SPK1通过调节Bcl-2/Bax途径干扰LLC细胞凋亡

SPK1 interferes with LLC cell apoptosis by regulating Bcl-2/Bax pathway

目的:研究鞘氨醇激酶1(SPK1)是否通过调节Bcl-2/Bax途径干扰小鼠Lewis肺癌(LLC)细胞的凋亡。方法:通过构建SPK1基因小干扰RNA(siRNA)真核表达载体,将siRNA转染至LLC细胞,在荧光显微镜下观察LLC细胞转染的情况。采用流式细胞术检测转染后LLC细胞的凋亡率,Western blot法检测转染后LLC细胞中SPK1、Bcl-2和Bax蛋白表达水平,ELISA法检测Bax和Bcl-2的蛋白表达。结果:转染后的LLC细胞在荧光显微镜下发出绿色荧光。siRNA-SPK1组细胞凋亡明显高于siRNA-SPK1-Neg组(P<0.01)。Western blot结果显示, siRNA-SPK1组中Bax蛋白表达明显较siRNA-SPK1-Neg组高,Bcl-2蛋白表达较siRNA-SPK1-Neg组低。ELISA结果显示siRNA-SPK1组中Bax表达水平显著高于siRNA-SPK1-Neg组(P<0.01), siRNA-SPK1组中Bcl-2表达水平显著低于siRNA-SPK1-Neg组(P<0.01)。结论:SPK1在LLC细胞中的表达与细胞凋亡率有关。SPK1可能是通过Bcl-2/Bax途径干扰LLC细胞凋亡。

AIM:To investigate whether sphingosine kinase 1(SPK1)interferes with apoptosis of Lewis lung cancer(LLC)cells by regulating the Bcl-2/Bax pathway.METHODS:The SPK1 gene siRNA eukaryotic expression vector was constructed,and transfected into the LLC cells.The transfected LLC cells was observed under a fluorescence microscope.The apoptotic rate of LLC cells after transfection was analyzed by flow cytometry.The expression levels of SPK1,Bcl-2 and Bax in LLC cells after transfection were detected by Western blot.The protein levels of Bax and Bcl-2 were measured by ELISA.RESULTS:Transfected LLC cells emitted green fluorescence under a fluorescence microscope.Apoptosis in siRNA-SPK1 group was significantly higher than that in siRNA-SPK1-Neg group(P<0.01).Western blot analysis showed that the expression of Bax in siRNA-SPK1 group was significantly higher than that in siRNA-SPK1-Neg group,and the expression of Bcl-2 was lower than that in siRNA-SPK1-Neg group.The ELISA results showed that the protein level of Bax in siRNA-SPK1 group was significantly higher than that in siRNA-SPK1-Neg group(P<0.01),and the protein level of Bcl-2 in siRNA-SPK1 group was significantly lower than that in siRNA-SPK1-Neg group(P<0.01).CONCLUSION:The expression of SPK1 in LLC cells is related to the apoptotic rate.SPK1 may interfere with the apoptosis of LLC cells via Bcl-2/Bax pathway.