The Study On The Biological Function And Related Mechanism Of RECQL4 In Serous Ovarian Cancer

BackgroundOvarian cancer is the most lethal gynecological malignant tumor.According to the statistics of the International Agency For Research On Cancer(IARC),there were 295414 new ovarian cancer cases and 184799 deaths in the world in 2018,ranking fifth in the cause of cancer death in the United States.Even if the response to initial treatment is good,70%of patients will relapse within 3 years,and the 5-year survival rate is still less than 50%.Late diagnosis,high recurrence rate and drug resistance are the main causes of high mortality in patients with ovarian cancer.According to histological types,epithelial ovarian cancer can be divided into four subtypes:mucinous,serous,clear cell and endometrioid.Serous ovarian cancer is the most common pathological type.The standard treatment is tumor cell reduction surgery and platinum-based chemotherapy and maintenance therapy.Most platinum-sensitive patients will eventually become platinum-resistant.Studies have shown that DNA repair pathway is involved in the formation of chemotherapy resistance.Serous ovarian cancer can be divided into high grade and low grade according to histological grade,and different subtypes have different molecular genotypes.The most common mutation gene in high-grade serous ovarian cancer is that about 50%of patients with TP53,have homologous recombination defects,and defects in NOTCH,RAS/MEK,PI3K and FOXM1 signal pathways are the most common signal transduction pathways.The frequency of BRAF and RAS mutations increased in low-grade serous ovarian cancer,and the genome was relatively stable.The whole-genome analysis of ovarian cancer published by the American Cancer Genome Map(TheCancerGenomeAtlas,TCGA)project shows that 96%of high-grade serous tumors have TP53 mutations,while the mutation frequency of other genes is much lower than that of TP53,such as BRCA1,which has a mutation rate of about 12.5%.The PTEN mutation rate is only 7%.In contrast,gene copy number mutations were more frequent,accounting for 23.1%(113max 489).The copy number amplification ratio of the most common ovarian cancer driving genes,such as MYC,CCNE1 and MECOM,is more than 20%.RECQL4 belongs to the DNA helicase family,which regulates the replication,transcription and recombination of DNA,maintains the stability of telomeres and genomes,and widely participates in many DNA repair pathways,such as homologous recombination,non-homologous terminal connection,base excision repair and so on.Recent studies have shown that RECQL4 plays a "double-edged sword" role in the occurrence and development of tumors.RECQL4 deficiency leads to an increase in the incidence of osteosarcoma or lymphoma.on the contrary,an increase in the expression of RECQL4 was observed in prostate,breast and hepatocellular carcinoma.However,the expression and biological function of RECQL4 in ovarian cancer have not been reported.The purpose of this research was to investigate the expression,function and clinical significance of RECQL4 in serous ovarian cancer,and further analyze the reasons for the high expression of RECQL4.By expounding the key downstream target genes and upstream regulatory molecules in which RECQL4 plays an important role in the role of oncogenes,it is proposed that miR-10a-5p/RECQL4/MAFB axis plays an important role in the diagnosis,occurrence,development,treatment and prognosis of serous ovarian cancer.The specific research content includes the following four parts:1.Study on the expression,biological function and clinical significance of RECQL4 in serous ovarian carcinoma.2.Study of the effect of RECQL4 on platinum and PARP inhibitor sensitivity of serous ovarian cancer cells.3.Study on the identification and biological functions of the downstream target gene of RECQL4 in serous ovarian cancer.4.Study on the upstream molecular regulation mechanism of RECQL4 in serous ovarian cancer.Part I:Study on the expression,clinical significance and biological function of RECQL4 in serous ovarian carcinoma.ObjectRECQL4 helicase not only participates in DNA replication,recombination,transcription and repair,and maintains genomic stability,but also participates in the occurrence and development of many malignant tumors such as prostate cancer,breast cancer and gastric cancer,and is related to the poor prognosis of tumors.Based on the biological information analysis results of TCGA and GEO database,this part intends to explore the difference of RECQL4 expression between serous ovarian cancer and normal fallopian tube fimbria tissues.The effect of RECQL4 on the prognosis and clinicopathological features of serous ovarian cancer was evaluated by immunohistochemical staining of tissue microarray combined with the clinical data of patients with microarray.Finally,the effects of RECQL4 expression on the proliferation,invasion,migration,cycle and apoptosis of ovarian cancer cells were evaluated by tumor formation in nude mice