ABSTRACT
Objective To evaluate the effect of small interfering RNA (siRNA)targeting survivin gene on the expression of survivin and proliferation,apoptosis,migration and invasion of a human cutaneous squamous cell carcinoma cell line A431 in vitro.Methods A survivin-specific siRNA was designed and synthesized.Cultured A431 cells were divided into 3 groups to be transfected with 50.0 nmol/L liposome complexes containing survivin-specific siRNA (survivin siRNA group),50.0 nmol/L liposome complexes containing unrelated siRNA (negative control group) and 50.0 nmol/L prepared vesicles (blank control group).Real-time quantitative reverse transcription-PCR (RT-PCR) and Western blot analysis were performed to determine the mRNA and protein expression of survivin in A431 cells,respectively.Methyl thiazolyl tetrazolium (MTT) assay was conducted to evaluate cellular proliferative activity,flow cytometry using annexin-V/propidium iodide (PI) staining to detect cell apoptosis,Transwell assay to estimate migratory and invasive activities of A431 cells,and flow cytometry to detect cell cycle changes.Results At 48 hours after transfection,the mRNA and protein expression of survivin both significantly differed among the survivin siRNA group,negative control group and blank control group (mRNA:0.56 ± 0.15,0.88 ± 0.37,0.90 ± 0.43,F =276.67,P ＜ 0.001;protein:0.59 ± 0.04,0.86 ± 0.05,0.91 ± 0.07,F =243.61,P ＜ 0.001),the survivin siRNA group showed significantly lower mRNA and protein expression of survivin compared with the negative control group and blank control group (all P ＜ 0.05),and there were no significant differences between the negative control group and blank control group (both P ＞ 0.05).Repeated measures analysis of variance showed that the transfection with survivin siRNA could significantly inhibit the proliferation of A431 cells (F =13.19,P =0.004),the proliferation inhibition rate was significantly higher in the survivin siRNA group than in the negative control group and blank control group (both P ＜ 0.05),and no significant difference was observed between the negative control group and blank control group (P ＞ 0.05).At 24 hours after transfection,the apoptosis rate significantly differed among the 3 groups (F =83.97,P =0.002).The survivin siRNA group showed a significantly higher apoptosis rate compared with the negative control group and blank control group (both P ＜ 0.05),and there was no significant difference between the negative control group and blank control group (P ＞ 0.05).At 48 hours after transfection,the survivin siRNA group showed a significantly higher proportion of cells at G2/M phase,but lower number of migratory cells and invasive cells compared with the negative control group and blank control group (all P ＜ 0.05).Conclusion Survivin-specific siRNA can inhibit the expression of survivin gene and the proliferation of A431 cells,promote cell apoptosis,and suppress cell migration and invasion,indicating that survivin may serve as a genetic target for the treatment of cutaneous squamous cell carcinoma.
ABSTRACT
Objective To evaluate effects of antisense oligonucleotides against microRNA-155 (miRNA-155) on the proliferation,apoptosis,migration and invasion of a human cutaneous squamous cell carcinoma cell line A431.Methods A431 cells were divided into 3 groups:nonsense oligonucleotide group transfected with nonsense control oligonucleotides using liposomes,antisense oligonucleotide group transfected with antisense oligonucleotides against microRNA-155 using liposomes,and blank control group treated with Dulbecco's minimum essential medium (DMEM) containing Lipofectamine 2000.Real-time quantitative polymerase chain reaction (qRT-PCR)was performed to determine the expression of miRNA-155 in A431 cells:Methyl thiazolyl tetrazolium (MTT) assay was conducted to estimate cellular proliferative activity at 24,36,72,96 and 120 hours after transfection,flow cytometry to detect apoptosis and cell cycle changes,and Transwell assay to evaluate the migration and invasion of A431 cells.Statistical analysis was carried out by one-way analysis of variance (ANOVA) for intergroup comparisons and by least significant difference (LSD)-t test for multiple comparisons.Results After transfection,there were significant differences in the expression of miRNA-155 among the nonsense oligonucleotide group,antisense oligonucleotide group and blank control group (0.98 ± 0.02,0.28 ± 0.18,1.00 ± 0.01 respectively,F =634.57,P ＜ 0.001),and the expression of miRNA-155 was significantly lower in the antisense oligonucleotide group than in the blank control group and nonsense oligonucleotide group (both P ＜ 0.05).At 72,96 and 120 hours,there were significant differences in the survival rate of A431 cells among the 3 groups (all P ＜ 0.05),and the antisense oligonucleotide group showed a significantly lower survival rate of A431 cells compared with the blank control group and nonsense oligonucleotide group (all P ＜ 0.05).Additionally,the proportions of cells at G0/G1 phase and at S phase,and the cellular proliferative index all significantly differed among the 3 groups (F =23.46,36.81,19.35,respectively,P ＜ 0.01).The antisense oligonucleotide group showed significantly higher proportion of cells at G0/G1 phase (74.63％ ± 2.13％),but lower proportion of cells at S phase (9.88％ ± 1.83％) and cellular proliferative index (25.36 ± 2.13) compared with the blank control group(62.92％ ± 2.56％,18.86％ ± 2.78％,37.08 ± 2.56,respectively,all P ＜ 0.05) and nonsense oligonucleotide group (63.75％ ± 3.06％,18.33％ ± 3.72％,36.25 ± 3.06,respectively,all P ＜ 0.05).Additionally,the antisense oligonucleotide group showed significantly lower numbers of migratory cells and invasive cells compared with the blank control group and nonsense oligonucleotide group (all P ＜ 0.05).Conclusion Transfection of A431 squamous cell carcinoma cells with antisense miRNA-155 oligonucleotides can decrease the expression of miRNA-155,effectively inhibit the proliferation,migration and invasion of A431 cells,and promote cell apoptosis.