| BackgroundOvarian cancer is the gynecological malignant tumor with the highest fatality rate.More than 70%of ovarian cancer patients are diagnosed with advanced stage,and the 5-year survival rate is less than 50%.Postoperative recurrence and drug resistance are still difficult problems in the treatment of ovarian cancer.The most common pathological type of ovarian cancer is serous ovarian carcinoma(SOC),which accounts for about 70%of ovarian cancer,which is highly malignant and has a poor prognosis.Therefore,to explore the new molecular targets for the occurrence and development of SOC and clarify its regulatory mechanism is an urgent problem to be solved.Recent studies have shown that RNA binding protein(RBP)is the main participant in the occurrence and development of cancer.The change of its expression and location will not only increase the expression of oncogenes,but also reduce the expression of tumor suppressor genes,thus promoting the occurrence and development of tumors.In recent years,people have paid more and more attention to the role of RBP in cancer,and studies have shown that RNA-binding protein RBM39 inhibitors have a potent pharmacological effect against leukemia;In triple-negative breast cancer,the RNA-binding protein YTHDF2 may be a target for new cancer drugs.Therefore,RBP is expected to become a new target for cancer treatment.We used bioinformatics methods to screen and identify the key RBP in ovarian cancer,and found that MEX3A is closely related to the clinical prognosis of ovarian cancer.MEX3A is a member of the MEX3 family.MEX3 is widely found in mammalian cells.It encodes four different genes,namely,MEX3A,MEX3B,MEX3C and MEX3D.MEX3A plays a variety of roles in biological processes.Recent studies have shown that MEX3A can promote the occurrence and development of many kinds of malignant tumors,which is related to poor clinical prognosis.However,the expression,biological role and related regulatory mechanisms of MEX3A in ovarian cancer have not been clearly explained.The purpose of this study was to explore the expression,clinical significance and biological function of MEX3A in serious ovarian carcinoma.Through the screening and identification of downstream target genes of MEX3A and the study of upstream molecular regulation mechanism,to explore the related mechanism of promoting the occurrence and development of serious ovarian carcinoma.The specific research content includes the following three parts:1.Study on the aberrant expression,clinical significances and biological functions of MEX3A in Serous Ovarian carcinoma.2.Study on the downstream regulatory mechanism of MEX3A in promoting the occurrence and development of ovarian cancer.3.Study of the regulatory mechanism of abnormally high expression of MEX3A in ovarian cancer.Part Ⅰstudy on the aberrant expression,clinical significances and biological functions of MEX3A in serous ovarian carcinomaObjectTo detect the expression of MEX3A in serous ovarian carcinoma and to reveal its relationship with clinicopathological features and clinical prognosis.To investigate the effect of MEX3A on malignant biological behaviors such as proliferation,migration and invasion of ovarian cancer cells.Contents and methodsThe expression of MEX3A in serous ovarian carcinoma was analyzed by GEPIA and TCGA-GTEX database.To analyze the differential expression of MEX3A in seriuos ovarian cancer,normal ovary and fallopian tube tissues in TCGA-GTEX database.The variation of MEX3A DNA copy number in serous ovarian carcinoma was analyzed by TCGA and CCLE database,and the correlation between MEX3A DNA copy number and its mRNA expression was analyzed.The data of TCGA were used to analyze the differential expression of MEX3A in different molecular subtypes of ovarian cancer.QRT-PCR and Western blot were used to analyze the differential expression of MEX3A in mRNA and protein levels between high-grade serous ovarian carcinoma(HGSOC)and normal fallopian tube fimbria tissue.The effect of MEX3A on the clinical prognosis of patients with ovarian cancer was evaluated by Kaplan-Meier Plotter.We used immunohistochemistry to detect the expression of MEX3A in HGSOC clinical samples of tissue microarray and analyzed its relationship with clinical parameters and clinical prognosis.Ovarian