Hsa-mir-106b Contribute To Cellcycle Progression In HEC-1B Human Endometrial Carcinomacellline

Objective: Cell cycle and apoptosis variation has been studied byconstruct over-expression and inhibition lentiviral vectors of miR-106b, andupgrade/downgrade its expression levels in HEC-1B human endometrialcancer cell line. We predicted one possible target gene by combinationanticipated algorithms and verification its expression level of mRNA andpotential by real time PCR reaction and indirect immunofluorescencerespectively. This study explored the molecule mechanism of miR-106b inhuman endometrial cancer.Methods:1) Construction of lentiviral vectors: The plasmids whichdown/up-regulation miR-106b has been constructed and purification byShanghai BioSun Sci&Tech Company.2) Flow cytometry: At72hours post-transfection, cell cycle andapoptosis were analyzed.3) Real-time quantitative RT-PCR: Reverse transcription and PCRprimer of miR-106b and TSG101were synthesized, and detected those variations before and after transfection respectively.4) Bioinformation analysis: We have predicted potential target gene ofmiR-106b by Pic Tar, TargetScan and miRGen Targets database.5) Indirect immunofluorescence: After sieved by puromycin, mouseanti human TSG101antibody and phycoerythrin dye were used to analyzeTSG101protein in HEC-1B cell line.Results:1) Positive cells were sieved by puromycin after transfected. GFP+ratio was increased to80%indicated that over-expression and inhibitionlentiviral vectors of miR-106b were successfully constructed.2) MiR-106b expression: We found miR-106b expression in HEC-1Bcell line. The Real-time quantitative RT-PCR results showed that comparedto blank group, miR-106b level in cells which transfected withover-expression vector was doubled, and inhibition vector group wasdown-regulation about50%. But control group were no statistics distinctioncompared to blank. These result demonstrated that lentiviral vectors weconstructed can change miR-106b expression in HEC-1B cell line.3) Cell cycle: compared to blank, G1phase cells were increased and Sphase cells were decreased in inhibition group, and arrested HEC-1B cellscycle at G1phase by decreasing the expression of miR-106b. Inover-expression group S phase cells were increased and with statisticalsignificance. Two control groups were no difference with blank. 4) Cell apoptosis: The apoptosis rate of inhibition group wassignificantly increased but the rest were no difference with blank.5) Bioinformation analysis: We have predicted Tumor SusceptibilityGene101was one potential target gene of miR-106b by Pic Tar, TargetScanand miRGen Targets database.6) TSG101mRNA expression: the mRNAexpression level of TSG101were no statistics distinction among all five groups, we demonstrated thatthe transcription level of TSG101can’t be controlled by miR-106b.7) Indirect immunofluorescence showed that: the TSG101protein wasboth expressed in nuclei and cytoplasm, but the former intensity was higherthan the latter. The fluorescence intensity in inhibition group was higher thanblank, but over expression group was lower. Two control groups were nostatistics difference with blank.Conclusion:1) Compared with control-treated cells, inhibition of miR-106b couldarrest cell cycle in G1phase, and over express it could consistently andsubstantially increase the S phase and reduce G1population. It suggests thatmiR-106b may contribute to cell cycle progression in HEC-1B cell line.2) TSG101protein level was significantly reduced by over expressionof miR-106b and increased by inhibit it, but the mRNA of TSG101have nodifference between those groups. Combination with bioinformationanalysis and these results we demonstrate that TSG101may be the potential target gene of miR-106b and miR-106b may contribute to cellcycle progression by regulating TSG101.