RBM14 enhances transcriptional activity of p23 regulating CXCL1 expression to induce EMT in lung cancer

RBM14 enhances transcriptional activity of p23 regulating CXCL1 expression to induce EMT in lung cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article RBM14 enhances transcriptional activity of p23 regulating CXCL1 expression to induce EMT in lung cancer Zhenlong Yu, Wen Zhang, Yulin Peng, Meirong Zhou, Yilin Che, Junlin Chen, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3842489/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 Metastasis serves as a malignant indicator and biological characteristic of pulmonary carcinoma. Epithelial-mesenchymal transition (EMT) plays a pivotal role in facilitating tumor invasion and metastasis, and enhances the aggressiveness of tumor cells. Prostaglandin E synthase 3 (PTGES3) functions as an HSP90 co-chaperone. Our previous study revealed its HSP90-independent role as a transcription factor involved in cancer-related inflammation. Our present study aims to investigate the impact and mechanism of p23 on lung cancer metastasis. By utilizing cell models in vitro and mouse tail vein metastasis models in vivo , our results provide solid evidences that p23 plays a crucial role in promoting lung cancer metastasis through regulating the downstream CXCL1 expression, which is not achieved independently, but rather through formatting a complex with RBM14, thereby facilitating the occurrence and progression of EMT in lung cancer. Therefore, our study demonstrates the potential therapeutic application of the RBM14-p23-CXCL1-EMT axis in targeting lung cancer metastasis. Biological sciences/Cancer Biological sciences/Molecular biology/Transcription EMT p23 CXCL1 RBM14 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Significance p23 is a transcription factor of CXCL1 to promote tumor metastasis. RBM14 assists the transcriptional regulation of p23 on CXCL1 thereby promoting EMT. Introduction Lung cancer has become a prominent contributor to global mortality rates associated with malignancies and a major public health burden globally [ 1 – 3 ] . Despite some advancements in diagnosis, surgical techniques, and radiotherapy and chemotherapy over the past decade [ 4 – 6 ] , the mortality rate of lung cancer remains alarmingly high with a mere 15% 5-year survival rate, which is primarily attributed to the widespread metastasis of lung cancer [ 7 , 8 ] . The efficacy of targeted therapy in tumor treatment is promising; however, the intricate interactions between tumor and stroma greatly impede the development of targeted therapy for tumor metastasis. Therefore, it is imperative to investigate novel molecular mechanisms and targets of lung cancer metastasis, in order to establish a theoretical foundation for developing the innovative treatment strategies or therapeutic agents, and ultimately enhance the quality of life for patients with lung cancer. The process of epithelial-mesenchymal transformation (EMT) in tumor cells is characterized by the loss of intercellular adhesion among epithelial tumor cells and the acquisition of migration and invasion properties of mesenchymal cells, thereby promoting metastasis and development of various cancers, such as lung cancer [ 9 ] . The acquisition of metastatic properties by tumor cells confers increased aggressiveness, bringing significant challenges to clinical management and cancer patient survival [ 10 ] . Therefore, it’ s urgent to investigate the molecular targets and pathways involved in EMT in tumor cells, which not only established a theoretical foundation for preventing EMT in tumor cells, but facilitated the identification of potential therapeutic agents. In the present study, we mainly focused on the EMT marker CXCL1 to reveal a new mechanism for its expression regulation. CXCL1, a chemokine of CXC family, is implicated in the pathogenesis of various inflammatory diseases and plays a crucial role in physiology and cancer progression, especially in EMT [ 11 – 13 ] . Elevated expression level of CXCL1 in tumor tissues and serum, have been associated with tumor metastasis and poor prognosis in ovarian cancer, lung adenocarcinoma, colorectal cancer and pancreatic ductal adenocarcinoma (PDA) [ 14 – 18 ] . Previous study reported that CXCL1 was positively correlated with the migration and invasive activity of osteosarcoma cell lines [ 19 ] , and it has become a key indicator of lung metastasis of osteosarcoma through paracrine release. Therefore, it was proposed that targeted inhibition of CXCL1 could exert a profound inhibitory effect on tumor growth and metastasis. Prostaglandin E synthase 3 (p23) is a highly conserved protein encoded by PTGES3 gene [ 20 , 21 ] , which is often overexpressed in various cancers, including prostate cancer and breast cancer, as well as lung cancer [ 22 – 27 ] . The current understanding of p23 mainly focuses on its role as a co-partner of heat shock protein 90 (Hsp90) [ 28 , 29 ] , that can stabilize the complex formed by Hsp90 and its client proteins, including estrogen receptor [ 30 ] , androgen receptor [ 31 ] and telomerase [ 32 ] . However, p23 could also play some functions independently of Hsp90, such as, enhancing the transcription factor of p53 [ 33 ] , and protecting human aryl hydrocarbon receptors from degradation [ 34 ] . Our previous study had re-defined p23 as an HSP90-independent transcription factor of COX-2 in promoting tumor growth [ 35 ] . This highlights the diversity and complexity of p23 functionality, emphasizing the importance of further studying its functional characteristics. At present, there are few studies on p23 and tumor metastasis. Therefore, it is of great significance to explore the role of p23 in tumor migration and its molecular mechanism. In this study, we discovered a novel function of p23 as a transcription factor to promote EMT in lung cancer and revealed the specific mechanism. Its activation on the CXCL1 promoter is not achieved directly, rather than requiring the assistance of cofactor RBM14 during EMT progression. Results p23 expression is upregulated in lung cancer exhibiting metastatic features. To evaluate the potential involvement of p23 in lymph node metastasis of lung cancer, we observed a significant upregulation of p23 expression in metastatic lung cancer tissues compared to primary counterparts by querying the TCGA database ( Fig. 1 A ) , and further confirmed in clinical samples (n = 133) by immunohistochemical staining ( Fig. 1 B ) , qPCR assays ( Fig. 1 C ) and western blotting ( Fig. 1 D ) . We next evaluated p23 expression on the prognosis of 93 lung adenocarcinoma patients from a tissue microarray. High p23 expression facilitated the metastasis of lung cancer to lymph nodes in patients (Fig. 1 E), and reduced the disease-free survival (DFS) of lung cancer metastasis patients ( Fig. 1 F ) . These results suggest that the elevated p23 expression is possibly associated with metastasis and predict the poor clinical outcomes of lung cancer metastasis patients. Knocking down p23 inhibits the invasion and migration of NSCLC cells. To further investigate the underlying mechanism of p23 in metastasis, we established stable A549 lung cancer cells with p23 knockdown (A549/shp23) (Supplementary Fig. S1 A). Real-time inhibitory analysis using the xCELLigence provided compelling evidence that depletion of endogenous p23 impeded the migratory capacity of lung cancer cells ( Fig. 2 A ) . The similar results were also obtained from scratch assay ( Fig. 2 B ) , transwell invasion ( Fig. 2 C ) and migration assay ( Fig. 2 D ) . Besides, several proteins involved in EMT were also evaluated by western blotting ( Fig. 2 E ) and qPCR analysis ( Fig. 2 F ) . p23 knock-down (shp23) greatly decreased protein levels of N-Cadherin and Vimentin, and increased the expression of E-Cadherin. By contrast, p23 stable overexpression (H1299/OEp23) altered the metastasis in an opposite fashion in lung cancer cells (Supplementary Figs. S1B-1D). Furthermore, a mouse model of lung cancer metastasis was also established by intravenous injection of tumor cells via tail vein to evaluate the effect of p23 on metastasis in vivo . Consistent with in vitro observations, shp23 dramatically decreased the number of lung metastatic nodules ( Figs. 2 G- 2 I ) . These data indicate that p23 played an oncogenic role in promoting lung cancer metastasis. The downstream target gene CXCL1 of p23 was found by RNA-seq sequencing. In order to determine the molecular pathways involved in p23-driven lung cancer metastasis, we performed RNA-seq sequencing analysis to explore the potential target genes related to p23. A total of 92 differentially expressed genes (DEGs) were found to be commonly shared among the three p23 knock-down groups ( Fig. 3 A ) . Then, 11 candidate DEGs for p23 knock-down were selected based on the analysis of typical lung cancer EMT marker genes from the EMTome database ( www.EMTome.org ) ( Fig. 3 B ) , and subsequently confirmed through qPCR detection. The consistent qPCR results validated the reliability of the RNA seq analysis findings ( Fig. 3 C ) . According to the abundance of these genes in cell samples, we selected CXCL1 with the highest content as the follow-up research focus ( Fig. 3 D ) . CXCL1 is a vital chemokine driver to promote tumor metastasis [ 14 , 19 , 36 ] . We then proposed that p23 is involved in lung cancer metastasis, at least partially, regulating CXCL1 expression. Importantly, both p23 and CXCL1 expressions were significantly increased in metastatic lung cancer tissues compared to non-metastatic counterparts ( Fig. 3 E and 3 F ) . In addition, CXCL1 expression was significantly downregulated by p23 depletion, and upregulated by p23 over-expression at both mRNA and protein levels in lung cancer cells ( Figs. 3 G- 3 J ) , indicating that p23 regulates CXCL1 expression in lung cancer cells. EMT induced by p23 is partially dependent on CXCL1. To test whether p23 achieves its metastatic regulation functions in a