草氨酸钠对胆管癌细胞自噬与凋亡的影响

Effects of Sodium Oxalate on Autophagy and Apoptosis of Cholangiocarcinoma Cells

目的观察乳酸脱氢酶A(lactate dehydrogenase A,LDHA)抑制剂草氨酸钠对人胆管癌细胞株Hucct-1自噬与凋亡的影响,并初步探讨其相关机制。方法用不同浓度的草氨酸钠处理Hucct-1细胞,或将LDHA小干扰RNA转染Hucct-1细胞后,CCK8法检测细胞增殖,流式细胞术检测细胞凋亡,划痕愈合实验检测细胞迁移,实时荧光定量PCR和蛋白质印迹法分别检测自噬相关mRNA和蛋白的表达水平,乳酸检测试剂盒检测细胞内乳酸的含量。结果草氨酸钠抑制Hucct-1细胞增殖(P<0.01),并呈剂量依赖性,其半数抑制浓度为50 mmol/L。用50 mmol/L草氨酸钠干预Hucct-1细胞或LDHA小干扰RNA转染Hucct-1细胞后,LDHA mRNA和蛋白表达水平均降低(均P<0.01),细胞内乳酸的含量减少(P<0.01),细胞凋亡率升高(P<0.01),细胞迁移距离缩短(P<0.01),细胞中自噬相关基因7(autophagy-related gene 7,ATG7)、Beclin-1和微管相关蛋白1轻链3Ⅱ(microtubule-associated protein 1 light chain 3Ⅱ,LC-3Ⅱ)表达水平升高(均P<0.01),P62蛋白表达水平降低(P<0.01)。结论草氨酸钠通过抑制Hucct-1细胞中LDHA表达来促进细胞自噬,诱导细胞凋亡。

Objective To observe the effects of sodium oxalate,an inhibitor of lactate dehydrogenase A(LDHA),on autophagy and apoptosis of cholangiocarcinoma cell line Hucct-1,and to explore the related mechanisms.Methods Hucct-1 cells were treated with different concentrations of sodium oxalate.And LDHA siRNA was transfected into Hucct-1 cells by LipofectAMINE 2000 and Opti-MEM medium.Then the cell proliferation was detected by CCK8 assay,the apoptosis was detected by flow cytometry,and the cell migration was detected by scratch healing test.Meanwhile,the expressions of autophagy-related mRNAs and proteins were detected respectively by RT-PCR and Western blotting.And the lactic acid detection kit was used to detect lactate content in cells.Results Sodium oxalate inhibited the proliferation of Hucct-1 cells in a dose-dependent manner,its half-inhibitory concentration increased was 50 mmol/L.After Hucct-1 cells were treated with 50 mmol/L sodium oxalate or transfected with LDHA siRNA,the mRNA and protein expression levels of LDHA were decreased(both P<0.01),and the intracellular lactic acid content was decreased(P<0.01).In the meantime,the apoptosis rate was increased(P<0.01),and the cell migration distance was decreased(P<0.01).And the expression levels of autophagy-related gene 7(ATG7),Beclin-1 and microtubule-associated protein 1 light chain 3Ⅱ(LC-3Ⅱ)were increased(all P<0.01).In addition,the expression of P62 protein was significantly decreased(P<0.01).Conclusion Sodium oxalate can promote autophagy and induce apoptosis by inhibiting the expression of LDHA in Hucct-1 cells.