Effects of a short hairpin RNA targeting STAT3 gene on the biological behavior of a human malignant melanoma cell line A375 / 中华皮肤科杂志

ABSTRACT

Objective To study the effects of short hairpin RNA (shRNA) targeting STAT3 gene on the proliferation and apoptosis of A375 human malignant melanoma cells.Methods Sense and antisense oligonucleotides with small hairpin structures targeting STAT3 gene were designed,synthesized and cloned into the plasmid vector psiRNA-hHlneo after annealing.Cultured A375 cells were divided into 3 groups control group receiving no treatment,psiRNA-H1 group transfected with empty plasmid,and psiRNA-H1/STAT3 group transfected with the recombinant plasmid containing the shRNA.After additional culture for different durations,reverse transcription PCR and Western blot were performed to detect the expression of STAT3 mRNA and protein,methyl thiazolyl tetrazolium (MTT) assay to evaluate cell proliferation,flow cytometry to assess cell cycle and apoptosis.Results The expression level of STAT3 mRNA and protein in A375 cells in psiRNA-H1/STAT3 group (0.2136 ± 0.0626,0.8214 ± 0.043,respectively) were significantly lower than that in the control group (0.7826 ± 0.0701,3.1693 ± 0.0846,respectively,both P ＜ 0.01) and psiRNA-H1 group (0.8518 ± 0.0783,3.218 ± 0.078,respectively,both P ＜ 0.01 ).The inhibition rates of cell proliferation at 24,48 and 72 hours were 21.35％ ± 2.12％,32.52％ ± 2.64％ and 40.4％ ± 3.08％ respectively in psiRNA-H1/STAT3 group,statistically different from those in the control group (1.39％ ± 0.53％,3.05％ ± 1.16％,4.41％± 1.42％,respectively,all P ＜ 0.01) and psiRNA-H1 group (2.63％ ± 1.38％,5.84％ ± 2.47％,10.32％ ±2.48％,respectively,all P ＜ 0.01).Flow cytometry showed a statistical increase in cell apoptosis rate in psiRNA-H1/STAT3 group compared with the control and psiRNA-H1 group (81.06％ ± 2.10％ vs.26.28％ ±0.47％ and 27.31％ ± 1.05％,both P ＜ 0.01 ).The psiRNA-H1/STAT3 group exhibited a higher percentage of cells at G0/G1 phase (68.43％ ± 4.00％) but a lower percentage of cells at S phase (17.4％ ± 2.05％) compared with the control group (60.07％ ± 2.47％,P ＜ 0.05; 28.40％ ± 2.09％,P ＜ 0.01 ) and psiRNA-H1 group (60.29％ ± 2.26％,27.34％ ± 3.63％,both P ＜ 0.05 ).Conclusions The small interference RNA targeting STAT3 gene can specifically down-regulate the expressions of STAT3 mRNA and proteins in,inhibit cellular proliferation of,and induce apoptosis in,A375 cells.