The Role And Mechanism Of MiR-424-5p Negatively Targeting CCNE-1 In The Development Of Epithelial Ovarian Cancer

Ovarian cancer(OC)is one of the most common malignant neoplasm in the female reproductive system with a high fatality rate.In 2016,cancer statistics showed that there were 22,280 new cases and 14,240 deaths in the United States.EOC is one of the most common ovarian cancer types,occupying 90%of the whole.If the early detection can find that tumors are still limited to the ovaries surface,about 70 to 90 percent of ovarian cancer patients can be cured in the end.However,due to the lack of specific markers,and atypical clinical symptoms such as abdominal cavity,the early detection of ovarian cancer is very difficuIt.Most patients were diagnosed with advanced stage,leading the overall 5-year survival rate of only 20%to 30%.Therefore,it is urgent to find new targets for early detection and the development and metastatic mechanism of ovarian cancer in order to find new therapeutic strategies just around the corner.MicroRNA(miRNA),is originally double-stranded which is composed of 19-25 nucleotide.In 1993,it was first discovered that miRNA lin-4 can downregulate expression of the protein lin-14 through sequence-specific hybridization to the 3'untranslated region(UTR)of messenger RNAs in Caenorhabditis elegans.It was identified as a small non-protein-coding RNA that are loaded onto the RNA-induced silencing complex(RISC)and induce the degradation or translation blocking of target mRNAs.miRNAs have been identified not only in plants,animals and some viruses,but almost every species,including humans.They are highly conserved,and more evidences have shown that microRNAs are aberrantly expressed in cancers and play an important regulatory role in multiple cancer cellular processes and different tumor onset,including EOC by altering expression of specific targets and fine-tuning of signaling pathways.miR-424-5p is presented in an intragenic miRNA clustered,located on human chromosome Xq26.3 and has been broadened into many types of tumors,such as gastric carcinoma,hepatocellular carcinoma,oral squamous cell carcinoma,esophageal squamous cell carcinoma,cervical carcinoma,pancreatic carcinoma,including ovarian carcinoma.However,its performance acts as oncogenes or tumor suppressors varies in different tissues.It was reported that the expression levels of miR-424-5p were decreased in hepatocellular carcinoma,esophageal squamous cell carcinoma,and cervical cancer,compared to the overexpression in pancreatic cancer.Although genome-wide integrative study of miRNAs expression profiling studies showed that miR-424-5p is downregulated in ovarian carcinoma,the detailed regulation mechanism of miR-424-5p in EOC remains unkown.Therefore,it is necessary to explore its role in epithelial ovarian cancer and find new target genes and regulatory mechanisms to provide a potential molecular targets for the gene therapy of ovarian cancer.The whole research includes the following three chapters:Chapter 1:The expression of miR-424-5p in epithelial ovarian cancer(EOC)and relationship with clinicopathological features of EOC patients.Chapter 2:The preciction and validation of miR-424-5p target gene CCNE1 in EOC.Chapter 3:The roles and preliminary mechanism for miR-424-5p and its target gene-CCNE1 in EOC biological activities.Chapter ? The expression of miR-424-5p in epithelial ovarian cancer(EOC)and relationship with clinicopathological features of EOC patientsBackground and Objective:At present,the molecular mechanism of the origin and progression of epithelial ovarian cancer is not fully understood.dayiThe study of MicroRNA(miRNAs)in cancer has been attracted more and more attention from domestic and foreign scholars,and has been proved to play an key role in ovarian cancer.miR-424-5p plays different roles in kinds of cancers,and its role and mechanism in the development of epithelial ovarian cancer have been rarely reported so far.This study is to test expression of miR-424-5p in both epithelial ovarian cancer tissues and cell lines,and to analyze the relationship between the clinical pathologic factors and prognosis in ovarian cancer in order to explore the possibility for miR-424-5p being as a new target in early diagnosis of the epithelial ovarian cancer.Materials and methods:Tissues,including 83 cancer cases(experimental group)and 19 normal ovarian epithelial cases(control group)were collected and confirmed by surgery and postoperative pathology in Qilu hospital of Shandong university.Each specimen was divided into two parts,one for real-time