CXCL1-depedent manner, we generated A549/shp23 lung cancer cells stably with over-expression of CXCL1 (A549/shp23 + OECXCL1) to detect the alterations in EMT. As shown in Figs. 4 A- 4 C, CXCL1 significantly reversed the migration inhibition caused by p23 knockdown, as well as the changes in expression of EMT-relative proteins (N-Cadherin, Vimentin and E-Cadherin) ( Fig. 4 D ) . Moreover, the similar results were also observed in vivo , that is, shp23 significantly reduced the number of lung metastatic nodules in NYG mice, and this reduction was completely reversed by CXCL1 overexpression ( Figs. 4 E- 4 G ) . These results indicated that p23 promotes lung cancer metastasis through CXCL1 expression. To support this notion, p23 overexpression largely enhanced lung cancer cell in vitro migration, and further CXCL1 knockdown diminished the lung cancer metastasis enhancement induced by p23 overexpression (Supplementary Fig. S2 A and 2B). p23 regulates the transcriptional expression of CXCL1 rather than affect its mRNA stability. To further assess the function of p23 on CXCL1 expression, we first examined the effect of p23 on the stability of CXCL1 mRNA. Although p23 depletion or overexpression could positively regulate the basal mRNA level of endogenous CXCL1, the half-life of CXCL1 mRNA was not affected by p23 alterations (Supplementary Fig. S3 A and 3B), indicating that p23 upregulated CXCL1 expression at transcription level rather than affecting mRNA stability. Furthermore, we constructed luciferase reporter plasmids containing the optimal CXCL1 promoter region (-874 to + 38). Indeed, p23 expression dramatically induced the CXCL1 luciferase activity ( Fig. 5 A ) . Through generating truncated the variants of CXCL1 promoter, we observed that deletion of the − 744 to -549 regions fully abolished p23-induced CXCL1 promoter activity ( Fig. 5 B ) , indicating that this region containing the binding sites for p23. Therefore, a biotin-labeled probe corresponding to the optimal CXCL1 promoter (from − 744 to + 38) was employed to evaluate the binding capability of p23, with the CXCL1 promoter (from − 549 to + 38) as negative control. As expected, p23 protein was indeed visualized in the pulldown of CXCL1 promoter region − 744 to + 38 from the nuclear extract of A549 cells, whereas no signal was detected in negative control region ( Fig. 5 C ) . To further identify the DNA binding motif of p23 in CXCL1 promoter, we truncated this region (-549 to -744 bp) into five fragments, including P1 (probe 1): -744 to -703, P2: -707 to -663, P3: -667 to -621, P4: -625 to -579, P5: -583 to -537. Nucleotide sequence of CXCL1-EMSA probe was listed in supplementary table 2 . The interaction between p23 and each probe was analyzed using EMSA assay with HEK-293T cell lysate overexpressing p23. It was found that p23 had binding affinities with P1, P2 and P3 ( Fig. 5 D ) , indicating that three fragments contained p23 binding motifs. This observation was further validated through a super-shift assay using anti-p23 antibody ( Fig. 5 E ) . Further DMEME analysis revealed a 6 bp p23-binding motif from three fragments with conserved locations at nucleotides 1C, 2A, 3C, 4T, 5G/T and 6A ( Fig. 5 F ) . Mutation of the motifs totally abolished the p23-induced CXCL1 reporter activity ( Fig. 5 G ) . These results further confirmed the transcriptional regulatory function of p23 on CXCL1 expression. To further investigate the direct binding activity of p23 to the CXCL1 promoter, we conducted EMSA assay using purified p23 protein. Surprisingly, purified p23 exhibited no binding affinity towards the CXCL1 promoter ( Fig. 5 H ) , suggesting that p23's transcriptional regulation of CXCL1 may not be solely dependent on its direct interaction with the promoter, but rather necessitates the involvement of other protein factors. RBM14 promotes the transcriptional regulation of p23 on CXCL1. In order to investigate the participants involved in the regulation of CXCL1 by p23, a total of 34 proteins interacting with p23 were identified through co-immunoprecipitation (Co-IP) combined with mass spectrometry (MS), of which 4 proteins were selected due to their transcriptional function ( Fig. 6 A ) . Co-IP assay further verify their interaction with p23 ( Fig. 6 B and supplementary Fig. S4 A ) , which also showed the accuracy of the identification results by mass spectrometry. However, only RBM14 could be pull downed by CXCL1 promoter probe ( Fig. 6 C and supplementary Fig. S4 B ) , and it can significantly promote p23 reporter activity of the optimal CXCL1 promoter ( Fig. 6 D ) . A similar result was also obtained from western blot analysis ( Fig. 6 E ) . Furthermore, we conducted EMSA assay to investigate the binding activity of purified p23 and RBM14 protein to the CXCL1 promoter. As expected, p23 or RBM14 individually exhibited no direct binding to the CXCL1 promoter; however, their complexes demonstrated binding activity ( Fig. 6 F ) . These results implied that the interaction between p23 and RBM14 plays a crucial role in the regulation of CXCL1 expression. We next explored the underlying mechanism of p23/RBM14 protein complex participating in the transcription regulation of CXCL1. Firstly, there was no reciprocal influence observed between p23 and RBM14 in terms of their protein and mRNA expression levels ( Supplementary Figs. S4C-4F ). Subsequently, we modulated intracellular complex production by conducting p23-knockdown and/or RBM14-overexpression as well as p23-overexpression and/or RBM14-knockdown ( Fig. 6 G and 6 H ) , to elucidate the impact of the complex on CXCL1 transcription. The results demonstrated a significant reduction in CXCL1 promoter activity upon destruction of the p23/RBM14 complex via knockdown of either p23 or RBM14 ( Fig. 6 I and 6 J ) . Similarly, the binding activity of p23 or RBM14 to CXCL1 promoter probe was significantly suppressed upon destruction of the p23/RBM14 complex ( Figs. 6 K and 6 L ). The above results show that the formation of the p23/RBM14 protein complex plays a pivotal role in mediating p23-induced CXCL1 expression. The influence of p23 on EMT is partially dependent on RBM14. Next, in order to explore whether the EMT process induced by p23 is RBM14-dependent, we constructed four stable cell lines by using lentivirus infection system: A549/NC, A549/OERBM14, A549/shp23, A549/shp23 + OERBM14. In vitro , the migration number of cells is monitored in real time by xCELLigence assay ( Fig. 7 A ) . The result implied RBM14 could promote the phenotype of EMT, but when p23 was knocked down at the same time, the phenotype of EMT could be reduced slightly. In addition, wound healing experiments ( Fig. 7 B ) and transwell invasion ( Fig. 7 C ) as well as migration ( Fig. 7 D ) experiments were performed and brought out similar conclusion. The changes of E-Cadherin, N-Cadherin and Vimentin protein levels were evaluated by western blotting ( Fig. 7 E ) , which also proved that p23 promoted EMT phenotype in a RBM14-dependent manner. In vivo , four stably transfected cell lines, A549/NC, A549/shp23, A549/OERBM14 and A549/shp23 + OERBM14, were injected into the tail vein respectively, and the number of lung metastatic nodules ( Fig. 7 F and G) and H&E staining ( Fig. 7 H ) of lung metastatic nodules were investigated. From above results, we can draw the conclusion that EMT induced by p23 is partially dependent on RBM14. Discussion Lung cancer has become the most prevalent and lethal malignant tumor in China and even in the world, with poor treatment outcomes [ 37 ] . The metastasis of lung cancer represents a malignant manifestation and biological characteristic, serving as the primary cause of mortality among patients with this disease [ 38 ] . The metastatic nature of cancer facilitates the dissemination of tumor cells from the primary site to distant tissues beyond the lungs via blood and lymphatic system [ 39 ] , resulting in a substantial attenuation of conventional therapeutic efficacy. Consequently, impeding lung cancer metastasis holds the paramount importance in terms of protracting patients' survival, augmenting their overall prognosis, enhancing their quality of life, and ameliorating treatment outcomes [ 40 ] . Metastasis of lung cancer is an intricate and multifaceted process involving multiple stages, steps, genes and factors. Therefore, elucidating the molecular mechanism underlying invasion and metastasis in lung cancer and identifying molecular targets for reversing or preventing these processes have become prominent areas to overcome lung cancer. In this topic, we found a novel pathway to regulate lung cancer metastasis, that is, RBM14 could assist p23 to implement transcription capabilities on CXCL1 to promote tumor metastasis. From the perspective of inhibiting lung cancer invasion and metastasis, which provided a theoretical basis for clinical treatment of lung cancer as well as screening its metastatic potential agents. PTGES3 (p23), namely prostaglandin E synthase 3, is a molecular chaperone protein and a known potential oncogene [ 22 – 24 ] . Previous studies have shown that [ 23 ] p23 is highly expressed in lung adenocarcinoma, and the overexpression of p23 is related to the short overall survival and poor prognosis of lung cancer patients. The relationship between PTGES3 and EMT was first reported to point out that p23 could cause invasion and migration of lung cancer cells [ 41 ] . However, the specific mechanism still remains unclear. The current research findings indicated that p23 played a significant role in promoting EMT in human lung cancer in vivo and in vitro . Our study has demonstrated that the primary mechanism underlying p23's promotion of EMT was dependent on its regulation of CXCL1 expression through binding to the promoter region of CXCL1. As the executor of vital biological processes [ 42 ] , the functionality of proteins is often manifested through their interactions with other proteins [ 43 ] . Investigating protein-protein interactions holds immense