NCLX介导的线粒体钙稳态对肝癌细胞生长的影响

Effect of NCLX-mediated mitochondrial calcium homeostasis on the growth of hepatocellular carcinoma cells

目的探讨线粒体钠钙交换体(mitochondrial Na+/Ca2+exchanger,NCLX)介导的线粒体钙稳态对肝癌细胞生长的影响。方法真核表达载体pSliencer3.0-shNCLX、pcDNA3.1(+)-NCLX分别转染肝癌细胞SNU-739和SNU-368,构建稳定干涉和过表达NCLX肝癌细胞。采用免疫印迹法检测转染后肝癌细胞中NCLX蛋白的表达,Rhod-2-AM染色检测肝癌细胞线粒体钙水平,MTS和EdU实验检测肝癌细胞增殖能力,流式细胞仪检测肝癌细胞凋亡情况。结果免疫印迹法检测结果显示成功构建干涉和过表达NCLX的SNU-739和SNU-368肝癌细胞。与转染空载体肝癌细胞比较,干涉NCLX后肝癌细胞SNU-739和SNU-368线粒体钙明显增多(P<0.01),细胞增殖率明显升高(P<0.05),细胞凋亡率明显下降(P<0.01);而过表达NCLX后肝癌细胞SNU-739和SNU-368线粒体钙明显减少(P<0.05),细胞增殖率明显下降(P<0.05),细胞凋亡率明显上升(P<0.01)。结论干涉NCLX诱导的线粒体钙水平升高可促进肝癌细胞增殖和抑制凋亡,而过表达NCLX诱导的线粒体钙水平下降抑制肝癌细胞增殖和促进凋亡,NCLX可能是肝癌的潜在治疗靶点。

Objective We performed this study to explore the effect of NCLX-mediated mitochondrial calcium on the growth of hepatocellular carcinoma(HCC)cell line. Methods Eukaryotic expression vector p Sliencer3.0-shNCLX and pcDNA3.1(+)-NCLX were transfected into HCC cells SNU-739 and SNU-368,respectively,and NCLX was stably knocked down or overexpressed. The expression level of NCLX protein was detected by Western blot assay. Rhod-2-AM staining was used to detect the level of mitochondrial calcium.The proliferation ability of cells was evaluated by MTS and EdU assay. The apoptosis of cells was assessed by apoptosis assay. Results Western blot assay showed that NCLX was stably knocked down or overexpressed in HCC cells SNU-739 and SNU-368. Compared with transfected empty vector,the mitochondrial calcium level was significantly increased after downregulation of NCLX expression(P<0.01),the cell proliferation rate was significantly increased(P<0.05),and the apoptosis rate was significantly decreased(P<0.01)in HCC cells SNU-739 and SNU-368. After upregulation of NCLX expression,the mitochondrial calcium level was significantly decreased(P<0.05),the cell proliferation rate was significantly decreased(P<0.05),and the apoptosis rate was significantly increased(P<0.01)in HCC cells SNU-739 and SNU-368. Conclusions In HCC cells,downregulation of NCLX-induced increase in mitochondrial calcium level may effectively promote cell proliferation and inhibit apoptosis. However,overexpression of NCLX-induced decrease in mitochondrial calcium levels inhibits cell proliferation and promotes apoptosis in HCC cells SNU-739 and SNU-368.These results suggest that NCLX may be a potential target for the treatment of hepatocellular carcinoma.