fluorescent quantitative PCR(qRt-PCR),one for immunohistochemistry of CCNE1 in the second part.None of the patients enrolled in the study received chemoradiotherapy before surgery.1.Real-time fluorescence quantitative PCR mothed was used to detect miR-424-5p relative expression in different ovarian cancer tissues and normal ovarian tissues,meanwhile,the relationship between miR-424-5p and clinicopathological parameters were analyzed.2.The survival curve was plotted by Kaplan-Meier survival analysis,and the correlation between miR-424-5p and the two indexes,the overall survival(OS)and the recurrence-free-survival(RFS)were analyzed for the prognosis of ovarian cancer.3.miR-424-5p expression in 3 different ovarian cancer cell lines HO8910,A2780,SKOV3 and normal ovarian epithelial cell line HOSEpiC were detected by real-time fluorescence quantitative PCR.Result:1.qRt-PCR Result showed that the relative expression of miR-424-5p in epithelial ovarian cancer tissues(0.3649±0.2709)was significantly lower than normal tissues(1.00000±0.1711,**p<0.01).Compared with 27 low grade serous epithelial ovarian cancer,miR-424-5p expression in 44 high grade serous epithelial ovarian cancers was significantly decreased(**p<0.01).Low miR-424-5p expression was strongly associated with poor differentiated pathological grade(p = 0.0334),advanced FIGO stage(p = 0.0036),residual tumor size(p = 0.0174),and lymphatic metastasis(p = 0.0398).However,we did not observe any obvious links between miR-424-5p levels and patients' ages,ascites and serum CA125 levels(all*p>0.05).2.Kaplan-Meier survival analysis revealed that downregulate miR-424-5p was remarkably associated with poor prognosis in 83 EOC patients using overall survival(OS)(p = 0.012)and recurrence-free survival(RFS)(p = 0.020).3.qRt-PCR Result showed that miR-424-5p expression in ovarian cancer cell lines HO8910,SKOV3,A2780 were all declined,compared with normal ovarian epithelial cell lines HOSEpiC(1.0000±0.04433)vs(0.6382±0.0437),(0.6382±0.0437),(0.2975±0.01251)(**p<0.01).Conclusion:miR-424-5p was significantly decreased in both epithelial ovarian cancer tissue and cell lines than in the normal controls.Meanwhile,miR-424-5p was greatly associated with differentiated pathological grade,TNM stage,lymphatic metastasis,residualtumor size.The OS and RFS of high miR-424-5p expression patients were greatly higher than those of low miR-424-5p expression patients,which suggested that miR-424-5p down regulation may play an important role in the process of occurrence and development,and the prognosis of epithelial ovarian cancer.Chapter ? The preciction and validation of miR-424-5p target gene CCNE1 in EOC.Background and Objective:miRNAs are those that single-stranded,endogenous non-coding small RNAs of 22 nt length which are capable of regulating gene expression.It can selectively inhibit the expression of target proteins through complementary.The studies have found that each mammalian miRNA may regulate more than 200 target genes.So far,although a lot of efforts have been consumed,the function of majority of miRNAs still remains unkonwn.The reason accounts for this may be the difficulty in the prediction and identification of target genes.Up to date,many public databases based on different algorithms can help to predict the seed binding sites of miRNAs,which are the most conserved fragments of miRNA from the 2nd to 8th nucleotides complementary targeting its genes 3'-UTR through the evolution.In Chapter 1,we have found that miR-424-5p is down-regulated in epithelial ovarian cancer,suggesting that miR-424-5p may be involved in the development of ovarian cancer as a tumor suppressor gene.So,which target gene does miR-424-5p exert its biological function in ovarian cancer?Therefore,in this chapter we hope to increase the success rate of target gene prediction by using the common intersection of multiple bioinformatics algorithms.According to the different miR-424-5p expression levels in different cell lines in the first chapter,appropriate cell lines were selected to to be transfected with miR-424-5p mimics or inhibitor,and the dual luciferase reporter gene system were conducted to verify the accuracy of the forecast results.Materials and methods:1.Three bioinformatics algorithms(miRDB,Targetscan and miRanda)were used to select the intersection of top 50s for the initial screening.Co-transfection miR-424-5p with mimics A2780 cells and miR-424-5p inhibitors with HO8910 cells to overexpresse or reduce miR-424-5p expression,transfection efficiency was predicted by qRT-PCR to verify these three common target