significance in comprehending and preventing diseases, targeting treatment for polygenic disorders, and elucidating the molecular mechanisms underlying complex biological phenomena [ 44 , 45 ] . Here, we initially demonstrated that p23 functions as a transcription factor indirectly binding to the promoter region of CXCL1, suggesting potential co-factors facilitating p23's transcription. Therefore, we identified this co-factor as RBM14 using mass spectrometry combined with Co-IP and DNA pull down experiments. RNA binding motif protein 14 (RBM14), also known as Co-Activator Activator (CoAA), functions as a transcriptional coactivator. RBM14 exhibits widespread expression in embryonic tissues and plays a crucial role in the regulation of early embryonic development [ 46 , 47 ] . Previous investigations have demonstrated that RBM14 is significantly upregulated in tumors, thereby promoting tumor proliferation and migration [ 48 , 49 ] ; however, its precise molecular mechanism remains a mystery. In this study, we discover that RBM14 has the ability to form a complex with p23, thereby augmenting the transcriptional regulation of p23 on CXCL1 expression. When there is a decrease or absence in the expression of either component, the formation of the complex would be decreased to result in inhibition of CXCL1's transcriptional expression, and subsequently weakens cellular invasion and migration abilities. Our findings propose that RBM14 served as a co-activator of p23 to promote EMT in lung cancer. In conclusion, our findings elucidate that p23 plays a crucial role in promoting lung cancer metastasis through the regulation of downstream CXCL1 expression. Mechanistically, the regulation of CXCL1 transcription by p23 is based on the formation of a complex with RBM14 during EMT progression in lung cancer. Therefore, our study demonstrates the potential therapeutic application of RBM14-p23-CXCL1-EMT axis in targeting tumor metastasis, particularly lung cancer metastasis. Materials and methods Reagents and antibodies Chemokine CXCL1 was purchased from Sigma (St. Louis, MO, USA) company, and the purity was over 98%. CXCL1 chemokine was dissolved in water containing 5% BSA, and the concentration of the stock solution was 25 mM, which stored at -80℃. The pure protein p23 and RBM14 were obtained from Invitrogen (USA). The primary antibody against HA was obtained from Cell Signaling Technology (USA). The primary antibodies against p23, Vimentin, hnRNPU, DDX5, RPS3, Flag, β-actin and all the secondary antibodies were obtained from Proteintech Group (USA). The primary antibodies against CXCL1 and RBM14 were obtained from Abclonal Technology. The primary antibodies against E-Cadherin, N-Cadherin were obtained from Jingjie PTM BioLab (Hangzhou). Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI 1640, fetal bovine serum (FBS), and trypsin were obtained from HyClone Laboratories (USA). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless specified otherwise. Cell lines and cell culture conditions Human lung cancer cell lines (A549, H460, H322, HCC827 and H1299), HLF and 293T cells were obtained from the American Type Culture Collection (ATCC Manassas, VA, USA) or Procell Life Science & Technology Co., Ltd (Wuhan, China), and preserved in our laboratory. The cells were cultured in F12K, DMEM or 1640 medium supplemented with 10% FBS, 100 μg/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere comprising 5% CO 2 . The authenticity of all cell lines was verified through genomic short tandem repeat profiling, and the cell lines were confirmed to be free of mycoplasma using the TransDetect® PCR Mycoplasma Detection Kit (TransGen, Beijing, China). qRT-PCR Total RNA was extracted using Trizol reagent (Invitrogen) from cells and tumor tissues. cDNA synthesis was performed with Oligo(dT) or random primers by using a Quantscript RT Kit (Tiangen). A SYBR RT–PCR kit (Tiangen) was used for transcript quantification with specific primers. Expression levels were quantified using the 2− ΔΔ Ct method with β-actin as an internal control. The primers are provided in Supplementary Table S1. Streptavidin-agarose pulldown assay for the detection of DNA-protein binding The binding affinity of p23 to the CXCL1 promoter probes was determined using a streptavidin-agarose pull-down assay. A 541-bp biotin-labeled double stranded probe corresponding to the CXCL1 promoter sequence (-541 to +38) was synthesized. Briefly, 50 ng of protein, biotinylated DNA probe (4 μg), streptavidin-conjugated agarose beads (40 μl) and PBSi (PBS buffer with 1 mM EDTA, 1 mM DTT and complete protease inhibitor cocktail) were incubated at room temperature for 5 h on a rotating rack. After washing with PBSi buffer, the beads were resuspended in SDS-PAGE loading buffer and boiled at 100 °C. The supernatant was analyzed by western blotting. Dual-luciferase reporter gene assay Luciferase activity was measured using the Dual-luciferase reporter gene assay system (Promega) according to the manufacturer’s instructions. Growth media were removed and cells were washed with PBS. Cell precipitates were collected and lysed by freezing and thawing twice in liquid nitrogen. Then, 10 μl of the lysate was transferred into a black 96-well plate (Thermo). Firefly and Renilla luciferase activity was assayed sequentially in the cell lysate in each well. Transcriptional activity was calculated as the ratio of firefly luciferase activity (reporter) to Renilla luciferase activity (control). Electrophoretic Mobility Shift Assay (EMSA) Biotin-labeled CXCL1 promoter probes were purchased from BGI Genomics (Beijing, China). For the DNA EMSA assay, 4 μg protein and 1 μg of biotin-labeled DNA probe were mixed in binding buffer and incubated for 20 min at 25 °C. DNA–protein complexes were separated in 6.5% acrylamide native PAGE gels at 4 °C. The specific bands were detected with HRP-conjugated streptavidin and visualized by enhanced chemiluminescence. Lentivirus preparation and transfection Lentivirus was produced in 293T cells using the second-generation packaging system plasmids psPAX2 (Addgene) and pMD2.G (Addgene). One 10 cm culture dish containing 5×10 6 293T cells was transfected using Lipofectamine 2000 (Invitrogen) with 15 μg lentiviral vector, 7.5 μg psPAX2 and 7.5 μg pMD2.G. After transfection, the supernatants were collected every 24 h between 24 and 72 h, and concentrated with PEG-8000, after which the viral titer was determined by serial dilution. The multiplicity of infection (MOI) during transfection was 5. Immunoprecipitation (IP) Take 800 µg of protein, and fix the volume to 500 µL in Binding buffer. Then 2 µg of flag antibody was added to the experimental group while 2 µg of IgG antibody was added to the blank group, and shake it on the rotary mixer for 1 h. After 1 h, 15 µL magnetic beads were added and shaked them overnight at 4 C on a rotary mixer. Magnetic separation is carried out the next day, and a proper amount of Binding buffer is added to wash the magnetic beads for three times after the supernatant is discarded. 50 μL 2×Loading Buffer was added to the magnetic beads and boiled for 10 minutes. After magnetic separation, the supernatant was transferred to for western blot analysis. Animal experiments Male NYG mice aged 5 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (China), and kept in the SPF Laboratory Animal Center of Dalian Medical University (Dalian, China). All animals were given free access to sterilized food and water and were habituated for 7 days and divided randomly before the experiments. The indicated tumor cells (1×10 6 in 100 μL PBS) were injected through tail vein of each mouse. After 4 weeks following injecting, the animals were sacrificed and the lungs from each mouse were excised while the number of pulmonary metastatic nodules was checked. Small parts of lung tissue were obtained for H&E staining. All procedures were carried out in strict accordance with the recommendations established by the Animal Care and Ethics Committee of Dalian Medical University as well as the guidelines promulgated in the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals. Statistical analysis Graphpad.9.0 was used for all statistical analyses. The Kaplan–Meier method was used to analyze survival. Pearson’s correlation test was used to examine the correlations among histochemical markers. Unpaired Student’s t-test to assess differences between two groups, while analysis of variance (ANOVA) followed by the least significant difference test was used for comparisons of multiple groups. Differences with P < 0.05 were considered statistically significant. Declarations Acknowledgments The authors thank the National Natural Science Foundation of China (82225048 and 82004089), Distinguished professor of Liaoning Province (XLYC2002008), and Sanming Project of Medicine in Shenzhen (No. SZZYSM202106004) for financial support. Author contributions M.-X.C. and Y.-Z.L. initiated the work and designed the experiments. Z.W., P.-Y.L., and Z.-M.R. performed the experiments, analyzed data, made figures and drafted the manuscript; C.-Y.L., C.-J.L., Z.-W.H., W.C., H.-C.J. T.-X.G., T.-M.M., F.L., W.Y. and H.-X.K. analyzed the data. M.-X.C., Y.-Z.L. and Z.W. supervised the study. All authors reviewed the manuscript. All authors read and approved the final manuscript. Conflict of Interest The authors declare no competing interests. Data Availability All experiments were repeated three times. 