Beclin1过表达对黑素瘤SK-MEL-2细胞生物学行为的影响

Effect of Beclin1 overexpression on biological behaviors of SK-MEL-2 human malignant melanoma cells

目的探讨自噬标志基因Beclin1过表达对黑素瘤SK-MEL-2细胞生物学行为的影响。方法Western印迹技术检测黑素瘤细胞A375、SK-MEL-2中Beclin1的蛋白表达水平,选取低表达Beclin1蛋白的SK-MEL-2细胞株作为研究对象,将细胞分为空白组、阴性对照组、实验组,空白组不作处理,阴性对照组转染pcDNA.3.1/myc-His(-)A,实验组转染pcDNA3.1-Beclin1质粒。培养一定时间后,采用CCK8法分析Beclin1对细胞增殖的影响;Transwell实验和细胞划痕实验观察Beclin1过表达对SK-MEL-2细胞侵袭和迁移能力的影响。采用重复测量方差分析和完全随机设计方差分析检验各组间的指标差异,组间比较采用LSD-t检验。结果SK-MEL-2细胞Beclin1蛋白相对表达水平(0.037±0.010)显著低于A375细胞(0.670±0.150),F=46.62,P<0.05。实验组Beclin1蛋白相对表达水平(0.32±0.04)显著高于阴性对照组(0.06±0.02)和空白组(0.07±0.02),均P<0.05。CCK8实验结果显示,不同组间、不同时间细胞增殖率差异均有统计学意义(F组间=1077.36,F时间=4903.04,均P<0.05),组别和时间之间有交互作用(F交互=205.20,P<0.05)。Transwell实验显示,24 h后实验组高倍(×200)视野下穿过小室的细胞数(18.67±1.19)明显低于阴性对照组(87.89±6.05)和空白组(86.78±5.93),均P<0.05;划痕实验中24、48 h时实验组细胞迁移距离均显著低于空白组和阴性对照组(P<0.05)。结论Beclin1过表达可显著抑制SK-MEL-2细胞的增殖、侵袭和迁移。

Objective To evaluate the effect of overexpression of the autophagy marker gene Beclin1 on biological behaviors of SK-MEL-2 human malignant melanoma cells.Methods Western blot analysis was performed to determine the protein expression of Beclin1 in melanoma cell lines A375 and SK-MEL-2.SK-MEL-2 cells with low Beclin1 protein expression were selected as research objects,and divided into 3 groups:blank group receiving no treatment,negative control group transfected with pcDNA.3.1/myc-His(-)A,and experimental group transfected with pcDNA3.1-Beclin1 plasmid.After 2-week culture,cell counting kit-8(CCK-8)assay was conducted to evaluate the effect of Beclin1 on cell proliferation at 24,48 and 72 hours,and Transwell assay and wound-healing assay were performed to assess the effect of Beclin1 overexpression on the invasion and migration abilities of SK-MEL-2 cells.Repeated measures analysis of variance and completely randomized analysis of variance were used to analyze differences in indices among groups,and least significant difference(LSD)-t test was used for multiple comparisons.Results The protein expression of Beclin1 was significantly lower in the SK-MEL-2 cells(0.037±0.010)than in the A375 cells(0.670±0.150,F=46.62,P<0.05).The experimental group showed significantly increased protein expression of Beclin1(0.32±0.04)compared with the negative control group(0.06±0.02,P<0.05)and blank group(0.07±0.02,P<0.05).CCK-8 assay revealed a significant difference in the cell proliferation rate among different groups and different time points(F=1077.36,4903.04 respectively,both P<0.05),and there was a significant interaction between the transfection treatment and time(F=205.20,P<0.05).Transwell assay showed that the number of SK-MEL-2 cells crossing the chamber per high-power field(×200)after 24-hour treatment was significantly lower in the experimental group(18.67±1.19)than in the negative control group(87.89±6.05,P<0.05)and blank group(86.78±5.93,P<0.05).In the wound-healing assay,the cell migration distance was significantly shorter in the experimental group than in the blank group and negative control group at 24 and 48 hours(all P<0.05).Conclusion Beclin1 overexpression can markedly inhibit the proliferation,invasion and migration of SK-MEL-2 cells.