cuixia\\u0026nbsp;Di,\\u0026nbsp;Caipeng\\u0026nbsp;Xu, Xiaohua Chen, Xuetian\\u0026nbsp;Zhang,\\u0026nbsp;Junfang\\u0026nbsp;Yan, Fang\\u0026nbsp;Wang, Hongyan Li \\u0026amp; Hong Zhang\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eLaboratory\\u0026nbsp;of\\u0026nbsp;Precision\\u0026nbsp;Medicine\\u0026nbsp;and\\u0026nbsp;Translational\\u0026nbsp;Medicine,\\u0026nbsp;Suzhou\\u0026nbsp;Hospital\\u0026nbsp;Affiliated\\u0026nbsp;to\\u0026nbsp;Nanjing\\u0026nbsp;Medical\\u0026nbsp;University,\\u0026nbsp;Suzhou\\u0026nbsp;Science\\u0026nbsp;and\\u0026nbsp;Technology\\u0026nbsp;Town\\u0026nbsp;Hospital,\\u0026nbsp;Suzhou,\\u0026nbsp;215153,\\u0026nbsp;China\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTuanjie\\u0026nbsp;Che\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eKey\\u0026nbsp;Laboratory\\u0026nbsp;of\\u0026nbsp;Functional\\u0026nbsp;Genomic\\u0026nbsp;and\\u0026nbsp;Molecular\\u0026nbsp;Diagnosis\\u0026nbsp;of\\u0026nbsp;Gansu\\u0026nbsp;Province,\\u0026nbsp;Lanzhou,\\u0026nbsp;730030, China\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTuanjie\\u0026nbsp;Che\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eDepartment of Biotherapy Center, Gansu Provincial Hospital, Lanzhou, China\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eGuoying Miao\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eMedical College of Soochow University, Soochow University, Suzhou, 215000, China\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eHongying\\u0026nbsp;Yang\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eContributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eYC, CD and HZ conceived the manuscript. YC, CD, CX and HC consulted the\\u0026nbsp;literature and wrote the initial draft of the manuscript. TC, GM, XZ, JY, FW, HL and HY participated in writing the manuscript. All authors reviewed the manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCorresponding author\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eCorrespondence to Hong\\u0026nbsp;Zhang.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\" \\u003cdiv id=\\\"CR1\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e1.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eBray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. Ca-a Cancer Journal for Clinicians. 2018;68(6):394\\u0026ndash;424.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR2\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e2.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eAasheim HC, Loukianova T, Deggerdal A, Smeland EB. Tissue specific expression and cDNA structure of a human transcript encoding a nucleic acid binding [oligo(dC)] protein related to the pre-mRNA binding protein K. Nucleic acids research. 1994;22(6):959\\u0026ndash;64.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR3\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e3.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eChaudhury A, Chander P, Howe PH. Heterogeneous nuclear ribonucleoproteins (hnRNPs) in cellular processes: Focus on hnRNP E1's multifunctional regulatory roles. RNA. 2010;16(8):1449\\u0026ndash;62.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR4\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e4.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eJi FJ, Wu YY, An Z, Liu XS, Jiang JN, Chen FF, et al. Expression of both poly r(C) binding protein 1 (PCBP1) and miRNA-3978 is suppressed in peritoneal gastric cancer metastasis. Sci Rep. 2017;7(1):15488.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR5\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e5.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eZhou M, Tong X. Downregulated Poly-C binding protein-1 is a novel predictor associated with poor prognosis in Acute Myeloid Leukemia. Diagnostic pathology. 2015;10:147\\u0026ndash;7.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR6\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e6.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eLiu Y, Gai L, Liu J, Cui Y, Zhang Y, Feng J. Expression of poly(C)-binding protein 1 (PCBP1) in NSCLC as a negative regulator of EMT and its clinical value. Int J Clin Exp Pathol. 2015;8(6):7165\\u0026ndash;72.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR7\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e7.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003ePillai MR, Chacko P, Kesari LA, Jayaprakash PG, Jayaram HN, Antony AC. Expression of folate receptors and heterogeneous nuclear ribonucleoprotein E1 in women with human papillomavirus mediated transformation of cervical tissue to cancer. J Clin Pathol. 2003;56(8):569\\u0026ndash;74.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR8\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e8.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eZhang W, Shi H, Zhang M, Liu B, Mao S, Li L, et al. Poly C binding protein 1 represses autophagy through downregulation of LC3B to promote tumor cell apoptosis in starvation. Int J Biochem Cell Biol. 2016;73:127\\u0026ndash;36.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR9\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e9.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eIshii T, Hayakawa H, Igawa T, Sekiguchi T, Sekiguchi M. Specific binding of PCBP1 to heavily oxidized RNA to induce cell death. Proc Natl Acad Sci U S A. 2018;115(26):6715\\u0026ndash;20.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR10\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e10.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eZhang T, Huang XH, Dong L, Hu D, Ge C, Zhan YQ, et al. PCBP-1 regulates alternative splicing of the CD44 gene and inhibits invasion in human hepatoma cell line HepG2 cells. Mol Cancer. 