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Additional Declarations (Not answered) Supplementary Files SupplementaryTableS1.xlsx Table 1 SupplementaryTableS2.xlsx Table 2 Fig.S1.jpg Fig.S2.jpg Fig.S3.jpg Fig.S4.jpg Supplementaryinformation.docx SupplementaryinformationXXXXXXwesternblot.docx 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-3842489","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":271728051,"identity":"e474eedd-1e04-4656-93f1-bad64e02ee8e","order_by":0,"name":"Zhenlong 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University","correspondingAuthor":false,"prefix":"","firstName":"Xiaokui","middleName":"","lastName":"Huo","suffix":""},{"id":271728065,"identity":"482345bb-4a2a-4d43-8607-4d1bebef7f9c","order_by":14,"name":"Xiaochi Ma","email":"","orcid":"https://orcid.org/0000-0003-4397-537X","institution":"Second Affiliated Hospital, Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaochi","middleName":"","lastName":"Ma","suffix":""}],"badges":[],"createdAt":"2024-01-07 12:35:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3842489/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3842489/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50909684,"identity":"59e06270-fe9c-44c9-8dda-802fba7a415a","added_by":"auto","created_at":"2024-02-09 11:50:09","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":291422,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ep23 expression is upregulated in lung cancer exhibiting metastatic features. (A)\u003c/strong\u003eTCGA clinical database analysis of the disparity in the expression of p23 between lung cancer tissues with lymph node metastasis and those without; \u003cstrong\u003e(B-D) \u003c/strong\u003eImmunohistochemical staining (B), qPCR(C) and western blotting (D) analysis of the expression of p23 in metastatic lung cancer tissues (M1 to M5) and non-metastatic lung cancer tissues (P1 to P5);\u003cstrong\u003e (E)\u003c/strong\u003e Tissue microarray analysis of the relationship between metastasis of lung cancer and p23 expression;\u003cstrong\u003e (F)\u003c/strong\u003e The overexpression of p23 leading to lung cancer metastasis is associated with a diminished disease-free survival rate among patients with lung cancer.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3842489/v1/8479b75a7367cf0ea5c783b3.jpg"},{"id":50909690,"identity":"39b55562-66d3-432b-bc92-f226a6c213da","added_by":"auto","created_at":"2024-02-09 11:50:10","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":455313,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnocking down p23 inhibits the invasion and migration of NSCLC cells. (A-D) \u003c/strong\u003eThe number of migrating cells determined in real time by\u003cstrong\u003e \u003c/strong\u003exCELLigence (A), wound healing assay (B), transwell invasion assay (C) and migration assay (D); \u003cstrong\u003e(E and F)\u003c/strong\u003e Western blotting (E) and qPCR (F) analysis the expression changes of invasion and migration proteins E-Cadherin, N-Cadherin and Vimentin after p23 knock-down;\u003cstrong\u003e (G-I) \u003c/strong\u003eRepresentative images of lung metastatic nodules (G), statistics of the number of lung metastatic nodules (H) and H\u0026amp;E staining representative images of metastatic nodules (I) after A549/wt or A549/shp23 cells were injected into the tail vein.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3842489/v1/2b803783d427e3b3b5b61aed.jpg"},{"id":50909696,"identity":"d0643de1-9893-4f83-86d7-216bf0be62f4","added_by":"auto","created_at":"2024-02-09 11:50:10","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":248715,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe downstream target gene CXCL1 of p23 was found by RNA-seq sequencing. (A) \u003c/strong\u003eWayne diagram shows the expression levels of all 92 differential genes in the sample;\u003cstrong\u003e (B-C) \u003c/strong\u003e11 EMT markers were found (B) and were selected to execute qPCR identification (C);\u003cstrong\u003e (D)\u003c/strong\u003e Heat map of the content of 11 EMT genes;\u003cstrong\u003e (E-F)\u003c/strong\u003eThe expression differences of p23 and CXCL1 in metastatic lung cancer tissues and non-metastatic lung cancer tissues at mRNA (E) and protein (F) levels; \u003cstrong\u003e(G-H)\u003c/strong\u003eChanges of CXCL1 expression at mRNA (G) and protein (H) level after p23-knock-down;\u003cstrong\u003e (I-J)\u003c/strong\u003e Changes of CXCL1 expression at mRNA (I) and protein (J) level after p23-overexpression.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3842489/v1/0dcfc73f7ffbb5f4864abd78.jpg"},{"id":50909691,"identity":"698fef4b-bc6f-4ee0-8c2c-24f8d16abb52","added_by":"auto","created_at":"2024-02-09 11:50:10","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":536669,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe EMT induced by p23 is partially dependent on CXCL1. (A)\u003c/strong\u003e The number of migrating cells were determined in real time by\u003cstrong\u003e \u003c/strong\u003exCELLigence; (\u003cstrong\u003eB-C\u003c/strong\u003e) Transwell invasion experiment (B) and migration experiment (C) investigated the influence of p23 and CXCL1 on the invasion and migration ability of A549 cells;\u003cstrong\u003e (D) \u003c/strong\u003eWestern blotting assay of the expression changes of invasion and migration proteins E-Cadherin, N-Cadherin and Vimentin and its quantitative graphs; \u003cstrong\u003e(E-G)\u003c/strong\u003eRepresentative images of lung metastatic nodules (E), statistics of the number of lung metastatic nodules (F) and representative images of H\u0026amp;E staining of metastatic nodules (G) after four cell lines (A549/wt, A549/OECXCL1, A549/shp23, A549/shp23+OECXCL1) were injected into the tail vein respectively .\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3842489/v1/d94f4ad5af63c93e74c8b818.jpg"},{"id":50909695,"identity":"ee9539b3-aae1-430c-8154-4be472ccfa03","added_by":"auto","created_at":"2024-02-09 11:50:10","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":251344,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ep23 regulates the expression of CXCL1 at the transcriptional level, but does not affect its mRNA stability. (A and B)\u003c/strong\u003eDual-luciferase reporter gene detection of CXCL1 reporter gene with the best promoter region \u003cstrong\u003e(A)\u003c/strong\u003e and truncated variant \u003cstrong\u003e(B); (C)\u003c/strong\u003e Pulldown assay of the binding of biotin-labeled CXCL1 promoter DNA (from -549 to +38 and from -744 to +38) and p23;\u003cstrong\u003e (D)\u003c/strong\u003e EMSA assay of the binding of 5 fragments of CXCL1 and p23; P1 to P5: 5 EMSA fragments of CXCL1 promoter;\u003cstrong\u003e (E)\u003c/strong\u003e Competitive binding analysis EMSA by using anti-p23 antibody; \u003cstrong\u003e(F) \u003c/strong\u003eThe binding motif of p23 and CXCL1; \u003cstrong\u003e(G) \u003c/strong\u003eChanges in the activity of p23 to CXCL1 dual-luciferase reporter gene after mutation of binding motif; \u003cstrong\u003e(H)\u003c/strong\u003e EMSA experiment of the binding of p23 pure protein and CXCL1 probe.\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3842489/v1/77be59ac025a4bb3b49656b6.jpg"},{"id":50909692,"identity":"00866ccb-cf12-4c4a-8385-bfeb06752c04","added_by":"auto","created_at":"2024-02-09 11:50:10","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":388957,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRBM14 promotes the transcriptional regulation of p23 on CXCL1. (A)\u003c/strong\u003e Twice identifying the results by mass spectrometry and selecting the protein with transcription activity;\u003cstrong\u003e (B)\u003c/strong\u003eCo-IP was used to verify the interaction with p23;\u003cstrong\u003e (C) \u003c/strong\u003ePulldown fishing with CXCL1 promoter probe, and only RBM14 can bind to CXCL1 probe; \u003cstrong\u003e(D)\u003c/strong\u003eThe dual-luciferase reporter gene assay proved that RBM14 can promote the transcriptional regulation of p23 on CXCL1; \u003cstrong\u003e(E)\u003c/strong\u003eWestern blot experiment verified that RBM14 promoted the transcriptional regulation of p23 on CXCL1; \u003cstrong\u003e(F)\u003c/strong\u003eThe transcription of CXCL1 can only be started when the coexistence of p23 and RBM14 is verified by EMSA experiment; \u003cstrong\u003e(G) \u003c/strong\u003eCO-IP revealed the complex of p23 and RBM14 was embellished by knocking down p23 and overexpressing RBM14; \u003cstrong\u003e(H) \u003c/strong\u003eCO-IP revealed the complex of p23 and RBM14 was embellished by knocking down RBM14 and overexpressing p23;\u003cstrong\u003e (I) \u003c/strong\u003eDual-luciferase reporter gene assay proved that knocking down p23 would reduce the exciting effect of RBM14 on CXCL1; \u003cstrong\u003e(J)\u003c/strong\u003e Dual-luciferase reporter gene assay confirmed that if RBM14 was knocking down, p23 would reduce the exciting effect of CXCL1;\u003cstrong\u003e (K) \u003c/strong\u003ePulldown assay proved that when p23 was knocked down, RBM14 caught in CXCL1 promoter region would decrease accordingly; \u003cstrong\u003e(L) \u003c/strong\u003ePulldown assay proved that when RBM14 was knocked down, p23 caught in CXCL1 promoter region would decrease accordingly.\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3842489/v1/ffc8ea8cd1fdc325d2e9e23a.jpg"},{"id":50910061,"identity":"52cc554c-ca34-4790-b793-441d3097309a","added_by":"auto","created_at":"2024-02-09 11:58:10","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":581968,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe influence of p23 on EMT is partially dependent on RBM14. (A) \u003c/strong\u003exCELLigence determined the number of migrating cells in real time; \u003cstrong\u003e(B-D)\u003c/strong\u003e Wound healing experiment \u003cstrong\u003e(B)\u003c/strong\u003e, transwell invasion \u003cstrong\u003e(C)\u003c/strong\u003e and migration experiment \u003cstrong\u003e(D)\u003c/strong\u003e of p23-overexpression and RBM14-knock-down of A549 cells; \u003cstrong\u003e(E)\u003c/strong\u003e The expression changes of invasion and migration proteins E-Cadherin, N-Cadherin and Vimentin and their quantitative graphs; \u003cstrong\u003e(F-H)\u003c/strong\u003eRepresentative images of lung metastatic nodules (F), statistics of the number of lung metastatic nodules (G) and H\u0026amp;E staining representative images of metastatic nodules (H) after four cell lines (A549/NC, A549/OEp23, A549/shRBM14, A549/OEp23+shRBM14) were injected into the tail vein respectively.