The Role And Mechanism Of Splicing Factor PQBP1 In Ovarian Cancer Initiation And Development

BackgroundOvarnan cancer is a common malignant tumor of the female reproductive system.and the mortality rate has always been the first in gynecological malignant tumors.High-grade Serous Ovarian Cancer(HGSOC)is the most malignant subfamily and accounts for 70%-80%of ovarian cancer due to its characteristics of occult occultation,strong invasion ability and poor prognosis.Most patients with ovarian cancer are diagnosed with advanced disease due to lack of specific symptoms and early screening methods and die from tumor recurrence and drug resistance.The 5-year survival rate of advanced patients is only 20%to 30%.Therefore,elucidating the molecular mechanism of ovarian cancer's development and progression,screening effective molecular targets is of great significance for the early diagnosis and treatment of ovarian cancer.Recent years it has caused extensive attention that abnormal alternative splicing plays an important role in the development of malignant tumors.Tumor cells produce splice isoforms that are beneficial to tumor proliferation,apoptosis,hypoxia,angiogenesis,immune escape and metastasis through abnormal alternative splicing.Our team found through transcriptome and proteomics analysis that among the signaling pathways involved in the development of ovarian cancer,spliceosome pathways is ranked on the first place.Further analysis shows that 33 out of 134 master splicing factors were up-regulated in ovarian cancer.It suggests that alternative splicing plays a key role in ovarian cancer but we don't know its specific molecular mechanism yet.There is many differential splicing of gene transcripts in ovarian cancer.For example,CD44s is highly expressed in ovarian cancer cells and CD44v is lowly expressed.CD44s mediates TGF?1-induced EMT pathway by down-regulating splicing factor ESRP1,which induces invasion and migration as well as stemness and drug resistance of ovarian cancer cells.Osteopontin(OPN)transcript OPNc is specifically expressed in ovarian cancer,but not OPNa or OPNb,promoting ovarian cancer cell proliferation,migration,invasion,tumor formation and other malignant behaviors.Bcl-x is a member of the bcl-2 family of apoptotic genes,the pre-mRNA alternative splicing of Bcl-x gene produces Bcl-xS and Bcl-xL,which are involved in the promotion and inhibition of apoptosis respectively.The overexpression of the Bcl-xL inhibits tumor cell apoptosis,promotes metastasis and chemoresistance and is associated with poor clinical outcomes,whereas Bcl-xS induces apoptosis and makes cells sensitive to chemotherapeutic drugs.PQBP1(polyglutamine binding protein 1,PQBP1)is one of the splicing factors found up-regulated in ovarian cancer.It is an evolutionarily highly conserved mental development-related protein,and its mutation leads to X chromosome-related"Renpenning syndrome".PQBP1 interacts with transcription factors such as RNA polymerase ? to participate in transcriptional regulation.In addition,PQBP1 interacts with multiple splicing factors and participates in the alternative splicing of many gene mRNAs in human cells.Missing PQBP1 reduces the association of SF3B1 with substrate mRNA,resulting in significant changes in alternative splicing patterns.The expression level of PQBP1 is up-regulated in ovarian cancer.It remains to be further studied of the role of PQBP1 in the development of ovarian cancer as well as the abnormal splicing events in which PQBP1 is involved.ObjectiveThis study aims to clarify the expression of PQBP1 in high-grade serous ovarian cancer;to explore the role of PQBP1 in the malignant biological behaviors such as proliferation,invasion,migration and apoptosis of ovarian cancer cells;to identify the downstream targets and alternative splicing events regulated by splicing factor PQBP1.MethodsqPCR,Western blotting and immunohistochemistry were used to detect the expression of PQBP1 in ovarian cancer;TCGA database was used to analyze the expression of PQBP1 and correlation between PQBP1 with clinical prognosis;molecular biological methods was used to construct PQBP1 lentiviral overexpression and knockdown plasmid;We used lentivirus to construct the up or down-regulated PQBP1 stable cell lines.Using the clonogenic assay,MTT,EdU experiments demonstrated the effect of PQBP1 on the proliferation of ovarian cancer cells.Transwell experiment was used to explore the effect of PQBP1 on the migration of ovarian cancer cells.The effect of PQBP1 on apoptosis of ovarian cancer cells was tested by flow cytometry.The selective splicing of