2010;9:72.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR11\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e11.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eJiang P, Li Z, Tian F, Li X, Yang J. Fyn/heterogeneous nuclear ribonucleoprotein E1 signaling regulates pancreatic cancer metastasis by affecting the alternative splicing of integrin beta1. Int J Oncol. 2017;51(1):169\\u0026ndash;83.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR12\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e12.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eNandal A, Ruiz JC, Subramanian P, Ghimire-Rijal S, Sinnamon RA, Stemmler TL, et al. Activation of the HIF prolyl hydroxylase by the iron chaperones PCBP1 and PCBP2. Cell Metab. 2011;14(5):647\\u0026ndash;57.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR13\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e13.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eWang H, Vardy LA, Tan CP, Loo JM, Guo K, Li J, et al. PCBP1 suppresses the translation of metastasis-associated PRL-3 phosphatase. Cancer Cell. 2010;18(1):52\\u0026ndash;62.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR14\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e14.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eShafabakhsh R, Reiter RJ, Mirzaei H, Teymoordash SN, Asemi Z. Melatonin: A new inhibitor agent for cervical cancer treatment. J Cell Physiol. 2019;234(12):21670\\u0026ndash;82.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR15\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e15.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eDejgaard K, Leffers H. Characterisation of the nucleic-acid-binding activity of KH domains. Different properties of different domains. Eur J Biochem. 1996;241(2):425\\u0026ndash;31.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR16\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e16.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eChkheidze AN, Liebhaber SA. A novel set of nuclear localization signals determine distributions of the alphaCP RNA-binding proteins. Mol Cell Biol. 2003;23(23):8405\\u0026ndash;15.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR17\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e17.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eLu J, Gao FH. Role and molecular mechanism of heterogeneous nuclear ribonucleoprotein K in tumor development and progression. Biomed Rep. 2016;4(6):657\\u0026ndash;63.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR18\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e18.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eBarboro P, Ferrari N, Balbi C. Emerging roles of heterogeneous nuclear ribonucleoprotein K (hnRNP K) in cancer progression. Cancer Lett. 2014;352(2):152\\u0026ndash;9.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR19\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e19.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eShi HS, Li H, Yuan RH, Guan W, Zhang XM, Zhang SY, et al. PCBP1 depletion promotes tumorigenesis through attenuation of p27(Kip1) mRNA stability and translation. Journal of Experimental Clinical Cancer Research. 2018;37:18.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR20\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e20.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eIshii T, Hayakawa H, Igawa T, Sekiguchi T, Sekiguchi M. Specific binding of PCBP1 to heavily oxidized RNA to induce cell death. Proc Natl Acad Sci USA. 2018;115(26):6715\\u0026ndash;20.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR21\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e21.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eGuo JH, Jia R. Splicing factor poly(rC)-binding protein 1 is a novel and distinctive tumor suppressor. J Cell Physiol. 2019;234(1):33\\u0026ndash;41.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR22\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e22.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eDi CX, Syafrizayanti QJ, Zhang YH, Chen YP, Wang XT, Zhang, et al. Function, clinical application, and strategies of Pre-mRNA splicing in cancer. Cell Death Differ. 2019;26(7):1181\\u0026ndash;94.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR23\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e23.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eMelino G, De Laurenzi V, Vousden KH. p73: Friend or foe in tumorigenesis. Nat Rev Cancer. 2002;2(8):605\\u0026ndash;15.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR24\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e24.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003ePrieto-Nieto MI, Pastor D, Rodriguez-Cobos J, Perez JP, Mendez C, Palacios E, et al. Delta Np73 status in peritoneal and ovarian dissemination of appendicular adenocarcinoids (goblet cells). Clinical Translational Oncology. 2019;21(10):1432\\u0026ndash;9.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR25\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e25.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eGomez LC, Sottile ML, Guerrero-Gimenez ME, Zoppino FCM, Redondo AL, Gago FE, et al. TP73 DNA methylation and upregulation of Delta Np73 are associated with an adverse prognosis in breast cancer. J Clin Pathol. 2018;71(1):52\\u0026ndash;8.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR26\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e26.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eZaika AI, Slade N, Erster SH, Sansome C, Joseph TW, Pearl M, et al. Delta Np73, a dominant-negative inhibitor of wild-type p53 and TAp73, is up-regulated in human tumors. J Exp Med. 2002;196(6):765\\u0026ndash;80.