\u003c/p\u003e","description":"","filename":"Fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3842489/v1/2c93c70b15b5faa635914617.jpg"},{"id":51188002,"identity":"fdb07cbc-69e2-4d7d-94b3-644a5fa991cf","added_by":"auto","created_at":"2024-02-15 16:25:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1560496,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3842489/v1/abb95258-a553-4f7e-b901-1727f48ee4ec.pdf"},{"id":50909685,"identity":"cbf25841-9614-4e98-9ab7-1bf099ae4b12","added_by":"auto","created_at":"2024-02-09 11:50:09","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10585,"visible":true,"origin":"","legend":"Table 1","description":"","filename":"SupplementaryTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3842489/v1/1bd76e2fb9a64fb36895b071.xlsx"},{"id":50909687,"identity":"a23a1388-bda9-4746-a7d8-44cd77052f05","added_by":"auto","created_at":"2024-02-09 11:50:09","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":9995,"visible":true,"origin":"","legend":"Table 2","description":"","filename":"SupplementaryTableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3842489/v1/add868349779e6a97b4ce566.xlsx"},{"id":50909686,"identity":"fd629e0e-7242-4d88-b637-b684fbae7d3a","added_by":"auto","created_at":"2024-02-09 11:50:09","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":269937,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig.S1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3842489/v1/683c134ec25bcac49b94c943.jpg"},{"id":50909689,"identity":"78c2da73-42e2-4c96-b667-e185a2afe5aa","added_by":"auto","created_at":"2024-02-09 11:50:10","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":226341,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3842489/v1/b2ff46ae320295a60febdbaf.jpg"},{"id":50909693,"identity":"8c5f750a-0ed7-4727-946f-7a6c0f332965","added_by":"auto","created_at":"2024-02-09 11:50:10","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":118051,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3842489/v1/0e40856ce20759986f14f41e.jpg"},{"id":50909698,"identity":"3ae0e985-b067-41a0-ab67-6dbb1655281c","added_by":"auto","created_at":"2024-02-09 11:50:11","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":151474,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3842489/v1/6a3baeae19a7507b3c0a6fb6.jpg"},{"id":50909699,"identity":"5168718a-cb19-41a4-9e6d-6cb7d3c28594","added_by":"auto","created_at":"2024-02-09 11:50:11","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":3874369,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-3842489/v1/762fa29dba24d3b808caf6d5.docx"},{"id":50909697,"identity":"6012f8ad-13ef-4fca-b59b-414891f4f813","added_by":"auto","created_at":"2024-02-09 11:50:10","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":4283447,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryinformationXXXXXXwesternblot.docx","url":"https://assets-eu.researchsquare.com/files/rs-3842489/v1/acc20c53643076a138d63590.docx"}],"financialInterests":"(Not answered)","formattedTitle":"RBM14 enhances transcriptional activity of p23 regulating CXCL1 expression to induce EMT in lung cancer","fulltext":[{"header":"Significance","content":"\u003col\u003e\n \u003cli\u003ep23 is a transcription factor of CXCL1 to promote tumor metastasis.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eRBM14 assists the transcriptional regulation of p23 on CXCL1 thereby promoting EMT.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Introduction","content":"\u003cp\u003eLung cancer has become a prominent contributor to global mortality rates associated with malignancies and a major public health burden globally\u003csup\u003e[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Despite some advancements in diagnosis, surgical techniques, and radiotherapy and chemotherapy over the past decade\u003csup\u003e[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e, the mortality rate of lung cancer remains alarmingly high with a mere 15% 5-year survival rate, which is primarily attributed to the widespread metastasis of lung cancer\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. The efficacy of targeted therapy in tumor treatment is promising; however, the intricate interactions between tumor and stroma greatly impede the development of targeted therapy for tumor metastasis. Therefore, it is imperative to investigate novel molecular mechanisms and targets of lung cancer metastasis, in order to establish a theoretical foundation for developing the innovative treatment strategies or therapeutic agents, and ultimately enhance the quality of life for patients with lung cancer.\u003c/p\u003e \u003cp\u003eThe process of epithelial-mesenchymal transformation (EMT) in tumor cells is characterized by the loss of intercellular adhesion among epithelial tumor cells and the acquisition of migration and invasion properties of mesenchymal cells, thereby promoting metastasis and development of various cancers, such as lung cancer\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. The acquisition of metastatic properties by tumor cells confers increased aggressiveness, bringing significant challenges to clinical management and cancer patient survival\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Therefore, it\u0026rsquo; s urgent to investigate the molecular targets and pathways involved in EMT in tumor cells, which not only established a theoretical foundation for preventing EMT in tumor cells, but facilitated the identification of potential therapeutic agents. In the present study, we mainly focused on the EMT marker CXCL1 to reveal a new mechanism for its expression regulation.\u003c/p\u003e \u003cp\u003eCXCL1, a chemokine of CXC family, is implicated in the pathogenesis of various inflammatory diseases and plays a crucial role in physiology and cancer progression, especially in EMT\u003csup\u003e[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Elevated expression level of CXCL1 in tumor tissues and serum, have been associated with tumor metastasis and poor prognosis in ovarian cancer, lung adenocarcinoma, colorectal cancer and pancreatic ductal adenocarcinoma (PDA)\u003csup\u003e[\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Previous study reported that CXCL1 was positively correlated with the migration and invasive activity of osteosarcoma cell lines\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e, and it has become a key indicator of lung metastasis of osteosarcoma through paracrine release. Therefore, it was proposed that targeted inhibition of CXCL1 could exert a profound inhibitory effect on tumor growth and metastasis.\u003c/p\u003e \u003cp\u003eProstaglandin E synthase 3 (p23) is a highly conserved protein encoded by PTGES3 gene \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e, which is often overexpressed in various cancers, including prostate cancer and breast cancer, as well as lung cancer\u003csup\u003e[\u003cspan additionalcitationids=\"CR23 CR24 CR25 CR26\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. The current understanding of p23 mainly focuses on its role as a co-partner of heat shock protein 90 (Hsp90)\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e, that can stabilize the complex formed by Hsp90 and its client proteins, including estrogen receptor\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, androgen receptor\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e and telomerase\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. However, p23 could also play some functions independently of Hsp90, such as, enhancing the transcription factor of p53\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e, and protecting human aryl hydrocarbon receptors from degradation \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Our previous study had re-defined p23 as an HSP90-independent transcription factor of COX-2 in promoting tumor growth\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. This highlights the diversity and complexity of p23 functionality, emphasizing the importance of further studying its functional characteristics. At present, there are few studies on p23 and tumor metastasis. Therefore, it is of great significance to explore the role of p23 in tumor migration and its molecular mechanism.\u003c/p\u003e \u003cp\u003eIn this study, we discovered a novel function of p23 as a transcription factor to promote EMT in lung cancer and revealed the specific mechanism. Its activation on the CXCL1 promoter is not achieved directly, rather than requiring the assistance of cofactor RBM14 during EMT progression.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ep23 expression is upregulated in lung cancer exhibiting metastatic features.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the potential involvement of p23 in lymph node metastasis of lung cancer, we observed a significant upregulation of p23 expression in metastatic lung cancer tissues compared to primary counterparts by querying the TCGA database \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e, and further confirmed in clinical samples (n\u0026thinsp;=\u0026thinsp;133) by immunohistochemical staining \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e, qPCR assays \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e and western blotting \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. We next evaluated p23 expression on the prognosis of 93 lung adenocarcinoma patients from a tissue microarray. High p23 expression facilitated the metastasis of lung cancer to lymph nodes in patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), and reduced the disease-free survival (DFS) of lung cancer metastasis patients \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. These results suggest that the elevated p23 expression is possibly associated with metastasis and predict the poor clinical outcomes of lung cancer metastasis patients.