endogenous Bcl-x gene influenced by PQBP1 was tested by RT-PCR and qPCR.We used Bcl-x minigene plasmid method to detect the alternative splicing of exogenous Bcl-x gene affected by PQBP1.Results1.PQBP1 is highly overexpressed in ovarian cancer and is associated with prognosisThe transcriptome and proteomic analysis revealed that the splicing factor PQBP1 was highly expressed in ovarian cancer.16 cases of normal fallopian tube tissue and 29 cases of high-grade serous ovarian cancer tissue were collected.The qPCR results showed that PQBP1 was highly expressed in high-grade serous ovarian cancer(*P<0.05).TCGA data analysis found that PQBP1 has genomic amplification and high mRNA variation(11%)in ovarian cancer.High expression of PQBP1 is associated with poor prognosis in ovarian cancer patients(p=0.025).2.PQBP1 promotes malignant behaviors such as ovarian cancer cells proliferation,invasion and migration,and tumor formation in vivo.The PQBP1 high-expression and low-expression stable cell lines were constructed using a lentiviral system for functional experimental studies.We showed that overexpression of PQBP1 can significantly enhance the clonality of ovarian cancer cells.Knockdown of PQB1 can significantly reduce the ability of ovarian cancer cells to form clone.MTT assay showed that overexpression of PQBP1 can promote ovarian cancer cell proliferation and knockdown of PQBP1 can reduce ovarian cancer cells proliferation.Detection of the expression of cycle-associated proteins,found that the expression of p53 and p21 proteins was increased in cancer cells.Knocking down PQBP1 found that the expression of CyclinD1 and CyclinE proteins was down-regulated.The above experimental results indicate that PQBP1 can significantly promote the proliferation of ovarian cancer cells in vitro.To further investigate the biological function of PQBP1 in ovarian cancer,we used Transwell experiment to detect the effect of PQBP1 on the invasiveness of ovarian cancer cells.It was found that overexpression of PQBP1 enhanced the invasiveness of ovarian cancer cells,while knockdown of PQBP1 decreased the invasiveness of ovarian cancer cells.It proves that PQBP1 promotes the invasion and metastasis of ovarian cancer cells and increases the malignant degree of cancer cells.Western blotting was used to detect the expression of EMT pathway-related proteins.It was found that overexpression of PQBPl promoted the expression of mesenchymal expression-related markers such as Vimentin,?-Catenin,N-Cadherin,Claudin-1,Snail,Slug and other proteins,and decreased epithelial expression-related markers such as E-Cadherin,ZO-1.The above results show that PQBP1 promotes the invasion and EMT progression of ovarian cancer cells.In vivo tumor formation experiments showed that compared with the control group,overexpression of PQBP1(HEY cell line)significantly enhanced the ability of subcutaneous tumor formation in nude mice as well as tumor growth rate and tumor weight were significantly higher than the control group.In summary,the splicing factor PQBP1 promotes the proliferation,invasion and tumorigenic ability of ovarian cancer cells and plays a role as an oncogene.3.PQBP1 inhibits apoptosis of ovarian cancer cells by regulating alternative splicing of Bcl-x geneApoptosis of Annexin/PI double staining revealed that overexpression of PQBP1 significantly inhibited apoptosis of ovarian cancer cells,and knockdown of PQBP1 promoted apoptosis of ovarian cancer cells.Western blotting was used to detect the expression of apoptosis-related proteins,and it was found that PQBP1 promoted the expression of Bcl-2 and inhibited the expression of Bax.qPCR detection revealed that knockdown of PQBP1 in ovarian cancer cell lines affected the expression of a series of apoptotic factors,including Bcl-x.RT-PCR detection found that PQBP1 affects the alternative splicing of the endogenous Bcl-x gene,promoting the expression of anti-apoptotic transcript Bcl-xL and inhibiting the expression of the pro-apoptotic transcript Bcl-xS.To further validate the regulation of Bcl-x gene by PQBP1,we constructed a Bcl-x minigene plasmid to detect the effect of PQBP1 on alternative splicing of exogenous Bcl-x gene,and found that PQBP1 promotes Bcl-xL expression and inhibits Bcl-xS expression.Conclusion1.PQBP1 is highly expressed in ovarian cancer and is associated with poor prognosis in ovarian cancer patients.2.PQBP1 promotes malignant behaviors such as ovarian cancer cell proliferation,invasion and migration,and tumor formation in vivo.3.PQBP1 regulates alternative splicing of Bcl-x gene to inhibit apoptosis of ovarian cancer cells.