cancer cell lines with stable knockout and overexpression of MEX3A were constructed by lentivirus transfection,and two small interfering(siRNA)were designed to interfere with the expression of MEX3A.The efficiency of interference and overexpression was verified by qRT-PCR and Western blot.The effects of MEX3A on the malignant biological behavior of ovarian cancer cells were studied by MTT,EdU Assays,plate cloning and Transwell Assays.We injected the constructed MEX3A stably knocked down HEY ovarian cancer cell lines and the control group cell lines subcutaneously into the armpit of the female nude mice.About 4 weeks later,when there is a tumor growing under the armpit of the nude mice,the nude mice were killed with the method of necking off under anesthesia,the subcutaneous transplanted tumor was taken out,and the tumor volume was calculated and weighed(n=6 for each group).Results1.High expression of MEX3A in serous ovarian carcinoma.GEPIA and TCGA-GTEX database showed that MEX3A was highly expressed in HGSOC(n= 426)compared with normal fallopian tube(n=5)and ovarian tissue(n=88).By analyzing the differential expression of MEX3A in four molecular types of ovarian cancer(proliferative type,n=135;mesenchymal type,n=106;immunoreactive type,n=108;fallopian type,n=134),we found that the expression of MEX3A was the highest in proliferative type,followed by mesenchymal type.These results suggest that MEX3A may be closely related to the proliferation and metastasis of ovarian cancer.MEX3A DNA copy number amplification occurred in 10%of HGSOC samples in TCGA database,and there was a linear positive correlation between MEX3A DNA copy number amplification and mRNA expression in ovarian cancer tissues and cell lines.We verified the expression of MEX3A mRNA in HGSOC tissue(n=24)and normal tubal umbrella tissue(n=12)by qRT-PCR,and found that the expression of MEX3A mRNA in HGSOC tissue was higher than that in normal tubal umbrella tissue.We verified the expression of MEX3A protein in HGSOC tissue(n=10)versus normal tubal umbrella tissue(n=6),as well as in tubal epithelial cell lines and ovarian cancer cell lines,using the Western blot method.The results showed that the expression of MEX3A protein in HGSOC was significantly higher than that in normal fallopian tube fimbria tissue,and the expression of MEX3A protein in A2780,HEY and SKOV3 ovarian cancer cells was significantly higher than that in normal fallopian tube epithelium FT187.2.High expression of MEX3 A is associated with poor clinical prognosis of serous ovarian carcinoma.We obtained the clinical information and follow-up data of all patients in tissue microarray from obstetrics and gynecology department follow-up office of Qilu Hospital.The immunohistochemical results of tissue microarray showed that the expression of MEX3A in HGSOC was mainly concentrated in the nucleus,and the expression in HGSOC was higher than that in normal fallopian tube.Among the HGSOC samples,the group with high expression of MEX3A accounted for 62.16%(69/111),while the group with high expression of MEX3A in normal fallopian tube samples accounted for 32.35%(11/34).Through survival analysis,it was found that the overall survival of HGSOC patients with low expression of MEX3A was significantly longer than that of patients with high expression of MEX3A(P<0.01).The high expression of MEX3A was associated with ascites volume(P=0.014),but not with age(P=0.845),blood CA125(P=0.256)and FIGO stage(P=0.558).3.MEX3A promotes the proliferation,migration and invasion of ovarian cancer cells in vitro.MTT results showed that after interfering with the expression of MEX3A,the proliferation efficiency of HEY,A2780 and SKOV3 ovarian cancer cell lines decreased significantly compared with the control group,and it was more significant after the third day.After overexpression of MEX3A,the proliferation efficiency of HEY and A2780 ovarian cancer cell lines was significantly higher than that of the control group,and it was the most significant after 3-4 days.EdU Assays results showed that the proportion of mitotic and proliferative cells in HEY,SKOV3 and A2780 ovarian cancer cells after knockdown of MEX3A was significantly lower than that in the control group.The results of plate cloning showed that the number of clones interfered