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eKnocking down p23 inhibits the invasion and migration of NSCLC cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the underlying mechanism of p23 in metastasis, we established stable A549 lung cancer cells with p23 knockdown (A549/shp23) (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Real-time inhibitory analysis using the xCELLigence provided compelling evidence that depletion of endogenous p23 impeded the migratory capacity of lung cancer cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. The similar results were also obtained from scratch assay \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e, transwell invasion \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e and migration assay \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Besides, several proteins involved in EMT were also evaluated by western blotting \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e and qPCR analysis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. p23 knock-down (shp23) greatly decreased protein levels of N-Cadherin and Vimentin, and increased the expression of E-Cadherin. By contrast, p23 stable overexpression (H1299/OEp23) altered the metastasis in an opposite fashion in lung cancer cells (Supplementary Figs. S1B-1D). Furthermore, a mouse model of lung cancer metastasis was also established by intravenous injection of tumor cells via tail vein to evaluate the effect of p23 on metastasis \u003cem\u003ein vivo\u003c/em\u003e. Consistent with \u003cem\u003ein vitro\u003c/em\u003e observations, shp23 dramatically decreased the number of lung metastatic nodules \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI\u003cb\u003e)\u003c/b\u003e. These data indicate that p23 played an oncogenic role in promoting lung cancer metastasis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe downstream target gene CXCL1 of p23 was found by RNA-seq sequencing.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn order to determine the molecular pathways involved in p23-driven lung cancer metastasis, we performed RNA-seq sequencing analysis to explore the potential target genes related to p23. A total of 92 differentially expressed genes (DEGs) were found to be commonly shared among the three p23 knock-down groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Then, 11 candidate DEGs for p23 knock-down were selected based on the analysis of typical lung cancer EMT marker genes from the EMTome database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.EMTome.org\" target=\"_blank\"\u003ewww.EMTome.org\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.EMTome.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e, and subsequently confirmed through qPCR detection. The consistent qPCR results validated the reliability of the RNA seq analysis findings \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. According to the abundance of these genes in cell samples, we selected CXCL1 with the highest content as the follow-up research focus \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCXCL1 is a vital chemokine driver to promote tumor metastasis\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. We then proposed that p23 is involved in lung cancer metastasis, at least partially, regulating CXCL1 expression. Importantly, both p23 and CXCL1 expressions were significantly increased in metastatic lung cancer tissues compared to non-metastatic counterparts \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. In addition, CXCL1 expression was significantly downregulated by p23 depletion, and upregulated by p23 over-expression at both mRNA and protein levels in lung cancer cells \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ\u003cb\u003e)\u003c/b\u003e, indicating that p23 regulates CXCL1 expression in lung cancer cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEMT induced by p23 is partially dependent on CXCL1.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo test whether p23 achieves its metastatic regulation functions in a CXCL1-depedent manner, we generated A549/shp23 lung cancer cells stably with over-expression of CXCL1 (A549/shp23\u0026thinsp;+\u0026thinsp;OECXCL1) to detect the alterations in EMT. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, CXCL1 significantly reversed the migration inhibition caused by p23 knockdown, as well as the changes in expression of EMT-relative proteins (N-Cadherin, Vimentin and E-Cadherin) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Moreover, the similar results were also observed \u003cem\u003ein vivo\u003c/em\u003e, that is, shp23 significantly reduced the number of lung metastatic nodules in NYG mice, and this reduction was completely reversed by CXCL1 overexpression \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG\u003cb\u003e)\u003c/b\u003e. These results indicated that p23 promotes lung cancer metastasis through CXCL1 expression. To support this notion, p23 overexpression largely enhanced lung cancer cell \u003cem\u003ein vitro\u003c/em\u003e migration, and further CXCL1 knockdown diminished the lung cancer metastasis enhancement induced by p23 overexpression (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA and 2B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ep23 regulates the transcriptional expression of CXCL1 rather than affect its mRNA stability.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further assess the function of p23 on CXCL1 expression, we first examined the effect of p23 on the stability of CXCL1 mRNA. Although p23 depletion or overexpression could positively regulate the basal mRNA level of endogenous CXCL1, the half-life of CXCL1 mRNA was not affected by p23 alterations (Supplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA and 3B), indicating that p23 upregulated CXCL1 expression at transcription level rather than affecting mRNA stability. Furthermore, we constructed luciferase reporter plasmids containing the optimal CXCL1 promoter region (-874 to +\u0026thinsp;38). Indeed, p23 expression dramatically induced the CXCL1 luciferase activity \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Through generating truncated the variants of CXCL1 promoter, we observed that deletion of the \u0026minus;\u0026thinsp;744 to -549 regions fully abolished p23-induced CXCL1 promoter activity \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e, indicating that this region containing the binding sites for p23. Therefore, a biotin-labeled probe corresponding to the optimal CXCL1 promoter (from \u0026minus;\u0026thinsp;744 to +\u0026thinsp;38) was employed to evaluate the binding capability of p23, with the CXCL1 promoter (from \u0026minus;\u0026thinsp;549 to +\u0026thinsp;38) as negative control. As expected, p23 protein was indeed visualized in the pulldown of CXCL1 promoter region \u0026minus;\u0026thinsp;744 to +\u0026thinsp;38 from the nuclear extract of A549 cells, whereas no signal was detected in negative control region \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further identify the DNA binding motif of p23 in CXCL1 promoter, we truncated this region (-549 to -744 bp) into five fragments, including P1 (probe 1): -744 to -703, P2: -707 to -663, P3: -667 to -621, P4: -625 to -579, P5: -583 to -537. Nucleotide sequence of CXCL1-EMSA probe was listed in supplementary table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The interaction between p23 and each probe was analyzed using EMSA assay with HEK-293T cell lysate overexpressing p23. It was found that p23 had binding affinities with P1, P2 and P3 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e, indicating that three fragments contained p23 binding motifs. This observation was further validated through a super-shift assay using anti-p23 antibody \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. Further DMEME analysis revealed a 6 bp p23-binding motif from three fragments with conserved locations at nucleotides 1C, 2A, 3C, 4T, 5G/T and 6A \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. Mutation of the motifs totally abolished the p23-induced CXCL1 reporter activity \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG\u003cb\u003e)\u003c/b\u003e. These results further confirmed the transcriptional regulatory function of p23 on CXCL1 expression. To further investigate the direct binding activity of p23 to the CXCL1 promoter, we conducted EMSA assay using purified p23 protein. Surprisingly, purified p23 exhibited no binding affinity towards the CXCL1 promoter \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e, suggesting that p23's transcriptional regulation of CXCL1 may not be solely dependent on its direct interaction with the promoter, but rather necessitates the involvement of other protein factors.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRBM14 promotes the transcriptional regulation of p23 on CXCL1.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn order to investigate the participants involved in the regulation of CXCL1 by p23, a total of 34 proteins interacting with p23 were identified through co-immunoprecipitation (Co-IP) combined with mass spectrometry (MS), of which 4 proteins were selected due to their transcriptional function \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Co-IP assay further verify their interaction with p23 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and supplementary Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e, which also showed the accuracy of the identification results by mass spectrometry. However, only RBM14 could be pull downed by CXCL1 promoter probe \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and supplementary Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e, and it can significantly promote p23 reporter activity of the optimal CXCL1 promoter \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. A similar result was also obtained from western blot analysis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. Furthermore, we conducted EMSA assay to investigate the binding activity of purified p23 and RBM14 protein to the CXCL1 promoter. As expected, p23 or RBM14 individually exhibited no direct binding to the CXCL1 promoter; however, their complexes demonstrated binding activity \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. These results implied that the interaction between p23 and RBM14 plays a crucial role in the regulation of CXCL1 expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next explored the underlying mechanism of p23/RBM14 protein complex participating in the transcription regulation of CXCL1. Firstly, there was no reciprocal influence observed between p23 and RBM14 in terms of their protein and mRNA expression levels \u003cb\u003e(\u003c/b\u003eSupplementary Figs. S4C-4F\u003cb\u003e).