siRNA沉默NHE1基因对MHCC97-H肝癌细胞侵袭迁移的影响

SiRNA-mediated silencing of the NHE1 gene inhibits cell invasion and migration in human hepatocellular carcinoma cell line MHCC97-H

目的:探讨RNA干扰沉默NHE1基因后对人肝癌细胞株MHCC97-H细胞侵袭迁移的影响.方法:应用NHE1基因小干扰RNA(small interferingRNA,siRNA)转染人肝癌细胞株MHCC97-H细胞,同时设立空白对照组和无关对照组.转染成功后采用RT-PCR和Westernblot分别从基因和蛋白水平检测RNA干扰沉默NHE1基因的效果.MTT法测定转染后三组细胞的增殖状况,Transwell小室检测沉默NHE1基因对MHCC97-H细胞侵袭、转移能力的影响,进一步采用免疫荧光观察其对MHCC97-H细胞骨架和伪足的影响.结果:与两对照组比较,转染NHE1-siRNA组NHE1mRNA和蛋白表达水平均明显降低(P<0.05);细胞侵袭、迁移实验结果显示,NHE1-siRNA组穿透人工基底膜的细胞数(34.1±5.2,120.2±12.8)较空白对照组(56.9±6.1,235.2±16.8)和转染无关对照组(57.2±6.1,231.9±14.7)均明显减少,差异均有统计学意义(P<0.05),而空白对照组与无关对照组比较差异无统计学意义;MTT结果显示,转染后48h和72h三组细胞间增殖活性差异无统计学意义;NHE1基因沉默后与两对照组相比,MHCC97-H细胞膜伪足形成减少,细胞内肌动蛋白网排列紊乱.结论:siRNA沉默NHE1基因后可能通过影响MHCC97-H细胞骨架的重组和伪足的形成,从而抑制MHCC97-H细胞的侵袭和迁移能力.

AIM： To investigate the effect of small interferingRNA （siRNA）-mediated silencing of the Na^＋/H^＋ exchanger 1 （NHE1） gene on cell invasion and migration in human hepatocellular carcinoma cell line MHCC97-H. METHODS： After MHCC97-H cells were transfected with NHE1-specific siRNA, the levels of NHE1 mRNA and protein were detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot, respectively. Cell proliferation was detected by MTT assay. The impact of NHE1 knockdown on cell invasion and migration was determined by Transwell chamber assay. The changes in morphology cytoskeleton and pseudopodia were observed by i mmunofluorescence. RESULTS： Compared to negative control and mock control cells, the levels of NHE1 mRNA and protein in MHCC97-H cells transfected with NHE1-specific siRNA decreased significantly （P 〈 0.05）. NHE1 knockdown significantly suppressed the invasion and migration of MHCC97-H cells compared to negative control and mock control cells （34.1 ± 5.2 vs 56.9 ± 6.1, 57.2 ± 6.1; 120.2 ± 12.8 vs 235.2 ± 16.8, 231.9 ± 14.7; all P 〈 0.05）. However, there were no significant differences in cell migration and invasion between the two control groups. Cell proliferation showed no significant differences among the three groups 48 or 72 h after transfection. Compared to the two control groups, deletion of NHE1 decreased the number of membrane pseudopodia and disrupted the cross-linked actin network in MHCC97-H cells. CONCLUSION： Deletion of NHE1 inhibits cell invasion and migration by influencing cytoskel- eton rearrangement and pseudopodia formation in human hepatocellular carcinoma cell line MHCC97-H.