SPK1通过调节Bcl-2/Bax途径干扰LLC细胞凋亡
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摘要
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.
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.
引文
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[3] 杨毅,官俏兵,郭丽,等.IL-1β促进nave T细胞向Th22细胞分化及在非小细胞肺癌中表达的相关性研究[J].中国病理生理杂志,2018,34(7):1301-1305.
[4] 朱明珍,徐海燕,蒋华.晚期非小细胞肺癌HIF-1α、ERCC1基因多态性与铂类药物疗效的关系[J].实用医学杂志,2016,32(3):385-388.
[5] Lu PH,Chen MB,Liu YY,et al.Identification of sphingosine kinase 1 (SphK1) as a primary target of icaritin in hepatocellular carcinoma cells[J].Oncotarget,2017,8(14):22800-22810.
[6] Furuya H,Shimizu Y,Tamashiro PM,et al.Sphingosine kinase 1 expression enhances colon tumor growth[J].J Transl Med,2017,15(1):120-128.
[7] Cai H,Xie X,Ji L,et al.Sphingosine kinase 1:a novel independent prognosis biomarker in hepatocellular carcinoma[J].Oncol Lett,2017,13(4):2316-2322.
[8] Chen J,Qi Y,Zhao Y,et al.Deletion of sphingosine kinase 1 inhibits liver tumorigenesis in diethylnitrosamine-treated mice[J].Oncotarget,2018,9(21):15635-15649.
[9] Zhu YJ,You H,Tan JX,et al.Overexpression of sphingosine kinase 1 is predictive of poor prognosis in human breast cancer[J].Oncol Lett,2017,14(1):63-72.
[10] Song L,Xiong H,Li J,et al.Sphingosine kinase-1 enhances resistance to apoptosis through activation of PI3K/Akt/NF-κB pathway in human non-small cell lung cancer[J].Clin Cancer Res,2011,17(7):1839-1849.
[11] Johnson KR,Johnson KY,Crellin HG,et al.Immunohistochemical distribution of sphingosine kinase 1 in normal and tumor lung tissue[J].J Histochem Cytochem,2005,53(9):1159-1166.
[12] Salinas NR,Lopes CT,Palma PV,et al.Lung Tumor Development in the Presence of Sphingosine 1-phosphate Agonist FTY720[J].Pathol Oncol Res,2009,15(4):549-554.
[13] Pchejetski D,B?hler T,Stebbing J,et al.Therapeutic potential of targeting sphingosine kinase 1 in prostate cancer[J].Nat Rev Urol,2011,8(10):569-678.
[14] 李银苹,张学铭,汪开诚,等.氢分子对高糖状态下肾小球系膜细胞凋亡相关蛋白及Nrf2信号通路的影响[J].中国病理生理杂志,2018,34(1):29-34.
[15] Wang Q,Zhang L,Yuan X,et al.The relationship between the Bcl-2/Bax proteins and the mitochondria-mediated apoptosis pathway in the differentiation of adipose-derived stromal cells into neurons[J].PLoS One,2016,11(10):e0163327.
[16] Lunavargas MPA,Chipuk JE.Physiological and pharmacological control of BAK,BAX,and beyond[J].Trends Cell Biol,2016,26(12):906-917.
[17] 王少敏,叶孟,倪曙民,等.bcl-3基因通过cyclin D1及Bax蛋白影响人结肠癌RKO细胞的凋亡[J].中国病理生理杂志,2017,33(5):939-943.
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