\u003c/b\u003e Subsequently, we modulated intracellular complex production by conducting p23-knockdown and/or RBM14-overexpression as well as p23-overexpression and/or RBM14-knockdown \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e, to elucidate the impact of the complex on CXCL1 transcription. The results demonstrated a significant reduction in CXCL1 promoter activity upon destruction of the p23/RBM14 complex via knockdown of either p23 or RBM14 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ\u003cb\u003e)\u003c/b\u003e. Similarly, the binding activity of p23 or RBM14 to CXCL1 promoter probe was significantly suppressed upon destruction of the p23/RBM14 complex \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL\u003cb\u003e).\u003c/b\u003e The above results show that the formation of the p23/RBM14 protein complex plays a pivotal role in mediating p23-induced CXCL1 expression.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe influence of p23 on EMT is partially dependent on RBM14.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNext, in order to explore whether the EMT process induced by p23 is RBM14-dependent, we constructed four stable cell lines by using lentivirus infection system: A549/NC, A549/OERBM14, A549/shp23, A549/shp23\u0026thinsp;+\u0026thinsp;OERBM14. \u003cem\u003eIn vitro\u003c/em\u003e, the migration number of cells is monitored in real time by xCELLigence assay \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. The result implied RBM14 could promote the phenotype of EMT, but when p23 was knocked down at the same time, the phenotype of EMT could be reduced slightly. In addition, wound healing experiments \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e and transwell invasion \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e as well as migration \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e experiments were performed and brought out similar conclusion. The changes of E-Cadherin, N-Cadherin and Vimentin protein levels were evaluated by western blotting \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e, which also proved that p23 promoted EMT phenotype in a RBM14-dependent manner. \u003cem\u003eIn vivo\u003c/em\u003e, four stably transfected cell lines, A549/NC, A549/shp23, A549/OERBM14 and A549/shp23\u0026thinsp;+\u0026thinsp;OERBM14, were injected into the tail vein respectively, and the number of lung metastatic nodules \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF \u003cb\u003eand G)\u003c/b\u003e and H\u0026amp;E staining \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e of lung metastatic nodules were investigated. From above results, we can draw the conclusion that EMT induced by p23 is partially dependent on RBM14.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eLung cancer has become the most prevalent and lethal malignant tumor in China and even in the world, with poor treatment outcomes\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. The metastasis of lung cancer represents a malignant manifestation and biological characteristic, serving as the primary cause of mortality among patients with this disease \u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. The metastatic nature of cancer facilitates the dissemination of tumor cells from the primary site to distant tissues beyond the lungs via blood and lymphatic system\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e, resulting in a substantial attenuation of conventional therapeutic efficacy. Consequently, impeding lung cancer metastasis holds the paramount importance in terms of protracting patients' survival, augmenting their overall prognosis, enhancing their quality of life, and ameliorating treatment outcomes\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. Metastasis of lung cancer is an intricate and multifaceted process involving multiple stages, steps, genes and factors. Therefore, elucidating the molecular mechanism underlying invasion and metastasis in lung cancer and identifying molecular targets for reversing or preventing these processes have become prominent areas to overcome lung cancer. In this topic, we found a novel pathway to regulate lung cancer metastasis, that is, RBM14 could assist p23 to implement transcription capabilities on CXCL1 to promote tumor metastasis. From the perspective of inhibiting lung cancer invasion and metastasis, which provided a theoretical basis for clinical treatment of lung cancer as well as screening its metastatic potential agents.\u003c/p\u003e \u003cp\u003ePTGES3 (p23), namely prostaglandin E synthase 3, is a molecular chaperone protein and a known potential oncogene\u003csup\u003e[\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Previous studies have shown that\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e p23 is highly expressed in lung adenocarcinoma, and the overexpression of p23 is related to the short overall survival and poor prognosis of lung cancer patients. The relationship between PTGES3 and EMT was first reported to point out that p23 could cause invasion and migration of lung cancer cells\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. However, the specific mechanism still remains unclear. The current research findings indicated that p23 played a significant role in promoting EMT in human lung cancer \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. Our study has demonstrated that the primary mechanism underlying p23's promotion of EMT was dependent on its regulation of CXCL1 expression through binding to the promoter region of CXCL1.\u003c/p\u003e \u003cp\u003eAs the executor of vital biological processes\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e, the functionality of proteins is often manifested through their interactions with other proteins\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Investigating protein-protein interactions holds immense significance in comprehending and preventing diseases, targeting treatment for polygenic disorders, and elucidating the molecular mechanisms underlying complex biological phenomena\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. Here, we initially demonstrated that p23 functions as a transcription factor indirectly binding to the promoter region of CXCL1, suggesting potential co-factors facilitating p23's transcription. Therefore, we identified this co-factor as RBM14 using mass spectrometry combined with Co-IP and DNA pull down experiments.\u003c/p\u003e \u003cp\u003eRNA binding motif protein 14 (RBM14), also known as Co-Activator Activator (CoAA), functions as a transcriptional coactivator. RBM14 exhibits widespread expression in embryonic tissues and plays a crucial role in the regulation of early embryonic development \u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. Previous investigations have demonstrated that RBM14 is significantly upregulated in tumors, thereby promoting tumor proliferation and migration \u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e; however, its precise molecular mechanism remains a mystery. In this study, we discover that RBM14 has the ability to form a complex with p23, thereby augmenting the transcriptional regulation of p23 on CXCL1 expression. When there is a decrease or absence in the expression of either component, the formation of the complex would be decreased to result in inhibition of CXCL1's transcriptional expression, and subsequently weakens cellular invasion and migration abilities. Our findings propose that RBM14 served as a co-activator of p23 to promote EMT in lung cancer.\u003c/p\u003e \u003cp\u003eIn conclusion, our findings elucidate that p23 plays a crucial role in promoting lung cancer metastasis through the regulation of downstream CXCL1 expression. Mechanistically, the regulation of CXCL1 transcription by p23 is based on the formation of a complex with RBM14 during EMT progression in lung cancer. Therefore, our study demonstrates the potential therapeutic application of RBM14-p23-CXCL1-EMT axis in targeting tumor metastasis, particularly lung cancer metastasis.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eReagents and antibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChemokine CXCL1 was purchased from Sigma (St. Louis, MO, USA) company, and the purity was over 98%. CXCL1 chemokine was dissolved in water containing 5% BSA, and the concentration of the stock solution was 25 mM, which stored at -80℃. The pure protein p23 and RBM14 were obtained from Invitrogen (USA). The primary antibody against HA was obtained from Cell Signaling Technology (USA). The primary antibodies against p23, Vimentin, hnRNPU, DDX5, RPS3, Flag, \u0026beta;-actin and all the secondary antibodies were obtained from Proteintech Group (USA). The primary antibodies against CXCL1 and RBM14 were obtained from Abclonal Technology. The primary antibodies against E-Cadherin, N-Cadherin were obtained from Jingjie PTM BioLab (Hangzhou). Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM), RPMI 1640, fetal bovine serum (FBS), and trypsin were obtained from HyClone Laboratories (USA). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless specified otherwise.