Effect On Proliferation Of Human Endometrial Cancer Cell By RNAi Inhibiting HMGB1Gene Expression

Objective:Use small interfering RNA(siRNA) for silence the expression of high mobility group box1(HMGB1) in endometrial cancer cell. To investigate the effects of HMGB1on proliferation, cell cycle and aopotosis of human endometrial cancer cell line HEC-1A, and to explore the molecular mechanism.Methods:Lentivirus vector with HMGB1shRNA was constructed and infected the endometrial cancer cell line HEC-1A. After viral infection for72h, Real time-PCR and Western Blot were performed to investigate HMGB1mRNA and protein expression. The cell proliferation was determined using methyl thiazolyl tetrazolium (MTT) method. Flow cytometry was performed to analyze the cell cycle progression of propidium iodide (P1)-stained HEC-IA cells and the apoptotic rate of annexinV/PI-stained cells. Western Blot was used to detect the protein expressions of AKT, pAKT and CyclinD1.Results:1、After transfected with lentivirus vector, endometrial cancer cell expressed strong fluorescence using fluorescence microscope. And the transfection efficiency was about80%. 2、After viral infection for72h, real time-PCR confirmed the expression of HMGB1mRNA in experimental group was lower than blank control group and negative control group (P<0.05). Western blot detected the expression of HMGB1protein in experimental group decreased significantly (P<0.01).3、The result of MTT assay indicated cell proliferation rate of experimental group was72.03%, and it was lower than other control groups (P<0.01). FCM results showed that the proportion GO/G1phase of experimental group was higher than blank control group and negative control group (P<0.01). However the proportion M phase and G2phase of experimental group were significantly lower (P<0.01).4、Annexin V-FITC&PI double staining method demonstrated that compared to blank control group and negative control group the early apoptosis rate, mid-late apoptosis rate and total apoptosis rate of experimental group were obviously increased (P<0.01).5、After HMGB1knockdown the protein expression of AKT had no difference in three groups(P>0.05). However the protein expressions of pAKT and CyclinD1were down-regulated after the HMGB1knockdown for72hours(P<0.01).Conclusion:Transfected lentivirus vector with HMGB1shRNA can effectively inhibit the expression of HMGB1mRNA and protein in endometrial cancer cell. After knockdown of HMGB1expression can inhibit cell proliferation, it blocks cell cycle in GO/G1phase. And it can induct cell apoptosis.HMGB1in endometrial cancer maybe regulate cell proliferation through PI3K/AKT signal pathway.