MiR-382对γδT细胞杀伤肺癌细胞活性的影响及机制研究

Effect and Mechanism of MiR-382 on cytotoxicity of γδT cells to lung cancer

关键词:  MiR-382  c-FLIP  γδT细胞  肺癌  caspase-8

Key words:  miR-382  c-FLIP  γδT cells  lung cancer  caspase-8

摘要:

目的 探讨gdT细胞对肺癌细胞系A549的杀伤活性，并研究miR-382在其中发挥的作用。方法 采用流式细胞术鉴别γδT细胞LDH释放实验检测γδT细胞和miR-382对A549的杀伤活性。荧光定量PCR检测miR-382在肺癌细胞系A549中的表达情况，western blot法检测miR-382对c-FLIP表达的影响。western blot试验检测miR-382及γδT细胞处理后A549细胞Smac/DIABLO和细胞色素c的释放及caspase-8、caspase-9和caspase-3的活化。结果 miR-382处理能显著增强γδT细胞对A549的杀伤活性，增强γδT细胞对A549细胞caspase-8、caspase-9和caspase-3的活化及细胞色素c和Smac/DIABLO的释放，下调A549细胞中c-FLIP蛋白的表达。转染c-FLIP质粒能显著抑制miR-382对γδT细胞的协同效应。结论 MiR-382通过下调肺癌细胞中c-FLIP的表达来增强γδT细胞对其的杀伤活性。

Abstract:

OBJECTIVE To investigate the effect of miR-382 on regulating the cytotoxicity of γδT cells to lung cancer cell line A549. METHODS γδT cells amplified in vitro were identified by flow cytometry. LDH release assays were performed to detect the cytotoxicity of γδT cells and miR-382 to A549. RT-qPCR assays were performed to detect the expression of miR-382 in lung cancer tissues and A549 cell line. Bioinformatics and western blot analysis were used to determine whether the expression of c-FLIP was regulated by miR-382. Western blot assays were performed to evaluate the release of Smac/DIABLO and cytochrome c and the activation of caspase-8, caspase-9 and caspase-3. RESULTS miR-382 significantly enhanced the cytotoxicity of γδT cells to A549. Furthermore, miR-382 enhanced the activation of caspase-8, caspase-9, caspase-3 and release of cytochrome C and Smac/DIABLO induced by γδT cells in A549 cells. In addition, transfection with miR-382 downregulated the expression of c-FLIP. Meanwhile, transfection with miR-382 abolished the synergistic effect of miR-382 on γδT cells. CONCLUSION MiR-382 enhances cytotoxicity of γδT cells to lung cancer through downregulating the expression of c-FLIP.