with MEX3A expression in A2780 and SKOV3 ovarian cancer cell lines decreased by about 50%compared with the control group,and that in HEY ovarian cancer cell lines by interfering with MEX3A expression decreased by about 70%compared with the control group.However,the number of HEY and A2780 ovarian cancer cell lines after overexpression of MEX3A was significantly higher than that of the control group.The results of Trans well assays showed that after interfering with MEX3A,the number of ovarian cancer cells penetrating the chamber of HEY,A2780 and SKOV3 decreased significantly,and the number of ovarian cancer cells of HEY,A2780 and SKOV3 with matrix colloid chamber was also significantly decreased after interfering with MEX3A.These results suggest that interference with the expression of MEX3A can decrease the proliferation,migration and invasion of ovarian cancer cell lines,while overexpression of MEX3A can significantly improve the proliferation,migration and invasion of ovarian cancer cell lines.4.MEX3A promotes the tumorigenicity of ovarian cancer cells in female nude mice in vivoThe results of subcutaneous tumor-formation experiment in nude mice showed that the average volume of tumors in the MEX3A knockdown group was reduced by more than 50%compared with the control group.Similarly,the average weight of tumors in the MEX3A knockdown group was reduced by about 50%compared with the control group.This indicates that interference with MEX3A significantly inhibited the tumor growth ability of ovarian cancer cells,resulting in a decrease in tumor weight and volume.SummaryMEX3A is highly expressed in serous ovarian carcinoma,which is related to poor overall survival.MEX3A plays the role of oncogenes in serous ovarian carcinoma and promotes the malignant biological behavior of serous ovarian carcinoma.Part ⅡStudy on the downstream Regulation Mechanism of MEX3A promoting the occurrence and development of serous ovarian carcinomaObjectTo explore the downstream target genes of MEX3A which play a malignant biological role in serous ovarian carcinoma,and to reveal the molecular mechanism of MEX3A regulating downstream target genes.Contents and methodsSiRNA was used to interfere with the expression of MEX3A in A2780 cells,and the interference efficiency was verified.The differentially expressed genes(DEGs)in ovarian cancer cells after MEX3A interference were analyzed by RNA-seq.The alternative splicing events of ovarian cancer cells after interfering with MEX3A were further analyzed.The alternative splicing events and DEGs changed after interfering with MEX3A were analyzed by overlap analysis and DEGs were screened.The correlation between DEGs and MEX3A was analyzed by TCGA ovarian cancer database,and DEGs in ovarian cancer,normal fallopian tube epithelium and ovarian tissues were analyzed by TCGA-GTEX database to obtain downstream target genes.The differential expressions of mRNA and protein of downstream target genes between HGSOC and normal control tissues were analyzed in TCGA-GTEX and CPTAC database.To analyze the correlation between the expression of MEX3A and downstream target genes in HGSOC.QRT-PCR and Western blot were used to evaluate the changes of mRNA and protein expression of downstream target genes in ovarian cancer cells after interfering with MEX3 A.Small interference RNA(siRNA)was used to down-regulate the expression of target genes in ovarian cancer cells,and qRT-PCR and Western blot were used to verify the interference efficiency.MTT proliferation assays,plate cloning and Transwell assays were used to verify the effect of downstream target genes on the malignant biological behavior of ovarian cancer cells.MTT assays,plate cloning,Transwell assays and subcutaneous tumorigenesis in nude mice were used to further verify whether MEX3A regulates the proliferation,migration and invasion of ovarian cancer cells through target genes.The alternative splicing events of downstream target gene mRNA after interfering with MEX3A were analyzed by sashimi map.By analyzing the mRNA transcripts of target genes in the middle and lower reaches of Ensembl database,the regulation mechanism of MEX3A on downstream target genes was explored in serous ovarian carcinoma.Results1.Screening and identification of MEX3A downstream target genes