in vivo and cytological experiments in vitro.Contents and methodsTCGA data were used to analyze the copy number variation in high-grade serous ovarian cancer and pan-cancer,as well as the copy number variation of RECQL4 in high-grade serous ovarian cancer.Furthermore,the database was used to analyze the effect of copy number variation of RECQL4 on its mRNA expression.TCGA and GEO database were used to analyze the difference of RECQL4 expression between serous ovarian cancer and normal ovary and fallopian tube fimbria tissue.qRT-PCR was used to detect the difference of RECQL4 mRNA expression between serous ovarian cancer and normal fallopian tube fimbria tissue,and Western Blot was used to detect the difference of RECQL4 protein expression between ovarian cancer cell line and normal fallopian tube fimbria epithelial cell.The tissue microarray of serous ovarian cancer constructed by our group was used for immunohistochemical staining to determine the expression of RECQL4 protein,and to check the relationship between clinical prognosis and its expression,and its correlation with clinicopathological features.Kaplan-Meier Plotter website was used to analyze the relationship between RECQL4 and PFS and OS in serous ovarian cancer.Ovarian cancer cell lines with stable overexpression and inhibitory expression of RECQL4 were constructed.The effect of RECQL4 on the proliferation of ovarian cancer cells was verified by CCK8 proliferation test,plate clone formation test in vitro and subcutaneous tumor formation in nude mice in vivo.The effects of RECQL4 on the invasion and migration of ovarian cancer cells were compared by Transwell test,scratch test in vitro and tumor formation in abdominal cavity of nude mice in vivo.The effect of RECQL4 on cell cycle and apoptosis of ovarian cancer cells was detected by flow cytometry.The effects of RECQL4 on epithelial-mesenchymal transformation,cell apoptosis and cell cycle related protein molecules of ovarian cancer cells were detected by Western Blot.ResultsTCGA data analysis showed that copy number variations occured in 100%of high-grade serous ovarian cancer and 97.8%of pan-cancer,and 19 of the top 20 genes were the same.RECQL4 showed abnormal copy number amplification in 27%of high-grade serous ovarian,cancer samples,and RECQL4 copy number amplification was positively correlated with its mRNA expression.TCGA data analysis showed that RECQL4 was highly expressed in more than 20 kinds of malignant tumors.GSE26712 and TCGA database analysis showed that the expression of RECQL4 in serous ovarian cancer was greater than that in normal fallopian tube fimbria tissue and ovary.The results of qRT-PCR showed that the expression of RECQL4 in high-grade serous ovarian carcinoma was higher than that in normal fallopian tube fimbria tissue.Western Blot showed that the expression of RECQL4 protein in ovarian cancer cell lines was obviously higher than that in normal fallopian tube epithelial cells.The results of immunohistochemical staining of tissue microarray consisting of 157cases of serous ovarian cancer and 54 cases of normal fallopian tube tissue showed that the overall survival time of patients with high expression of RECQL4 was significantly lower than that of patients with low expression of RECQL4.Kaplan-Meier Plotter website analysis showed that the overall survival and progression free survival of sufferer with high expression of RECQL4 were shorter than those with low expression of RECQL4.The expression of RECQL4 was associated with blood CA125 level,ascites and omental metastasis,but not with age,FIGO stage and lymph node metastasis.And the expression level of RECQL4 in platinum-resistant ovarian cancer patients was remarkably higher than that in platinum-sensitive patients.BRCA wild type ovarian cancer cell lines A2780,HEY,SKOV3 and BRCA deletion cell line UWB 1.289,were selected to construct cell lines with stable interference and overexpression of RECQL4.The results of CCK8 proliferation test,clone formation test,Transwell and scratch test showed that the ability of proliferation,invasion and migration of cells after overexpression of RECQL4 was significantly higher than that of the control group,while the ability of proliferation,invasion and migration of cells after interfering with the expression of RECQL4 was significantly lower.The abdominal tumorigenesis ability and subcutaneous tumorigenesis test of nude mice showed that the number and size of metastatic nodules formed in pelvic and abdominal cavity and the volume and weight of subcutaneous tumor formation in the group of high expression of RECQL4 were higher than those in the control group.The epithelial-mesenchymal transformation-related proteins detected by Western Blot showed that after interfering with the expression of RECQL4,the expression of stromal phenotypic markers N-cadherin,β-Catenin,Vimentin,Slug,Snail and MMP7 was down-regulated,while the corresponding interstitial phenotypic markers were significantly up-regulated after