Short Hairpin RNA Silences HMGB1 Gene And Affets Proliferation In Endometrial Cancer Cell Line

Objective:The main purpose of this study is to investigate the effects of short hairpin RNA(shRNA) interference on expression of HMGB1 and cell proliferation in RL95-2 cell lines.Methods:The recombinant plasmid pGPU6-shRNA expressing HMGB1-targeted shRNA was transfected into endometrial cancer cell lines by lipofectamine.Six groups of research objects namely RL95-2 HMGB1/p-shRNA,RL95-2/p,RL95-2/mock-control,RL95-2 GAPDH/p-shRNA,RL95-2 HMGB1/p-NC and RL95-2 were enrolled in this study.The expression of HMGB1 mRNA and protein were detected by RT-PCR and Western-blot.The cell proliferation after transfection was detected by MTT.Results:(1)The DNA sequence analysis of the recombinant plasmid pGPU6-HMGB1 shRNA revealed that the fragment template of shRNA targeting HMGB1 gene has been inserted into the exclusive RNAi plasmid namely pGPU6/GFP/Neo Express and the results of the sequence and design were absolutely together.(2) The fluorescent showed that the recombinant plasmid pGPU6-shRNA expressing HMGB1-targeted shRNA had been transfected.(3) RT-PCR show that,after the recombinant plasmid pGPU6-shRNA expressing HMGB1-targeted shRNA was transfected,the level of HMGB 1mRNA in group RL95-2 HMGB1/p-shRNA was 0.05 times of RL95-2,and there was significantly difference between the above two groups(2^{-ΔΔCt}<0.5).Meanwhile,the level of HMGB1mRNA in group RL95-2/p,RL95-2/mock-control,RL95-2 HMGB1/p-NC were respectively 0.78,1.01 and 0.64 times of RL95-2;There was no significantly difference between group RL95-2 HMGB1/p-shRNA and RL95-2 GAPDH/p-shRNA;And there was no significant difference among the group RL95-2/p,RL95-2/mock-control,RL95-2 HMGB1/p-NC and RL95-2.(4) Western-blot revealed that,after the recombinant plasmid pGPU6-shRNA expressing HMGB1-targeted shRNA was transfected,the relative volume of HMGB1 protein in group RL95-2 HMGB1/p-shRNA and group RL95-2 respectively were 0.153±0.004 and 0.568±0.005,the difference was statistically significant between the above groups(p＜0.05);and the relative volume of GAPDH protein in RL95-2 GAPDH/p-shRNA was 0.158±0.006,There was no significant difference between the group RL95-2 HMGB1/p-shRNA and the group RL95-2 GAPDH/p-shRNA;the relative volume of HMGB1 protein in group RL95-2/p,RL95-2/mock-control,RL95-2 HMGB1/p-NC were 0.566±0.006,0.573±0.004,0.563±0.004,and there were respectively no significant difference compared to the group RL95-2;There was no significant difference among the group RL95-2/p,RL95-2/mock-control and RL95-2 HMGB1/p-NC;(5) MTT show that compared to RL95-2/p,RL95-2 HMGB1/mock control,RL95-2 HMGB1/p-NC and RL95-2,The proliferation of RL95-2 HMGB1/p-shRNA and RL95-2 GAPDH/p-shRNA were obviously decrease,and the difference was statistically significant(p＜0.05);There was no significantly difference between group RL95-2 HMGB1/p-shRNA and RL95-2 GAPDH/p-shRNA;And there was no significant difference among the group RL95-2/p,RL95-2/mock-control,RL95-2 HMGB1/p-NC and RL95-2.Conclusions:Short hairpin RNA(shRNA) interference could effectively suppress the expression of HMGB1,and inhibit the proliferation of endometrial cancer cell,would provide new ideas for the treatment of endometrial cancer.