Beclin1基因在SW620细胞自噬和凋亡中作用

Role of Beclin 1 gene in autophagy and apoptosis of SW620 cells

目的探讨Beclin1基因在SW620细胞自噬和凋亡中作用。方法采用RNA干扰技术抑制Beclin1表达,Beclin1被抑制后细胞的存活能力通过MTT实验评估;结直肠癌细胞SW620在血清剥夺状态下自噬活性通过LC3蛋白水平评估;分别经血清剥夺、星孢菌素及依托泊苷处理的SW620凋亡率通过流式细胞仪检测;Beclin1表达情况经Western blot评估。结果 pSUPERBecl组细胞血清剥夺后存活能力显著降低(P<0.05),在血清剥夺24 h后,空白对照组和p SUPER-non组抗凋亡能力比p SUPERBecl组更强(P<0.05),星孢菌素处理后,pSUPER-Becl组细胞凋亡最为显著(P<0.05),用依托泊苷处理后得到相似的结果(P<0.05);pSUPER-non组与空白对照组细胞中Beclin1的表达随血清剥夺时间的延长呈现上调趋势(P<0.05),这与LC3的表达变化相一致;而pSUPER-Becl组LC3的表达显著减少(P<0.05)。结论 Beclin1是结直肠癌细胞中自噬和凋亡的关键调节因素,并且维持凋亡和自噬的平衡;Beclin1通过调节细胞自噬活性,在结直肠癌发展与演进中作为一种保护机制,使肿瘤细胞免受到低营养和化疗所致的损伤而持续生存。

Objective To study the role of Beclin1 gene in autophagy and apoptosis of SW620 cells. Methods RNA interference was used to knockdown the expression of Beclin 1 in SW620 cells,and the cell viability was measured by MTT assays. The autophagic activity in serum-starved SW620 CRC cells was evaluated by assessing endogenous microtubule-associated protein1 light chain 3(LC3) protein levels using immunofluorescence assay. Flow cytometry was used to measure the apoptosis rate of SW620 cells treated with serum deprivation,staurosporine or etoposide,and the protein expression of Beclin1 was detected using Western blotting. Results The viability of cells in p SUPER-Becl group was significantly reduced after serum deprivation(P<0.05). Serum deprivation for 24 h resulted in a stronger apoptotic resistance in cells in the control and p SUPER-non groups than in p SUPER-Becl group(P<0.05). Treatment with staurosporine the most significantly increased the cell apoptosis in p SUPER-Becl group(P<0.05),and similar effect was observed with etoposide treatment(P<0.05). As the time of serum deprivation extended,the expression of Beclin 1 in control group and p SUPER-non group increased progressively(P<0.05),which was consistent with the changes in LC3 expression; LC3 expression in p SUPER-Becl group decreased significantly with a prolonged serum starvation(P<0.05). Conclusion Beclin 1 is a crucial regulator of autophagy and apoptosis in colorectal cancer cells to maintain the balance between autophagy and apoptosis. Beclin 1 may serve as a protective mechanism to protect colorectal cancer cells from injury caused by low nutrition and chemotherapy byregulating cell autophagy.