ABSTRACT
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 = 1 077.36, 4 903.04 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.
ABSTRACT
Objective To evaluate the effect of overexpression of the autophagy marker gene Beclin I on biological behaviors of SK-MEL-2 human malignant melanoma cells.Methods Western blot analysis was performed to determine the protein expression of Beclin 1 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 Beclin 1 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 =1 077.36,4 903.04 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 Beclin 1 overexpression can markedly inhibit the proliferation,invasion and migration of SK-MEL-2 cells.