overexpression of RECQL4.Cell cycle analysis by flow cytometry showed that after interfering with the expression of RECQL4,the proportion of G1 phase of ovarian cancer HEY and A2780 cells raised signally,while the proportion of S phase decreased.Correspondingly,apoptosis analysis showed that reducing the expression of RECQL4 could increase the number of early and late apoptotic ovarian cancer cells.Western Blot detection of cell cycle-related proteins showed that after interfering with the expression of RECQL4,the expression of CCND1,CCNE1,CDK4 and CDK6 decreased,while the expression of cell cycle arrest proteins P21,P27 and p53 increased.Overexpression of RECQL4 had the opposite result.The detection of Western Blot showed that the apoptosis-related proteins Bax,Cleaved-caspase9,and Cleaved-caspase3 were significantly up-regulated after interfering with the expression of RECQL4,while the expression of corresponding proteins was down-regulated after overexpression of RECQL4.ConclusionRECQL4 is highly expressed in serous ovarian cancer,which is related to its poor clinical prognosis and is expected to become a new prognostic marker of serous ovarian cancer.RECQL4 promotes the proliferation,invasion and migration of serous ovarian cancer;interference with RECQL4 can cause cell cycle G1/S phase arrest,and lead to an increase in the number of early apoptotic and late apoptotic cells,.So RECQL4 is prospected to become a new drug target for the treatment of ovarian cancer.Part Ⅱ:Study of the effect of RECQL4 on platinum and PARP inhibitor sensitivity of serous ovarian cancer cells.ObjectThe standard treatment of epithelial ovarian cancer is cytoreductive surgery plus adjuvant chemotherapy and maintenance therapy.Platinum and paclitaxel is the first choice for adjuvant chemotherapy.Although 80%of ovarian cancer patients respond well to first-line chemotherapy,about 70%of patients will relapse,and most of platinum-sensitive relapse will eventually progress to platinum-resistant relapse.The application of PARP inhibitors is the biggest progress in the treatment of ovarian cancer in recent years.A variety of PARP inhibitors have been recommended by the NCCN guidelines for ovarian cancer,but the problem of drug resistance has gradually emerged.There has been evidence that abnormal DNA repair in epithelial ovarian cancer is involved in response to chemotherapy and drug resistance.In view of the fact that RECQL4 plays an important role in multiple DNA repair pathways,and the immunohistochemical staining of tissue microarray shows that the expression of RECQL4 in platinum-resistant patients is significantly higher than that in platinum-sensitive patients,the purpose of this part is to study the effect of RECQL4 expression on cisplatin and olapalide sensitivity of serous ovarian cancer cells in vitro.Contents and methodsThe protein expression of RECQL4 in ovarian cancer A2780 cells and platinum-resistant A2780/DDP cells was detected by Western Blot.Western Blot was used to identify the alter of RECQL4 protein level in ovarian cancer cells following different concentrations of cisplatin and olaparib.BRCA wild-type ovarian cancer cell lines A2780,HEY and SKOV3 with low expression of RECQL4 and their control groups were selected.RECQL4 siRNA was used to transiently transfect platinum-resistant A2780/DDP cells and BRCA mutant UWB 1.289 cells to reduce the expression of RECQL4.The ovarian cancer cells obtained above were treated with different concentration gradients of cisplatin and olaparib.CCK8 proliferation test was used to detect the growth activity of ovarian cancer cells after adding different concentrations of cisplatin and olaparib,and plate clone formation test was used to detect the changes of ovarian cancer cell clone formation ability.ResultsThe results of Western Blot detection showed that the expression of RECQL4 in platinum-resistant A2780/DDP cells was significantly higher than that in A2780 cells.The protein expression level of RECQL4 in ovarian cancer cells increased with the increase of cisplatin and olaparib concentration.The growth status of cells was observed under microscope.It was found that the cells died in different degrees after adding cisplatin and olaparib,and the number of dead cells increased gradually with the increase of drug concentration.The results of CCK8 proliferation assay showed that the IC50 of ovarian cancer cells with low expression of RECQL4 was significantly lower than that of control cells after adding different concentrations of cisplatin and olaparib.The clone formation experiment showed that the number of clones decreased with the increase of cisplatin and olaparib concentrations.At the same drug concentration,the number of clones formed by the cells with low expression of RECQL4 was less than that of the control group.ConclusionIn ovarian cancer cells,interfering with the expression of RECQL4 can enhance the sensitivity of cisplatin and olaparib.RECQL4 can be used as a biomarker to predict the