FUNDC1调控肺癌A549细胞及其干细胞的生长、自噬、凋亡和细胞干性的研究

FUNDC1 regulates the growth,autophagy,apoptosis and cell stemness of lung cancer A549 and its stem cells

目的:本实验旨在阐明线粒体自噬受体蛋白FUNDC1(FUN 14 domain containing 1)在肺癌病理进程中对肿瘤细胞生长死亡以及细胞干性的调控作用。方法:使用RT-qPCR分析FUNDC1在肺癌组织和细胞中的表达情况。siRNAs和过表达载体转染A549细胞以及诱导形成的A549干细胞团(A549/CSC)。采用CCK-8增殖试剂盒及集落形成实验分析不同处理对肺癌细胞增殖的影响。Western blotting检测FUNDC1、细胞自噬相关蛋白(Beclin-1、LC3-Ⅱ、p62)、凋亡相关蛋白(Bax、Bcl-2、caspase3)和细胞干性相关蛋白(CD133、Sox2、Oct4)的表达。细胞凋亡试剂盒(Elisa)用于测定细胞凋亡水平。流式细胞术分析CD133细胞阳性率。结果:FUNDC1在肺癌组织及细胞中表达上调,干扰FUNDC1抑制A549细胞增殖和保护性自噬,同时诱导细胞凋亡。此外,FUNDC1与CD133表达显著正相关,干扰FUNDC1可显著抑制细胞干性相关基因CD133、Sox2、Oct4的表达。A549/CSC细胞的FUNDC1表达高于正常A549细胞。干扰FUNDC1同样会抑制A549/CSC细胞增殖和保护性自噬,促进细胞凋亡。结论:FUNDC1调控肺癌细胞及CSC细胞的生长、自噬、凋亡及细胞干性。

Objective:To clarify the regulatory effects of FUNDC1(FUN 14 domain containing 1)exerted on the growth and death,as well as cell stemness,of tumor cells in the pathological process of lung cancer.Methods:The expression of FUNDC1 in lung cancer tissues and cells was analyzed by RT-qPCR.siRNAs and overexpression vectors were transfected into A549 cells and A549 cancer stem cells(A549/CSCs).Cell growth was analyzed using CCK-8 method and colony formation experiment.Western blotting was conducted to measure the expressions of FUNDC1,autophagy related proteins(Beclin-1,LC3-Ⅱ,p62),apoptosis related proteins(Bax,Bcl-2,caspase3)and stemness related proteins(CD133,Sox2,Oct4).Cell Death Elisa kit was used to determine the apoptosis rates.The rate of CD133+cells was analyzed by Flow cytometry.Results:FUNDC1 expression was up-regulated in both lung cancer tissues and cells.Inhibiting FUNDC1 depressed the proliferation and protective autophagy of A549 cells,while apoptosis was promoted.Additionally,FUNDC1 was positive-correlated with CD133 expression,and FUNDC1 inhibition could significantly suppress CD133,Sox2,and Oct4.The expression of FUNDC1 in A549/CSC cells was elevated compared with normal A549 cells.FUNDC1 inhibition also decreased the proliferation and protective autophagy of A549/CSC cells and promote cell apoptosis.Conclusion:FUNDC1 regulates the growth,autophagy,apoptosis and cellular stemness of lung cancer cells as well as their CSC cells.