自噬基因Beclin-1通过食管癌相关基因4通路影响A549肺癌细胞自噬和凋亡

Effects of autophagy - related gene Beclin - 1 on autophagy and apoptosis of A549 lung cancer cells via esophageal cancer related gene 4 pathway

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.