chemo-sensitivity of serous ovarian cancer and a potential molecular target for tumor therapy.Part Ⅲ:Study on the identification and biological functions of the downstream target gene of RECQL4 in serous ovarian cancer.ObjectThe structure of RECQL4 protein is different from other human RECQ helicases.At present,it is not clear which functional domains of RECQL4 affect its role in tumors.Understanding the interacting proteins of RECQL4 plays an important role in inference of its function in cancer cells,understanding of its mechanism,and influence in the early diagnosis and treatment of tumors.Although it has been found that RECQL4 interacts with TP53,MCM10 and survivin in different tumors,malignant tumors are highly heterogeneous and the downstream target genes of RECQL4 in serous ovarian cancer have not been fully elucidated.The purpose of this part is to screen the key downstream target genes of RECQL4 in serous ovarian cancer by high-throughput transcriptome sequencing,and to explore the biological function of the target gene in ovarian cancer.Contents and methodsOvarian cancer A2780 cells were transiently transfected with RECQL4 siRNA to reduced RECQL4 expression.RECQL4 mRNA was extracted and qRT-PCR was used to verify the transfection efficiency.The downstream target genes that may be regulated by RECQL4 in serous ovarian cancer were analyzed by high throughput sequencing(RNAseq).qRT-PCR was used to verify the mRNA level of downstream target genes obtained by RNAseq.TCGA database and qRT-PCR were used to analyze the expression differences of downstream target genes in serous ovarian cancer and normal fallopian tube fimbria tissues.The correlation between the changes of target gene mRNA and RECQL4 mRNA in serous ovarian cancer was verified by qRT-PCR The protein expression of target gene after change of RECQL4 protein was detected by Western Blot,and finally the target gene was selected for study.The small interference RNA of the target gene was transiently transfected into ovarian cancer cells to interfere the expression of the target gene.The effect of the target gene on the proliferation of ovarian cancer cells was detected by CCK8 proliferation test and colony formation assay,and the invasive ability was detected by Transwell assay.siRNA was used to interfere the expression of target gene in stablely high expression of RECQL4 and its control cells,then Rescue experiment was carried out.CCK8 proliferation test,plate cloning test and Transwell test were used to distinguish the proliferation,invasion and migration of ovarian cancer cells promoted by RECQL4 after reducing the expression of target gene.ResultsThe results of high-throughput sequencing transcriptome analysis showed that there were 302 differentially expressed genes,including 179 up-regulated genes and 123 down-regulated genes.The candidate target genes were verified by qRT-PCR.It was found that the mRNA expression levels of CBLN2,MAFB,ITGB3,PGPEP1L,GPR78,SKAP2,AHRR,IGFBP3 and AREGB decreased after interfering with RECQL4 expression.Through the analysis of the expression of the above genes in serous ovarian cancer by TCGA data,it was found that the expression of MAFB was obviously higher than that in normal fallopian tube.qRT-PCR detection showed that the expression of MAFB in 40 cases of serous ovarian carcinoma was higher than that in 12 normal fallopian tube fimbria tissues,and the mRNA expression level of MAFB was positively correlated with the mRNA expression level of RECQL4.Western Blot assay showed that the expression of MAFB protein was up-regulated after overexpression of RECQL4,while it was down-regulated after interfering with the expression of RECQL4.The cytological function of MAFB was studied,and the proliferation experiment of CCK8 showed that the growth rate of ovarian cancer cells was significantly lower than that of the control group after reducing the expression of MAFB.Plate colony formation assay showed that the number of ovarian cancer cells with low expression of MAFB was evidently fewer than the control group.Transwell assay showed that after decreasing the expression of MAFB,the count of trans-membrane cells decreased significantly.Rescue experiment found that,CCK8 proliferation test and plate cloning test showed that reducing the expression of MAFB could weaken the ability of RECQL4 to promote the proliferation of ovarian cancer cells.Transwell assay showed that decreasing the expression of MAFB could reverse the enhancement of invasion and migration caused by overexpression of RECQL4.ConclusionMAFB is highly expressed in serous ovarian cancer and can promote the proliferation and metastasis of ovarian cancer cells.Decreasing the expression of MAFB can weaken the ability of RECQL4 to promote the proliferation and metastasis of ovarian cancer cells.RECQL4 plays a tumor-promoting role by regulating MAFB in serous ovarian cancer.Part Ⅳ:Study on the upstream molecular regulation mechanism of RECQL4 in serous ovarian cancer.ObjectWhile