摘要
目的探讨自噬基因Beclin-1表达沉默对A549肺癌细胞自噬和凋亡的影响以及其作用机制。方法应用实时荧光定量聚合酶链反应(FQ—PCR)和Westernblot的方法分别检测转染后各组A549肺癌细胞Beclin-1mRNA和蛋白的表达;噻唑蓝(MTT)法检测各组A549细胞生长增殖;单丹磺酰戊二胺(MDC)法检测各组A549细胞自噬的变化;用流式细胞仪检测各组A549细胞凋亡;Westernblot方法检测各组A549细胞食管癌相关基因4(ECRG4)蛋白表达变化。结果与对照组比较,感染Beclin-1小干扰RNA(siaNa)慢病毒颗粒后,A549细胞中Beclin-1mRNA和蛋白表达显著减少(P〈0.05),Beclin-1mRNA的沉默效率分别为35.56%、89.22%和66.78%;感染Beclin-1siRNA慢病毒颗粒后,A549细胞凋亡率为(4.31±0.49)%,和对照组比较显著降低(P〈0.05);感染Beclin-1siRNA慢病毒颗粒后,A549细胞群体平均倍增时间为(37.7±2.6)h,和对照组比较,平均倍增时间缩短(P〉0.05);感染Beclin—1siRNA慢病毒颗粒后,和对照组比较,A549细胞自噬细胞阳性数量减少(P〉0.05);感染Beclin-1siRNA慢病毒颗粒后,和对照组比较,ECRG4蛋白表达下降(P〈0.05)。结论自噬基因Beclin-1通过ECRG4通路影响A549肺癌细胞自噬和凋亡。
Objective To investigate the effect and molecular mechanism of autophagy - related gene Beclin - 1 down - regulation on autophagy and apoptosis of A549 lung cancer cells. Methods Real - time fluorescent quantitative polymerase chain reaction ( FQ - PCR) and Western blotting were used to detect Beclin - 1 mRNA and protein expression levels of transfected cells in each group. The growth and proliferation of A549 lung cancer cells were observed by methyl thiazol tetrazolium (MT'F) method. Mono- dansylcadaverine (MDC) method was used to detect autophagy. Flow cytometry was used to detect apopto- sis. Western blotting was used to detect esophageal cancer related gene 4 (ECRG4) protein expression lev- els of transfected cells in each group. Results After A549 cells were infected with Beclin - 1 small inter- feting RNA (siRNA) lentiviral particles, the expression levels of Beclin -1 mRNA and protein were statis- tically decreased (P 〈0. 05) with gene silencing efficacy being 35.56% , 89. 22% and 66. 78% respec- tively. After A549 cells were infected with Beclin - 1 siRNA lentiviral particles, the cell apoptosis rate in siRNA silencing group [ (4. 31 ± 0. 49 ) % ] was significantly reduced ( P 〈 O. 05 ). After A549 cells were infected with Beclin - 1 siRNA lentiviral particles, the population doubling time of A549 cells [ ( 37. 7 ± 2. 6) h] was decreased (P 〉0. 05). After A549 cells were infected with Beelin - 1 siRNA lentiviral parti- cles, the number of autophagy positive cells was decreased ( P 〉 0. 05 ). After A549 cells were infected with Beclin - 1 siRNA lentiviral particles, the ECRG4 expression was decreased ( P 〈 0. 05 ). Conclusion Autophagy - related gene Beclin - 1 can influence autophagy and apoptosis of A549 lung cancer cells via the ECRG4 pathway.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2016年第3期691-694,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金(U1304817)
郑州市科技攻关计划(141PPTGHG298)