MiR-101’s Regulative Effect On Cellular Behaviors Of Cervical Cancer Hela Cells By Targeting C-Fos

ObjectiveMicroRNAs are a series of short single-stranded non-coding RNAs thatfunction in post-transcriptional regula tion of gene exp ression and playsignificant roles in biological progresses. The chief objective of this study isto verify the microRNAs targeting c-Fos (a proto-oncogene commonlyover-expressed in cervical cancer) and explore underlying impacts on thecellular behaviors in cervical cancer.Materials and MethodsWe initially analyzed the3’UTR of c-Fos and predicted the targetingmicroRNAs using three bio-informatical websites： pictar(http://www.pictar.org), miRanda (http://microrna.org) and TargetScanrelease6.2(http://www.targetscan.org). It was predicted that many miRNAscan potentially regulate c-Fos through a post-transcriptional way. After that,we selected miR-101as the candidate to do further research. In order toexplore whether miR-101can bind to the3’UTR of c-Fos mRNA directly,the dual-luciferase reporter assay was conducted. After confirmation ofmiR-101that could post-transcriptionally regulate c-Fos, t h e mimic o fmiR-101was transfected into HeLa cell (human cervical cancer cell lines).The mRNA l ev el and protein level of c-Fos were measured to clarify thepotential mechanism of miR-101’s regulation. Furthermore, FlowCytometry along with the c-Fos rescue strategy was applied to detect theregulative effect of chosen microRNAs on cell-cycle and apoptosis of HeLa cells; EdU proliferation assay and wound healing assay were used toevaluate the candidate microRNA’s effect of regulation on proliferation andmetastasis of HeLa cell respectively.ResultsAmong these predicted microRNAs, miR-101is the microRNAspredicted by all the three used websites, The3’UTR of c-Fos, appears to behighly conserved among mammal species such as human, monkey, cow,mouse and rabbit indicated by TargetScan.Thesedata showed that3’UTR ofc-Fos is highly conserved and is most likely targeted by miR-101.The results of luciferase reporter assay showed that the relative activityof luciferase reporter co-transfected with the miR-101andpcDNA3.1-luciferase-c-Fos-3’UTR was decreased significantly below thatof the negative control, which means that MiR-101directlytarget the3’UTRof c-Fos.Real-time PCR and Western-blotting demonstrated that miR-101directly targeted the3’UTR of c-Fos and down-regulate the expression of c-Fos at mRNA and protein levels.Furthermore, Flow Cytometry along with the c-Fos rescue strategyshowed that miR-101regulates the cell-cycle of HeLa cells by targetingc-Fos in part.EdU proliferation assay and Flow cytometry identified that over-expression of miR-101inhibits the proliferation of HeLa cell line. MiR-101has no influences on the apoptosis of HeLa cell line by targeting c-Fos.The Wound Healing assay demonstrated that over-expression ofmiR-101has effect of regulation on the metastasis of HeLa cell line.ConclusionIn this study, we validated that miR-101plays important roles inregulation of cell behaviors such as cell cycle, proliferation, metastasis in cervical cancer and elucidated a potential pathway may that contribute to thisphenomenon. The new-insights offer novel-mechanisms for regulation ofcell behaviors as well as potential-therapeutic targets for cervical-cancer. Ofcourse, miR-101possesses many other target genes and other more functions,which need further-exploration. Furthermore, it is worth to search newmicroRNAs targeting c-Fos, and to explore novel cell functions for c-Fos incervical cancer. The future works should also focus on investigation in thefunctions of the miR-101and the miR-101-c-Fos relationships in vivo.