in serous ovarian carcinoma.RNA-seq results were used to screen 543 upregulated genes and 712 downregulated genes in ovarian cancer cells after MEX3A knockdown.The alternative splicing events of ovarian cancer cells after down-regulation of MEX3A were further analyzed.DEGs after the variable splicing event and the MEX3A interference were over-analyzed and 182 DEGs were identified.Using TCGA ovarian cancer database to analyze the correlation between MEX3A and 182 DEGs,7 DEGs(CSNKE1,OBSL1,DNASE1,ZNF711,TIMELESS,SAMD11 and BCL9)were found to be positively correlated with MEX3A expression.The changes of 7 DEGs after MEX3A knockdown were analyzed by RNA-seq,and the differences of 7 DEGs in HGSOC,normal ovary and tubal epithelial tissues were analyzed by TCGA-GTEX database.The results showed that only TIMELESS was highly expressed in HGSOC.Therefore,we consider that TIMELESS is the downstream target of MEX3A in ovarian cancer.2.High expression of TIMELESS in serous ovarian carcinoma.By analyzing the difference of TIMELESS mRNA expression between HGSOC and normal control tissues(HGSOC,n=426;fallopian tube,n=5;ovary,n=88)in TCGA-GTEX database,it was found that the expression of TIMELESS in HGSOC was higher than that in normal fallopian tube and ovarian tissue.The data of 77 cases of HGSOC and 17 cases of normal ovarian tissue in CPTAC database were analyzed.The results showed that the expression of TIMELESS protein in HGSOC was significantly higher than that in normal ovarian tissue.By analyzing the correlation between MEX3 A and TIMELESS in 489 HGSOC samples from TCGA database,it was confirmed that the expression of TIMELESS mRNA was positively correlated with the expression of MEX3A mRNA in HGSOC tissues.Two small interfering RNA(siRNA)were used to down-regulate the expression of MEX3A in ovarian cancer cells,and the changes of TIMELESS mRNA and protein expression were verified by qRT-PCR and Western blot.QRT-PCR results showed that the expression of TIMELESS mRNA decreased by more than 75%in HEY and SKOV3 ovarian cancer cell lines by knocking down the expression of MEX3A by small interference RNA.Western blot results showed that the expression of TIMELESS protein decreased significantly after interfering with MEX3A in HEY,A2780 and SKOV3 cell lines,which indicated that the down-regulation of MEX3A mediated by siRNA decreased the expression of mRNA and protein in TIMELESS.3.TIMELESS promotes the proliferation,migration and invasion of ovarian cancer cells in vitro.The results of MTT assays showed that interference with the expression of TIMELESS could inhibit the proliferation of HEY,SKOV3 and A2780 ovarian cancer cell lines.The results of plate cloning showed that interference with the expression of TIMELESS could decrease the clone formation ability of HEY,SKOV3 and A2780 ovarian cancer cell lines.The results of Transwell assays showed that after interfering with TIMELESS,the number of ovarian cancer cells penetrating the chamber of HEY,A2780 and SKOV3 decreased significantly.It is suggested that interference with TIMELESS can inhibit the migration and invasion of ovarian cancer cells.4.Interference with TIMELESS weakens the ability of MEX3A to promote the proliferation,migration and invasion of ovarian cancer cells.The results of MTT experiment showed that the proliferation ability of MEX3A+siTIMELESS group and PCMV+siTIMELESS group decreased significantly after interfering with TIMELESS compared with the control group.The results of plate cloning showed that compared with the control group,the number of single clones in MEX3A+siTIMELESS group and PCMV+siTIMELESS group decreased significantly after interfering with TIMELESS.The results of Transwell showed that the number of ovarian cancer cells penetrating the chamber after interfering with TIMELESS in MEX3A+siTIMELESS group and PCMV+siTIMELESS group was significantly lower than that in control group.These results suggest that interference with TIMELESS partially reverses the ability of overexpression of MEX3A to promote the proliferation,clone formation,migration and invasion of ovarian cancer cells.5.Interference with TIMELESS weakens the ability of MEX3A to promote the tumorigenesis of ovarian cancer cells in nude mice.Experimental results in nude mice showed that the tumor