Effects of lncRNA AFAP1-AS1 on proliferation and invasion of thyroid cancer cells and its mechanisms / 国际肿瘤学杂志

ABSTRACT

Objective:To explore the effects of long noncoding RNA (lncRNA) actinfilament-associated protein 1-antisense RNA1 (AFAP1-AS1) on proliferation and invasion of thyroid cancer cells and its mechanisms.Methods:Quantitative real time fluorescence PCR (qRT-PCR) was performed to assess the expression of AFAP1-AS1 in normal thyroid cells and thyroid cancer cells. The thyroid cancer cell line WRO was divided into three groups, AFAP1-AS1 silencing group (AFAP1-AS1-siRNA group), negative control group (NT-siRNA group) and blank control group (Blank group). AFAP1-AS1-siRNA group and NT-siRNA group were transfected with AFAP1-AS1 siRNA and NT-siRNA respectively using Lipofectamine? 3000, and Blank group was treated with PBST. The proliferation ability was measured by CCK-8. The invasion ability was measured by Transwell assay. The expression levels of Rho A, Cyclin D1 and MMP-9 protein were measured by Western blotting.Results:The relative expressions of AFAP1-AS1 in normal thyroid cell line FRTL-5, thyroid cancer cell lines SW579, CAL-62, FRO and WRO were 1.03±0.04, 2.95±0.17, 5.31±0.35, 7.26±0.49 and 9.67±0.53 respectively, and the difference among the five groups was statistically significant ( F=16.932, P=0.027). The expression of AFAP1-AS1 was highest in WRO cells, therefore, the WRO cells were selected for subsequent experiments. The relative expressions of AFAP1-AS1 in AFAP1-AS1-siRNA group, NT-siRNA group and Blank group were 0.23±0.02, 1.02±0.04 and 1.03±0.05 respectively, and the difference among the three groups was statistically significant ( F=13.590, P=0.006). Compared with NT-siRNA group, the expression of AFAP1-AS1 in AFAP1-AS1-siRNA group was significantly lower ( P<0.001). The A450 values in the three groups were 0.68±0.06, 1.17±0.09, 1.22±0.09, and 0.96±0.08, 1.69±0.11, 1.72±0.12 respectively 3 and 4 days after transfection, and the differences were statistically significant ( F=7.318, P=0.016; F=10.351, P=0.004). The differences between AFAP1-AS1-siRNA group and NT-siRNA group 3 and 4 days after transfection were statistically significant ( P=0.043; P=0.013). The numbers of invasive cells in the three groups were 72.8±5.7, 145.6±8.9, 148.4±7.3, and the difference was statistically significant ( F=37.273, P=0.034). The number of invasive cells in AFAP1-AS1-siRNA group was significantly less than that in NT-siRNA group ( P=0.021). The expressions of Rho A protein in the three groups were 0.34±0.03, 1.02±0.04 and 1.04±0.03 respectively, and the difference was statistically significant ( F=9.667, P=0.013). The expression of Rho A protein in AFAP1-AS1-siRNA group was significantly lower than that in NT-siRNA group ( P=0.018). The expressions of Cyclin D1 protein in the three groups were 0.52±0.04, 1.03±0.02 and 1.05±0.04 respectively, with a statistically significant difference ( F=15.464, P=0.010). The expression of Cyclin D1 in AFAP1-AS1-siRNA group was significantly lower than that in NT-siRNA group ( P=0.023). The expressions of MMP-9 protein in the three groups were 0.42±0.04, 1.05±0.03 and 1.02±0.04 respectively, and the difference was statistically significant ( F=10.328, P=0.032). The expression of MMP-9 in AFAP1-AS1-siRNA group was significantly lower than that in NT-siRNA group ( P=0.035). Conclusion:The silencing of lncRNA AFAP1-AS1 can inhibit the proliferation and invasion of thyroid cancer cells, and the mechanism may be related to the down-regulation of Rho A, Cyclin D1 and MMP-9 proteins.