FBXL20对人非小细胞肺癌A549细胞生长的影响

目的 观察FBXL20(F-box and leucine rich repeat protein 20) 对人非小细胞肺癌A549细胞生长的影响并初步探究其机制.方法 采用慢病毒感染A549细胞,构建对照组(FBXL20-vector组)及干扰组(FBXL20-RNAi组),通过Western blot验证慢病毒敲减效率.利用CCK-8及集落形成实验观察FBXL20的下调对A549细胞增殖能力的影响,流式细胞术检测细胞周期的变化,Western blot分析周期蛋白的改变,基因集富集分析(gene set enrichment analysis,GSEA) 探究FBXL20可能参与的生物学过程.结果 干扰组FBXL20的蛋白表达(0. 36 ± 0. 02) 显著低于对照组(0. 58 ± 0. 01)(P＜0. 01) .与对照组相比,干扰组的增殖能力增强(P＜0. 05),形成的集落数目也显著增加(100 ± 20 vs 53 ± 6)(P＜0. 05),且停留在G2 /M期的细胞比例明显降低(5. 65 ± 1. 35 vs 9. 94 ± 1. 44)(P＜0. 05) .Western blot结果显示Cyclin D1、ERK1 /2蛋白表达无明显变化(P＞ 0. 05),CDK2、Cyclin E、CDK1、Cyclin A、Cyclin B1蛋白表达显著增加,而p-ERK1 /2则显著降低(P＜0. 05) .GSEA结果显示FBXL20的低表达与G2 /M检查点呈正相关(P＜0. 01) .结论 下调FBXL20可以加快A549细胞G2 /M期的进程,提高其生长能力.其机制可能是通过抑制p-ERK的激活,使得其下游周期蛋白CDK1、Cyclin A、Cyclin B1表达增加而实现的.

Effects of FBXL20 on proliferation in human non-small cell lung cancer A549 cells

Objective To detect the effect of F-box and leucine rich repeat protein 20 (FBXL20) on the proliferation of human non-small cell lung cancer A549 cells and mine the underlying mechanism. Methods Lentiviral vector FBXL20-RNAi was used to transfect A549 cells to knock down the expression of FBXL20, and blank lentiviral vector (FBXL20-vector) was used for control cells. Knockdown efficiency was verified by Western blotting. CCK-8 assay and colony formation assay were used to determine the effects of silenced FBXL20 on the proliferation. Flow cytometry was performed to explore cell cycle alteration. Expression changes of cell cycle proteins were measured by Western blotting. Gene set enrichment analysis(GSEA) was conducted to investigate the biological process involved with FBXL20.Results The protein level of FBXL20 was remarkably lower in the interference cells than the control cells (0.36 ± 0.02 vs 0.58 ± 0.01, P＜0.01). Compared with the control cells, the interference cells had greater proliferation ability(P＜0.05) and increased colony count (100 ± 20 vs 53 ± 6, P＜0.05), and less cells arrested in G2 /M phase (5. 65 ± 1. 35 vs 9. 94 ± 1. 44, P＜0.05). Western blot results showed there were no significant differences in protein levels of Cyclin D1 and ERK1 /2 (P＞0.05), and the protein levels of CDK2, Cyclin E, CDK1, Cyclin A and Cyclin B1 were notably increased while p-ERK1 /2 was distinctly decreased (P＜0.05). GSEA revealed that low level of FBXL20 was positively correlated with G2 /M checkpoint (P＜0.01). Conclusion Down-regulation of FBXL20 can expedite the G2 /M phase of A549 cells and enhance the growth ability, which may due to the suppression of p-ERK activation, and therefore enhancement of the expression of its downstream cell cycle proteins CDK1, Cyclin A and Cyclin B1.