volume and weight of the MEX3A+ shTIMELESS group were significantly reduced compared with the MEX3A group.and the tumor volume and weight of PCMV+shTIMELESS group was significantly smaller than that of PCMV group.The results showed that the volume and weight of subcutaneous tumors in nude mice after TIMELESS knockdown were significantly smaller than those in the control group,and interfering with TIMELESS could partially reverse the effect of MEX3A overexpression on the tumorigenesis of ovarian cancer cells in vivo.6.MEX3A regulates the expression of TIMELESS mRNA through regulating the retention of introns.The analysis of Ensembl database and RNA-seq results showed that,compared with the protein-coding transcripts TIMELESS-201 and TIMELESS-203,the non-coding TIMELESS-205 transcripts retained intron 23 between exons 23 and 24.RT-PCR results showed that after interfering with MEX3A,the expression of transcripts TIMELESS-201/203 of coding proteins decreased,while the expression of TIMELESS-205 of non-coding proteins increased.QRT-PCR also showed that the proportion of transcripts TIMELESS-205 of non-coding proteins increased significantly after interfering with MEX3A.Preserving the TIMELESS transcript of intron 23 can cause the normal coding region of TIMELESS to contain early termination codons,which destroys the open reading frame and leads to nonsense-mediated decay(NMD).After we interfered with UPF1,the key factor in NMD,the expression of TIMELESS mRNA increased significantly.The NMD pathway depends on protein translation,so translation inhibitor cycloheximide(CHX)inhibits NMD-mediated mRNA degradation.QRT-PCR results showed that the inhibition of NMD by CHX led to the increase of relative expression of TIMELESS mRNA.RIP-qPCR results showed that MEX3A protein could bind to TIMELESS mRNA.SummaryTIMELESS is highly expressed in serious ovarian carcinoma,and MEX3A regulates the expression of TIMELESS mRNA through nonsense-mediated decay(NMD).The MEX3A/TIMELESS axis promotes the malignant biological behavior of ovarian cancer cells.Part IIIStudy on the regulatory mechanism of abnormal overexpression of MEX3A in serous ovarian carcinomaObjectThis part aims to clarify the upstream regulatory molecules of MEX3A,explore the impact of upstream regulatory molecules on the proliferation,invasion and migration of serous ovarian carcinoma,and verify the impact of upstream regulatory molecules on the biological behavior of MEX3A in ovarian cancer.Contents and methodsUse the ENCORI database to predict miRNAs that can bind to MEX3A 3’-UTR.The miRNA data of differential expression between ovarian cancer and normal ovarian epithelium were downloaded from GSE47841(logFC ≤-1).The miRNA was screened by overlapping analysis of the above data sets.The effect of overexpression of these miRNAs on MEX3A mRNA expression was verified one by one through qRT-PCR,and the upstream miRNAs that negatively regulate MEX3A were screened.The expression changes of MEX3A mRNA and protein in ovarian cancer cells transfected with upstream miRNA were verified by qRT-PCR and Western blot.We found the potential binding sites of MEX3A 3’-UTR region and upstream miRNA through the database.The wild type plasmid containing MEX3A binding site was constructed based on pmirGLO,and the wild type potential binding sequence was changed to mutant by deleting the seed sequence.Double luciferase reporting test was used to verify whether miRNA directly binds to MEX3A.MTT proliferation assays,plate cloning and Transwell assays verified the biological function of miRNA in ovarian cancer.We constructed ovarian cancer MEX3 A overexpression cell line by lentivirus transfection,transiently transfected miRNA in MEX3A overexpression cell line,and verified the effect of miRNA on the biological function of MEX3A in ovarian cancer by MTT,plate cloning and Transwell.Results1.Screening and identification of upstream regulatory miRNAs of MEX3A.The ENCORI database predicted that 272 miRNAs could bind to MEX3A 3’-UTR.The miRNA data of differential expression between ovarian cancer and normal ovarian epithelium were downloaded from GSE47841.Overlapping analysis of the two data sets identified seven miRNAs,which were down regulated in ovarian cancer and may bind to 3’-UTR of MEX3A mRNA.The effect