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR27\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e27.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eRodriguez N, Pelaez A, Barderas R, Dominguez G. Clinical implications of the deregulated TP73 isoforms expression in cancer. Clinical Translational Oncology. 2018;20(7):827\\u0026ndash;36.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR28\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e28.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eTomasini R, Tsuchihara K, Wilhelm M, Fujitani M, Rufini A, Cheung CC, et al. TAp73 knockout shows genomic instability with infertility and tumor suppressor functions. Genes Dev. 2008;22(19):2677\\u0026ndash;91.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR29\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e29.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eMuller M, Schilling T, Sayan AE, Kairat A, Lorenz K, Schulze-Bergkamen H, et al. TAp73/Delta Np73 influences apoptotic response, chemosensitivity and prognosis in hepatocellular carcinoma. Cell Death Differ. 2005;12(12):1564\\u0026ndash;77.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR30\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e30.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eLucena-Araujo AR, Kim HT, Thome C, Jacomo RH, Melo RA, Bittencourt R, et al. High DeltaNp73/TAp73 ratio is associated with poor prognosis in acute promyelocytic leukemia. Blood. 2015;126(20):2302\\u0026ndash;6.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR31\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e31.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eRaisova M, Hossini AM, Eberle J, Riebeling C, Wieder T, Sturm I, et al. The Bax/Bcl-2 ratio determines the susceptibility of human melanoma cells to CD95/Fas-mediated apoptosis. Journal of Investigative Dermatology. 2001;117(2):333\\u0026ndash;40.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR32\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e32.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eDi CX, Han L, Zhang H, Xu S, Mao AH, Sun C, et al. Diallyl disulfide attenuated carbon ion irradiation-induced apoptosis in mouse testis through changing the ratio of Tap73/DeltaNp73 via mitochondrial pathway. Sci Rep. 2015;5:16020.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR33\\\" class=\\\"Citation\\\"\\u003e \\u003cspan class=\\\"EditNotAllowed\\\" name=\\\"CitationNumber\\\"\\u003e33.\\u003c/span\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eZhang Q, Di C, Yan J, Wang F, Qu T, Wang Y, et al. Inhibition of SF3b1 by pladienolide B evokes cycle arrest, apoptosis induction and p73 splicing in human cervical carcinoma cells. Artificial Cells Nanomedicine Biotechnology. 2019;47(1):1273\\u0026ndash;80.\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"CR34\\\" class=\\\"Citation\\\"\\u003e \\u003cdiv class=\\\"BibAuthorName\\\"\\u003e \\u003cdiv class=\\\"NoInitials\\\"\\u003e\\u003c/div\\u003e \\u003cdiv class=\\\"FamilyName\\\"\\u003eDeclarations\\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"BibUnstructured\\\"\\u003eDeclarations.\\u003c/div\\u003e \\u003c/div\\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\":\"info@researchsquare.com\",\"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\":\"PCBP1, p73, splicing, apoptosis, cancer, therapy\",\"lastPublishedDoi\":\"10.21203/rs.2.24804/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.2.24804/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e\\u003cstrong\\u003eBackground\\u003c/strong\\u003e Splicing factor poly(rC)-binding protein 1 (PCBP1) is a novel tumor suppressor that is downregulated in many cancers thereby regulates tumor formation and metastasis. However, to date, little information has been available on the molecular mechanisms by which PCBP1 evokes apoptosis.\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eResults\\u003c/strong\\u003e Here, we explored the molecular mechanism by which PCBP1 triggers apoptosis in human cervical cancer cells. We testified that overexpression of PCBP1 greatly repressed proliferation of HeLa cells in time-dependent manner. It also induced a significant increase in G2 / M phase arrest and apoptosis. Furthermore, it was shown that overexpression of PCBP1 caused p73 splicing, and thus efficiently downregulated the ratio of Bax / Bcl-2, the release of cytochrome c and the expression of caspase-3.\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eConclusion \\u003c/strong\\u003eOur results revealed that PCBP1 played a vital role in cycle arrest, apoptosis induction, and p73 splicing in human cervical carcinoma cells and targeting PCBP1 may be a promising approach in cervical cancer therapy.\\u003c/p\\u003e\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\",\"manuscriptTitle\":\"Overexpression of splicing factor poly(rC)-binding protein 1 elicits cycle arrest, apoptosis induction, and p73 splicing in human cervical carcinoma cells\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2020-02-28 15:41:49\",\"doi\":\"10.21203/rs.2.24804/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"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\":\"51a0d1ba-f4e6-4302-9456-ee19b839389d\",\"owner\":[],\"postedDate\":\"February 28th, 2020\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[{\"id\":63578,\"name\":\"General Cell Biology \\u0026 Physiology\"}],\"tags\":[],\"updatedAt\":\"2020-05-30T14:50:38+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2020-02-28 15:41:49\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-15362\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"identity\":\"rs-15362\",\"version\":[\"v1\"]},\"buildId\":\"_2-kVJe1T_tPrBINL-cwx\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}