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell lines and cell culture conditions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman lung cancer cell lines (A549, H460, H322, HCC827 and H1299), HLF and 293T cells were obtained from the American Type Culture Collection (ATCC Manassas, VA, USA) or Procell Life Science \u0026amp; Technology Co., Ltd (Wuhan, China), and preserved in our laboratory. The cells were cultured in F12K, DMEM or 1640 medium supplemented with 10% FBS, 100 \u0026mu;g/ml penicillin and 100 \u0026mu;g/ml streptomycin at 37\u0026deg;C in a humidified atmosphere comprising 5% CO\u003csub\u003e2\u003c/sub\u003e. The authenticity of all cell lines was verified through genomic short tandem repeat profiling, and the cell lines were confirmed to be free of mycoplasma using the TransDetect\u0026reg; PCR Mycoplasma Detection Kit (TransGen, Beijing, China).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using Trizol reagent (Invitrogen) from cells and tumor tissues. cDNA synthesis was performed with Oligo(dT) or random primers by using a Quantscript RT Kit (Tiangen). A SYBR RT\u0026ndash;PCR kit (Tiangen) was used for transcript quantification with specific primers. Expression levels were quantified using the 2\u0026minus;\u003csup\u003e\u0026Delta;\u0026Delta;\u003c/sup\u003eCt method with \u0026beta;-actin as an internal control. The primers are provided in Supplementary Table S1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStreptavidin-agarose pulldown assay for the detection of DNA-protein binding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe binding affinity of p23 to the CXCL1 promoter probes was determined using a streptavidin-agarose pull-down assay. A 541-bp biotin-labeled double stranded probe corresponding to the CXCL1 promoter sequence (-541 to +38) was synthesized. Briefly, 50 ng of protein, biotinylated DNA probe (4 \u0026mu;g), streptavidin-conjugated agarose beads (40 \u0026mu;l) and PBSi (PBS buffer with 1 mM EDTA, 1 mM DTT and complete protease inhibitor cocktail) were incubated at room temperature for 5 h on a rotating rack. After washing with PBSi buffer, the beads were resuspended in SDS-PAGE loading buffer and boiled at 100 \u0026deg;C. The supernatant was analyzed by western blotting.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDual-luciferase reporter gene assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLuciferase activity was measured using the Dual-luciferase reporter gene assay system (Promega) according to the manufacturer\u0026rsquo;s instructions. Growth media were removed and cells were washed with PBS. Cell precipitates were collected and lysed by freezing and thawing twice in liquid nitrogen. Then, 10 \u0026mu;l of the lysate was transferred into a black 96-well plate (Thermo). Firefly and Renilla luciferase activity was assayed sequentially in the cell lysate in each well. Transcriptional activity was calculated as the ratio of firefly luciferase activity (reporter) to Renilla luciferase activity (control).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElectrophoretic Mobility Shift Assay (EMSA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBiotin-labeled CXCL1 promoter probes were purchased from BGI Genomics (Beijing, China). For the DNA EMSA assay, 4 \u0026mu;g protein and 1 \u0026mu;g of biotin-labeled DNA probe were mixed in binding buffer and incubated for 20\u0026thinsp;min at 25\u0026thinsp;\u0026deg;C. DNA\u0026ndash;protein complexes were separated in 6.5% acrylamide native PAGE gels at 4\u0026thinsp;\u0026deg;C. The specific bands were detected with HRP-conjugated streptavidin and visualized by enhanced chemiluminescence.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLentivirus preparation and transfection\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLentivirus was produced in 293T cells using the second-generation packaging system plasmids psPAX2 (Addgene) and pMD2.G (Addgene). One 10 cm culture dish containing 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e 293T cells was transfected using Lipofectamine 2000 (Invitrogen) with 15 \u0026mu;g lentiviral vector, 7.5 \u0026mu;g psPAX2 and 7.5 \u0026mu;g pMD2.G. After transfection, the supernatants were collected every 24 h between 24 and 72 h, and concentrated with PEG-8000, after which the viral titer was determined by serial dilution. The multiplicity of infection (MOI) during transfection was 5.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoprecipitation (IP)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTake 800 \u0026micro;g of protein, and fix the volume to 500 \u0026micro;L in Binding buffer. Then 2 \u0026micro;g of flag antibody was added to the experimental group while 2 \u0026micro;g of IgG antibody was added to the blank group, and shake it on the rotary mixer for 1 h. After 1 h, 15 \u0026micro;L magnetic beads were added and shaked them overnight at 4 C on a rotary mixer. Magnetic separation is carried out the next day, and a proper amount of Binding buffer is added to wash the magnetic beads for three times after the supernatant is discarded. 50 \u0026mu;L 2\u0026times;Loading Buffer was added to the magnetic beads and boiled for 10 minutes. After magnetic separation, the supernatant was transferred to for western blot analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale NYG mice aged 5 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (China), and kept in the SPF Laboratory Animal Center of Dalian Medical University (Dalian, China). All animals were given free access to sterilized food and water and were habituated for 7 days and divided randomly before the experiments. The indicated tumor cells (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e in 100 \u0026mu;L PBS) were injected through tail vein of each mouse. After 4 weeks following injecting, the animals were sacrificed and the lungs from each mouse were excised while the number of pulmonary metastatic nodules was checked. Small parts of lung tissue were obtained for H\u0026amp;E staining. All procedures were carried out in strict accordance with the recommendations established by the Animal Care and Ethics Committee of Dalian Medical University as well as the guidelines promulgated in the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGraphpad.9.0 was used for all statistical analyses. The Kaplan\u0026ndash;Meier method was used to analyze survival. Pearson\u0026rsquo;s correlation test was used to examine the correlations among histochemical markers. Unpaired Student\u0026rsquo;s t-test to assess differences between two groups, while analysis of variance (ANOVA) followed by the least significant difference test was used for comparisons of multiple groups. Differences with P \u0026lt; 0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the National Natural Science Foundation of China (82225048 and 82004089), Distinguished professor of Liaoning Province (XLYC2002008),\u0026nbsp;and\u0026nbsp;Sanming Project of Medicine in Shenzhen (No. SZZYSM202106004) for financial support.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.-X.C. and Y.-Z.L. initiated the work and designed the experiments. Z.W., P.-Y.L., and Z.-M.R. performed the experiments, analyzed data, made figures and drafted the manuscript; C.-Y.L., C.-J.L., Z.-W.H., W.C., H.-C.J. T.-X.G., T.-M.M., F.L., W.Y. and H.-X.K. analyzed the data. M.-X.C., Y.-Z.L. and Z.W. supervised the study. All authors reviewed the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were repeated three times. All data and associated protocols are included in the manuscript and available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eThai A A, Solomon B J, Sequist L V, et al. Lung cancer [J]. Lancet (London, England), 2021, 398(10299): 535-554.\u003c/li\u003e\n\u003cli\u003eSchabath M B, Cote M L. Cancer Progress and Priorities: Lung Cancer [J]. 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Human Cell, 2022, 35(6): 1838-1855.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"EMT, p23, CXCL1, RBM14","lastPublishedDoi":"10.21203/rs.3.rs-3842489/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3842489/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMetastasis serves as a malignant indicator and biological characteristic of pulmonary carcinoma. Epithelial-mesenchymal transition (EMT) plays a pivotal role in facilitating tumor invasion and metastasis, and enhances the aggressiveness of tumor cells. Prostaglandin E synthase 3 (PTGES3) functions as an HSP90 co-chaperone. Our previous study revealed its HSP90-independent role as a transcription factor involved in cancer-related inflammation. Our present study aims to investigate the impact and mechanism of p23 on lung cancer metastasis. By utilizing cell models\u003cem\u003e in vitro\u003c/em\u003e and mouse tail vein metastasis models\u003cem\u003ein vivo\u003c/em\u003e, our results provide solid evidences that p23 plays a crucial role in promoting lung cancer metastasis through regulating the downstream CXCL1 expression, which is not achieved independently, but rather through formatting a complex with RBM14, thereby facilitating the occurrence and progression of EMT in lung cancer. Therefore, our study demonstrates the potential therapeutic application of the RBM14-p23-CXCL1-EMT axis in targeting lung cancer metastasis.\u003c/p\u003e","manuscriptTitle":"RBM14 enhances transcriptional activity of p23 regulating CXCL1 expression to induce EMT in lung cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-09 11:50:04","doi":"10.21203/rs.3.rs-3842489/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c2589b38-399b-41c8-8a52-d138336209db","owner":[],"postedDate":"February 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":28649589,"name":"Biological sciences/Cancer"},{"id":28649590,"name":"Biological sciences/Molecular biology/Transcription"}],"tags":[],"updatedAt":"2024-02-15T16:17:13+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-09 11:50:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3842489","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3842489","identity":"rs-3842489","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}