Molecular Mechanisms Of Regulation Cell Proliferation Of "Tamoxifen/Let-7g/Target Protein" Pathway On Endometrial Cancer RL-95-2Cell

ObjectiveTAM (Tamoxifen) is a selective regulator of estrogen receptor, foundthat could increase the risk of endometrial hyperplasia and malignant tumorswhen treated for ER-positive breast cancer patients. It’s considered thatTAM roled as a weak estrogen-like effects in the endometrial tissues. Let-7gis one of the endogenous non-coding RNA (MicroRNA, miRNA). In recentyears, some studies have showed that let-7g plaied an important role in thedevelopment of malignant tumors. In this study, we choosed endometrialcancer cell RL-95-2as the experimental subjects, to observe the effect ofTAM on cell growth and expression let-7g, to proven the regulation of therelationship between TAM and let-7g. Then with the help of bioinformaticsand laboratory methods to predict the potential target genes of let-7g, toexplore the possible molecular mechanisms of TAM and let-7g. So toprovide a new theoretical basis for further cognition on the pathogenesis ofendometrial cancer. Methods1. Estrogen Receptor(ER) was detected by Immunofluorescencestaining.2. After different concentrations of TAM treated. Cell proliferation wasdetermined by CCK-8assay；Cell cycle was analyzed by flow cytometry(FCM); the expression of let-7g was detected by Real-time quantitativePolymerase Chain Reaction (RT-qPCR).3. With the help of bioinformatics tools to predict the let-7g target genesand analysis their biological functions.4. After let-7g was up-regulated or down-regulated by let-7g mimics orlet-7g inhibitor, cell proliferation of RL-95-2cells was detected by CCK-8assay, expression of RB1mRNA and protein was determined by RT-qPCRand Western Blot respectively.Results1. RL-95-2cells express ER.2. Compared with the control group:(1) after treated for48h, TAM(1×10-7mol/L) could stimulated the proliferation of RL-95-2cells; Aftertreated for72h, TAM (1×10-9～1×10-5mol/L) could significantly stimulatethe growth of cells, and increased the expression of let-7g, especially at theconcentration of1×10-7mol/L group; at the concentration of1×10-4mol/L, itinhibited cell growth both after treated for48h or72h, and reduced the let-7gexpression.(2)TAM (1×10-7mol/L) treated for72h, cells at G0/G1phase was significantly reduced, while increased at S-phase.3. The analysis of bioinformatics indicated UHRF2, RB1, GAS7,PLAGL2, NAB1and ZNF282as the cancidate targets for let-7g, then weanalysised focus on the RB1.4. Up-regulated let-7g by transfected with let-7g mimic, can promotecell proliferation, and make the RB1protein level significantly decreased,we also observed the opposite results in cells that let-7g down-regulated bytransfected with let-7g inhibitor, but not in cells transfected with mimicscontrol or inhibitor control, however, we found no significant difference onRB1mRNA level.ConclusionSuitable concentration of tamoxifen has a significantly effect ofproliferation on RL-95-2cells and promote the cells from G0/G1phase intoS phase, and this effect may be achieved by up-regulated let-7g expressionthen negative regulation of RB1.