MiR-17-5p Targets YES1 And CHAF1A In Human Ovarian Cancer Cells

[Background]MicroRNAs(miRNAs)are a class of small non-coding RNAs transcripted by RNA polymerase II and III,regulating genes expression by targeting 3’UTR of mRNA.MiRNAs are involved in many physiological and pathological progresses.Evidence indicates that special miRNAs play a critical role in cancer initiation and progression by regulating their target genes.Recent data suggest that up-regulation of miR-17-5p level in human ovarian cancer cells.In this study,we focused on the effects of miR-17-5p on the cell growth activity,cell proliferation ability,apoptosis and cell cycle.We also predicted and confirmed that YES1 and CHAF1A are direct target gene of miR-17-5p and illustrate its molecular mechanism.[Methods]Previous studies indicate that miR-17-5p expressed a high level in ovarian cell.Base on that we observed effect of cell growth activity,cell proliferation ability,apoptosis and cell cycle after over-expressing or blocking miR-17-5p by performing MTT assay,cell growth curve assay,TUNEL and flow cytometry(FACS).Then we predicted several candidates target gene of miR-17-5p by bioinformatics analysis and cDNA Array,confirming them through fluorescent reporter experiment.Real-time PCR and Western Blot will be performed to detect endogenous mRNA and protein level of target gene of miR-17-5p.Finally,the target gene was knocked down using RNA interference and changes of cell phenotypes were detected with MTT assay,cell growth curve assay,TUNEL and FACS.[Results]There are dramatic enhancement of cell activity and cell proliferation ability,the rate of apoptosis declined aslo accelerated the cell cycle from S phase to G2 phase after over-expressing miR-17-5p in both ovarian cell lines.While blocking of miR-17-5p takes the other way round.Bioinformatics analysis indicates that oncogene YES1(V-yes-1 Yamaguchi sarcoma viral oncogene homolog 1)and tumor suppressor gene CHAF1A(chromatin assembly factor 1,subunit A,p150)are candidates of miR-17-5p target gene.The YES1 and CHAF1A mRNA 3’ untranslated region(3’UTR)contains the potential binding site of miR-17-5p.The fluorescent reporter assay also confirmed that miR-17-5p can directly bind to the specific site of YES1 and CHAF1A mRNA 3’UTR,positively regulate the expression of YES 1 and negatively regulate CHAF1A.When miR-17-5p function was overexpressed in human ovarian cancer cells,mRNA level and protein level of YES1 were both enhanced,while mRNA level and protein level of CHAF1A were both depressed.When miR-17-5p function was blocked in human ovarian cancer cells,mRNA level and protein level of YES 1 were both depressed,while mRNA level and protein level of CHAF1A were both enhanced.We also discovered that when the target gene YES1 was knocked down,the cell growth activity and cell proliferation ability were depressed,the rate of apoptosis increased and inhibited the cell cycle from S phase to G2 phase.When the target gene CHAF1A was knocked down,the cell growth activity and cell proliferation ability were enhanced,the rate of apoptosis reduced,and accelerated the cell cycle from S phase to G2 phase.[Conclusions]Our results show that in human ovarian cells,miR-17-5p functions as tumor suppressors and accelerated cell proliferation.The miR-17-5p affected the progression of human ovarian cells by up-regulate YES1 and down-regulate CHAF1A.The elucidation of the mechanisms of miR-17-5p in human ovarian cells helps us to further understand the mechanism of human ovarian initiation and progression,and pave a way for clinical diagnosis and therapy of human ovarian.