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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.
Objective: To investigate the effects of silencing NHE1 gene by RNA interference on the invasion and migration of human hepatocellular carcinoma cell line MHCC97-H. Methods: Transfect the human hepatocellular carcinoma cell line MHCC97-H with NHE1 small interfering RNA (siRNA). The control group and the unrelated control group. After transfection, RT-PCR and Western blot were used to detect the effect of silencing NHE1 gene by RNA interference from the gene and protein levels. The proliferation of three groups of cells after transfection was measured by MTT assay, and the silence was detected by Transwell chamber. The effect of NHE1 gene on the invasion and metastasis of MHCC97-H cells was further investigated by immunofluorescence. The results showed that the expression of NHE1 mRNA and protein in the transfected NHE1-siRNA group compared with the two control groups. The average level was significantly lower (P<0.05); cell invasion and migration experiments showed that the number of cells that penetrated the artificial basement membrane in the NHE1-siRNA group was (34.1±5.2, 120.2±12.8) compared with the blank control group (56.9±6.1, 235.2± 16.8) and transfection-irrelevant control group (57.2 ± 6.1, 231.9 ± 14.7) were significantly reduced, the difference was statistically significant (P <0.05), while the blank control group and the unrelated control group had no statistically significant difference; MTT results Obvious There was no significant difference in cell proliferation activity between the three groups at 48h and 72h after transfection. Compared with the two control groups, the formation of pseudopodia of MHCC97-H cells was decreased and the intracellular actin network was disordered after NHE1 gene silencing. siRNA silencing NHE1 gene may inhibit the invasion and migration of MHCC97-H cells by affecting the reorganization of the MHCC97-H cytoskeleton and the formation of pseudopods.