RECQL4 plays a role in promoting cancer by regulating downstream target genes,it is also regulated by a variety of factors.In the first part of this study,it has been confirmed that the amplification of RECQL4 gene copy number can lead to its high expression.microRNA is a large family of non-coding RNA which can negatively regulates the expression of target genes at the post-transcriptional level.In recent years,more and more attention has been paid to the role of microRNA in the occurrence and development of ovarian cancer.In order to explore the upstream regulatory molecules of RECQL4 in serous ovarian cancer,this part uses the miRNA prediction database to screen the upstream miRNA,that may regulate the expression of RECQL4,so as to provide further basis for explaining the mechanism of RECQL4 in ovarian cancer.Contents and methodsBased on the results of microRNA target gene prediction database TargetScan7.1 and miRDB,the upstream miRNA which may regulate the expression of RECQL4 was selected.The mRNA expression level of upstream regulatory molecules was detected by qRT-PCRWestern Blot assay was used to detect the protein level of RECQL4 in ovarian cancer cells after overexpression of miRNA.qRT-PCR assay was used to detect the changes of RECQL4 mRNA level after overexpression and inhibition of miRNA expression.The database TargetScan7.1 looked up the possible binding sites between RECQL4 and upstream regulatory molecules,constructed wild type and mutant type pmirGLO vectors,and carried out double luciferase reporter gene experiments to verify whether the selected miRNA was directly bound to RECQL4.In the ovarian cancer cells with stablely high expression of RECQL4 and the control group,mimics of miRNA was transiently transfected.CCK8 proliferation test,plate cloning test and Transwell assay were used to detect the effect of miRNA on the proliferation and invasion of ovarian cancer cells,and the effect of over-expression of miRNA on the proliferation and metastasis of ovarian cancer cells promoted by RECQL4.ResultsBased on the online prediction results of microRNA target gene prediction database TargetScan7.1 and miRDB,miR-10a-5p was selected as the research object.qRT-PCR assay showed that the expression of miR-10a-5p in serous ovarian carcinoma was lower than that in normal fallopian tube fimbria tissue.Western Blot assay showed that the protein expression of RECQL4 decreased after overexpression of miR-10a-5p in ovarian cancer cells.qRT-PCR experiment showed that the mRNA expression of RECQL4 decreased after overexpression of miR-10a-5p,while the inhibition of miR-10a-5p expression led to the increase of RECQL4mRNA level.TargetScan7.1 database found that there were sites in the 3’UTR region of RECQL4 that could directly bind to miR-10a-5p.The results of double luciferase report showed that the relative luciferase activity of miR-10a-5p over-expression group was significantly lower after transfected of wild type vector.After the binding site on the vector was mutated,the relative luciferase activity of the over-expressed miR-10a-5p group was not significantly different from that of the control group.In SKOV3 cells with stable high expression of RECQL4 and control group,transient transfection of mimics overexpressed miR-10a-5p.The results of CCK8 proliferation assay,plate cloning test and Transwell assay showed that over-expression of miR-10a-5p inhibited the proliferation and invasion of ovarian cancer cells.In addition,the over-expression of miR-10a-5p could weaken the ability of RECQL4 to promote the proliferation and metastasis of ovarian cancer cells.ConclusionMiR-10a-5p is down-regulated in serous ovarian cancer and can inhibit the proliferation,invasion and migration of ovarian cancer cells.MiR-10a-5p can directly bind to RECQL4 and negatively regulate its expression and biological function.The innovation of this study1.This study systematically analyzed the expression,biological function and clinical significance of RECQL4 in serous ovarian cancer.2.It is confirmed for the first time that interfering with RECQL4 can enhance the sensitivity of ovarian cancer cells to cisplatin and olaparib,which is a potentially drug target for ovarian cancer.3.It is the first time to confim that RECQL4 plays a tumor-promoting function by regulating MAFB in serous ovarian cancer.4.It is clear that miR-10-5p can directly negatively regulate the expression of RECQL4 in serous ovarian cancer.The unusual expression of RECQL4 is related to its copy number amplification and miR-10a-5p.The limitation of this study1.In this study,we only analyzed the effects on the sensitivity of cisplatin and olaparib respectively after inhibit the expression of RECQL4,but there was no analysis of the effect of cisplatin and olaparib combination,nor was it verified by in vivo models such as PDX model.2.In this study,we found that RECQL4 can promote cancer by regulating MAFB,but it has not been proved that RECQL4 protein is directly related to MAFB protein.