of overexpression of seven miRNAs on the expression of MEX3 A mRNA was verified by qRT-PCR.It was found that only miR-532-5p negatively regulated the expression of MEX3A mRNA.The expression of MEX3A mRNA and protein in ovarian cancer cells transfected with miR-532-5p was significantly reduced by qRT-PCR and Western blot.2.Double luciferase report experiment verifies that miR-532-5p can directly bind to MEX3A.The results of double luciferase reporter gene detection showed that the luciferase activity of the two wild type report plasmids co transfected with miR-532-5p mimics in HEK293T cells was significantly reduced,while the luciferase activity of the two mutant report plasmids co transfected with miR-532-5p mimics was not significantly reduced.This indicates that miR-532-5p can specifically bind to the two binding sites of MEX3A3 ’-UTR,and inhibit the expression of MEX3A gene at the post transcriptional level.3.MiR-532-5p inhibits proliferation,migration and invasion of ovarian cancer cells.The results of MTT cell proliferation assays in vitro showed that overexpression of miR-532-5p in SKOV3 and A2780 ovarian cancer cell lines could significantly reduce the proliferation ability of ovarian cancer cells,especially on the first 2-3 days.The results of plate cloning experiment showed that after overexpression of miR-532-5p in SKOV3 and A2780 ovarian cancer cell lines,the number of monoclonal colonies decreased significantly compared with the control group.The results of Transwell showed that SKOV3 and A2780 ovarian cancer cell lines overexpressing miR-532-5p significantly reduced the number of cells penetrating the chamber.The above results indicate that miR-532-5p can inhibit the proliferation,migration and invasion of ovarian cancer cells.4.MiR-532-5p can weaken the ability of MEX3A to promote the proliferation,migration and invasion of ovarian cancer cells.The results of MTT showed that the proliferation ability of MEX3A+miR-532-5p group and PCMV+miR-532-5p group decreased significantly after overexpression of miR-532-5p compared with the control group,and it was more significant from the third day.The results of plate cloning showed that compared with the control group,the number of monoclonal colonies in MEX3A+miR-532-5p group and PCMV+miR-532-5p group decreased significantly after overexpression of miR-532-5p.the results of Transwell assays showed that:compared with the control group,the number of ovarian cancer cells penetrating the chamber in MEX3A+miR-532-5p group and PCMV+miR-532-5p group significantly decreased after overexpression of miR-532-5p.The above results indicate that miR-532-5p partially reverses the effects of MEX3A overexpression on the proliferation,cloning,migration and invasion of ovarian cancer cells.SummaryMiR-532-5p can inhibit the proliferation,migration and invasion of ovarian cancer cells.MiR-532-5p can directly combine with MEX3A and negatively regulate the expression and biological function of MEX3A.Conclusion1.MEX3A is highly expressed in serous ovarian carcinoma,which is related to poor survival and promotes the malignant biological progress of serous ovarian carcinoma.2.MEX3A promotes the occurrence and development of serous ovarian carcinoma by regulating the retention of TIMELESS introns.3.MiR-532-5p negatively regulates MEX3A expression and malignant biological behavior in serous ovarian carcinoma.The innovation of this study1.We found RNA binding proteins are closely related to the development of serous ovarian carcinoma,and confirmed that the high expression of MEX3A is related to the poor prognosis of patients.2.It is the first time to find that MEX3A regulates TIMELESS mRNA expression through nonsense-mediated decay(NMD)to promote the occurrence and development of serous ovarian carcinoma.3.It is confirmed that miR-532-5p can directly and negatively regulate the expression of MEX3A in serous ovarian carcinoma,and it is an upstream regulator of MEX3A.Inadequacies of this study1.There is no study on the effect of MEX3 A on drug resistance of ovarian cancer cells.2.The motif of MEX3A-binding RNA has not yet been clarified,and the molecular mechanism of MEX3A regulating TIMELESS can be further clarified by eCLIP method in the future. |
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