小干扰RNA抑制HMGB1表达对子宫内膜癌细胞侵袭与迁移的影响

Effect of expression of high mobility group box-1 inhibited by small hairpin RNA on the invasion and migration of human endometrial carcinoma HEC-1A cells

目的:探讨靶向HMGB1的shRNA对HMGB1表达改变的影响及其与子宫内膜癌细胞侵袭与迁移力改变的关系。方法:运用RNA干扰技术,构建HMGB1短发夹RNA(pshRNA-1/HMGB1,pshRNA-2/HMGB1,pshRNA-3/HMGB1),同时设置转染非特异性质粒的阴性对照组(HMGB1/p-NC)和脂质体转染组(Lipo组),用脂质体法转染人子宫内膜癌细胞(HEC-1A),利用反转录-聚合酶链反应(RT-PCR)和Western印迹法检测转染后48 h各组细胞HMGB1在mRNA和蛋白水平的表达,Transwell小室法观察转染HMGB1 shRNA后HEC-1A细胞的侵袭情况,细胞划痕实验观察转染HMGB1 shRNA后HEC-1A细胞的迁移情况。结果:RT-PCR显示:3组重组质粒HMGB1-pshRNAs转染后,HMGB1 mRNA相对表达水平分别是0.192±0.006,0.055±0.002和0.123±0.086,较Lipo组(0.268±0.008)和HMGB1/p-NC组(0.270±0.004)明显减少(P<0.05),最大抑制率分别达28.4%,79.5%和54.1%。Western印迹显示:HMGB1蛋白相对表达水平分别是0.259±0.129,0.032±0.002和0.104±0.007,较Lipo组(0.347±0.007)和HMGB1/p-NC组(0.349±0.007)明显减少(P<0.05),最大抑制率分别达25.4%,90.8%和70.0%。实验组在接种后48 h,穿过Transwell小室膜的细胞数显著少于HMGB1/p-NC组和Lipo组(P<0.05)。划痕愈合实验亦显示实验组划痕愈合率明显低于HMGB1/p-NC组和Lipo组(P<0.05)。结论:靶向干扰HMGB1的shRNA可以抑制HMGB1的表达,转染后的子宫内膜癌细胞侵袭与迁移能力明显下降。

Objective:To investigate the effect of inhibiting high mobility group box-1(HMGB1) gene expression on the invasion and migration of endometrial carcinoma HEC-1A cells by small hairpin RNA.Methods:Three specific recombinant plasmids of HMGB1(pshRNA-1 /HMGB1,pshRNA-2 /HMGB1,and pshRNA-3/HMGB1) were transfected into the endometrial cancer cell lines HEC-1A by lipofectamine TM 2000.The expression of HMGB1 mRNA and protein was decteted by RTPCR and Western blot.The invasion and migration abilities of transfected HEC-1A cells were evaluated using Transwell assay and wound healing assay.Results:RT-PCR and Western blot revealed that the expression of HMGB1 at both mRNA and protein levels was significantly inhibited by HMGB1-pshRNA targeting sequence 1,2,and 3(P<0.05),and the levels of 3 mRNAs in the transfection group were 0.192±0.006,0.055±0.002,and 0.123±0.086,respectively,which were significantly lower than those in the Lipo group(0.268±0.008) and the HMGB1/p-NC group(0.270±0.004).The maximum inhibiton rates of the 3 mRNAs were 28.4%,79.5%,and 54.1%.The levels of 3 HMGB1 proteins in the transfection group were 0.259±0.129,0.032±0.002,and 0.104±0.007,significantly lower than those in the Lipo group(0.347±0.007) and the HMGB1/p-NC group(0.349±0.007),and the maximum inhibitory rates were 25.4%,90.8%,and 70.0%.The transwell chamber assay and wound healing assay showed that the invasion and migration of HEC-1A cells were effectively suppressed by inhibiting HMGB1 expression(P<0.05).Conclusion:pshRNA-HMGB1 can effectively inhibit HMGB1 expression at both mRNA and protein levels,and decrease the invasion and migration of endometrial cancer cells.