genes(SLC9A6,AN03 and CCNE1).2.3'-UTR luciferase plasmid pGL3-CCNEl was conducted and co-transfect A2780 cells or HO8910 cells with the miR-424-5p mimic or inhibitor to observe the wild group(WT)and mutations.compared luciferase activity.3.qRT-PCR and Western Blot were used to analyze differences for CCNE1 expression in both mRNA and protein level.4.CCNE1 expression was measured by immunohistochemistry(IHC)and qRT-PCR to further verify the correlation between CCNE1 and miR-424-5p in epithelial ovarian cancer.Result:1.SLC9A6,AN03 and CCNE1 were the intersection of top 50s predicted common target genes through bioinformatics algorithms(miRDB,Targetscan and miRanda)2.miR-424-5p mimics,inhibitor and its control groups were transfected into A2780 and HO8910 cells respectively,after 4 hours up to90%positive green fluorescence cells can be seen by fluorescence microscopy.qRT-PCR analysis showed that compared with miR-NC group,miR-424-5p expression in miR-424-5p mimics transfected A2780 significantly increased to 7.559±0.428 folds(if normalized miR-NC group as 1)(**P<0.01);While in miR-424-5p inhibitor transfected HO8910 cells,miR-424-5p expression was significantly down-regulated to 0.558 ± 0.060 folds(**P<0.01).3.qRT-PCR analysis showed that SLC9A6 and AN03 mRNA levels didn't significantly change in both miR-424-5p mimics transfected A2780 cells and miR-424-5p inhibitors transfected HO8910 cells,compared with its control groups.Western Blot and qRT-PCR analysis both showed that CCNE1 mRNA and protein levels were significantly downregulated(1.00±0.077 vs 0.384±0.002),(1.001 ±0.019 vs 0.62±0.019)in miR-424-5p mimics transfected A2780 cells,While its expressions were significantly up-regulated in miR-424-5p inhibitor transfected HO8910 cells(1.001 ±0.057 vs 1.815±0.049)and(1.001 ±0.063 vs 1.422±0.034).P<0.01).4.The sites of miR-424-5p and target gene CCNE1 are located at 247-254 and 485-492,respectively.The dual luciferase reporter assay results showed the fluorescence intensity of the 3'UTR-WT co-transfection group(miR-424-5p inhibitor+pGL-CCNE-1)in HO8910 cells were significantly stronger than the inhibitor NC control group(**p<0.01).The fluorescence intensity of the 3'UTR-WT co-transfection group(miR-424-5p mimics+pGL-CCNE-1)in A2780 cells were significantly weaker than in the NC control group(**p<0.01).However,for the mutant vector(pGL-CCNE-1 3'UTR-MU),there was no significant difference between the two groups of fluorescence intensity(*p>0.05).5.IHC data showed that CCNE1 expressed in both epithelial ovarian cancer and normal ovarian epithelium in varying degrees,and mainly located in cytoplasm,and nucleus with brownish-yellow staining.CCNE1 was overexpressed in the epithelial ovarian cancer,compared with low expression or no expression in the normal ovary epithelial cells.qRt-PCR data indicated CCNE1 in epithelial ovarian cancer tissues was significantly higher than in normal ovarian epithelial tissues(3.187 ± 0.1519 vs 1 ± 0.2219,**p<0.01).6.The higher CCNEI of IHC score,the lower miR-424-5p expression(**p<0.01),qRT-PCR results showed that CCNE1 relative expression were significantly higher in low miR-424-5p expression patients.There was a significant negative correlation between miR-424-5p and CCNE1 in EOC(**p<0.01)Conclusion:1.CCNE1 was the target gene of miR-424-5p in EOC.2.miR-424-5p can affect CCNE1 mRNA and protein expression level by regulating CCNE1 3'-UTR3.miR-424-5p was negatively correlated with CCNE1 in EOC.Chapter III The roles and preliminary mechanism for miR-424-5p and its target gene CCNE1 in EOC biological activities.Background and Objective:The cell growth is based on cell cycle.Both the external environment and activation of intracellular oncogenes or inactivation of tumor suppressor genes trigger a cascade reaction to induce cell proliferation.miRNAs acte as oncogenes and tumor suppressor genes to participate in the regulation of cell cycle progression.In chapter 2,we predicted and identified that CCNE1 was miR-424-5p target gene in the epithelial ovarian cancer.CCNE1 is a cell cycle-associated protein and expressed during the cell cycle which plays an important role during cell's transition from the G1 phase to the S phase.It is encoded by cell cycle regulatory protein El and then interacted with the cell protein-dependent kinase 2(CDK2)to form a complex which promoted cell's ability to regulate mitosis in eukaryotic cells through the G1/S restriction point in the cell cycle.Under normal circumstances,CCNEI conducted nuclear fission in an orderly manner.In abnormal circumstances,unstable expression can lead to uncontrolled activities,interfering with cell mitosis which lead to chromosome instability,and promote tumor growth.In this chapter,we observed the effect of miR-424-5p on the biological behavior,in particular,the effects of cell growth,cycle,and apoptosis.We tried to explore the new mechanism of miR-424-5p in EOC.Materials and methods:1.Western Blot was used to detect small interference CCNE1 and pcDNA3.1-CCNE1 plasmid to knock down and overexpress CCNE12.Transfection miR-424-5p mimics with A2780 cells and miR-424-5p inhibitors with HO8910 cells to overexpresse or reduce miR-424-5p expression,transfection efficiency was examined by qRT-PCR to verify these three common target genes(SLC9A6,AN03 and CCNE1),and CCK-8 analysis was used to find miR-424-5p effect on cell proliferation.Co-transfected si-CCNE1 and miR-424-5p inhibitor or pcDNA3.1-CCNE1 plasmid and miR-424-5p mimics into A2780 or HO8910 were conducted to observe the effect of miR-424-5p on cell proliferation.3.Flow cytometry was used to observe the effect of miR-424-5p on cell cycle and apoptosis when miR-424-5p was overexpressed or downregulated,and roles of CCNE1 on cell cycle.4.Western Blot was used to analyze E2F1 and pRb expression.Result:1.Western Blot results showed that CCNE1 expression in HO8910 cells transfected with si-CCNE1 was significantly lower,compared with si-NC control group(0.022±0.0009 folds,**p<0.01).Compared with empty vector group,CCNEl expression was significantly higher in A2780 cells co-transfected with pcDNA3.1-CCNE1 plasmid(1.428± 0.2034 folds,**p<0.01).2.CCK-8 results showed that cell viability at 48-96hours were significantly decreased in miR-424-5p mimics transfected A2780 cells compared with its NC groups,0.5731± 0.041 vs 0.396±0.042,1.030±0.054 vs 0.626±0.052,1.453±0.045 vs 0.939±0.056;Co-transfected pcDNA3.1-CCNE1 plasmids and miR-424-5p mimics into A2780 cells,48-hour OD value was significantly increased,0.308±0.025.Vs0.469±0.042,72-hour OD was 0.478±0.026 vs 0.744±0.055,96-hour OD was 0.739±0.046 vs 1.052±0.069,compared to the empty plasmid group.Compared with inhibitor NC group,miR-424-5p inhibitor transfected H08910 cells after 48-96 hours showed a significant increase in cell viability(429±0.031 vs 0.57±0.032 and 0.615±0.042 vs 0.842±0.043,1.020±0.041vs 1.545±0.047).Co-transfected si-CCNE1 and miR-424-5p inhibitor into HO8910 cells,cell viability was significantly decreased(*p<0.05,**p<0.01),48hours?72hours?96hours OD were 0.637 ± 0.048,0.989 ±0.070,vs0.455 ± 0.029,compared with si-NC controls(0.687 ± 0.049,0.744±0.055,1.052±0.069.),3.Flow cytometry results showed that miR-424-5p up-regulation in A2780 arrests cell cycle in G0/G1 phase,and miR-424-5p downregulation promotes transition from GO/G1 phase to S phase in H0 8910,compared with the corresponding controls(G0/G1 phase:57.32%±0.286%vs 72.883%±1.419%,S phase:34.62%± 0.563%vs 24.873%±0.86%).Compared with NC group(G0/G1 phase:63.923%±0.309%,S phase:30.162%±0.57%),G0/G1 phase in miR-424-5p inhibitor transfected HO8910 cells was significantly decreased(55.23%±1.367%),S phase was significantly increased(38.97%± 1.302%),(*p<0.05,**p<0.01).The results of the recovery experiment showed that the co-transfected pcDNA3.1-CCNE1 plasmid can resist the blocking effect of miR-424-5p on G0/G1 phase;co-transfected si-CCNEl can reverse the miR-424-5p inhibitor on cell cycle from G0/G1 to S phase.We didn't see obvious changes in up or down-regulation of miR-424-5p expression in both miR-424-5p overexpressed A2780 or miR-424-5p down-regulated HO8910 cells in cell apoptosis(p =0.1173,0.0596).4.Western Blot results showed that compared with control group,E2F1 and pRb expression were significantly decreased in miR-424-5p mimics transfected A2780 cells(1.0±0.047 vs 0.416±0.013,1.0±0.026 vs 0.81 ±0.039).Recovery experimentally co-transfected pcDNA3.1-CCNE1 plasmid could reverse the down-regulation of E2F1 and pRb expression level(.416±0.013 vs.0.72±0.057,0.81 ±0.039 vs 1.147±0.032).Compared with the inhibitor NC control group,the E2F1 and pRb expression were significantly increased in HO8910 cells transfected with miR-424-5p inhibitor(1.0±0.043 vs 1.25±0.041,1.0±0.112 vs 1.582±0.069).Silencing CCNE1 attenuated upregulation of E2F1 and pRb expression(1.25±0.041 vs 0.419±0.041,1.582±0.069 vs 0.866±0.112).Conclusion:1.miR-424-5p can inhibit proliferation in EOC;2.CCNE1 overexpression can promote cell proliferation in EOC,and can block miR-424-5p inhibition effect on cell proliferation;3.miR-424-5p inhibits cell proliferation in4.epithelial ovarian cancer by targeting the CCNEl/E2F1/pRb pathway.