长非编码RNA AFAP1-AS1对甲状腺癌细胞增殖和侵袭的影响及其机制

doi:10.3760/cma.j.cn371439-20191129-00030

摘要/Abstract

摘要：

目的研究长非编码RNA(lncRNA)肌动蛋白纤维相关蛋白1-反义RNA1(AFAP1-AS1)对甲状腺癌细胞增殖和侵袭的影响,并探讨其机制。方法采用实时荧光定量PCR(qRT-PCR)检测正常甲状腺和甲状腺癌细胞株中AFAP1-AS1的表达。将甲状腺癌细胞株WRO分成3组,AFAP1-AS1沉默表达组(AFAP1-AS1-siRNA组)、阴性对照组(NT-siRNA组)及空白对照组(Blank组),AFAP1-AS1-siRNA组和NT-siRNA组采用Lipofectamine^TM 3000分别转染AFAP1-AS1-siRNA、NT-siRNA,Blank组加入PBST对照。CCK-8检测3组细胞增殖能力,Transwell实验检测侵袭能力,Western blotting检测Rho A、Cyclin D1和MMP-9蛋白的表达情况。结果正常甲状腺细胞株FRTL-5、甲状腺癌细胞株SW579、CAL-62、FRO、WRO的AFAP1-AS1相对表达量分别为1.03±0.04、2.95±0.17、5.31±0.35、7.26±0.49、9.67±0.53,5组间比较差异具有统计学意义(F=16.932,P=0.027);AFAP1-AS1在WRO细胞中表达量最高,因此,选择WRO细胞进行后续实验。AFAP1-AS1-siRNA组、NT-siRNA组、Blank组3组细胞AFAP1-AS1的相对表达量分别为0.23±0.02、1.02±0.04、1.03±0.05,差异具有统计学意义(F=13.590,P=0.006),与NT-siRNA组相比,AFAP1-AS1-siRNA组AFAP1-AS1表达量显著降低(P<0.001)。3组细胞转染后3、4 d的A₄₅₀值分别为0.68±0.06、1.17±0.09、1.22±0.09,0.96±0.08、1.69±0.11、1.72±0.12,差异均具有统计学意义(F=7.318,P=0.016;F=10.351,P=0.004),第3、4 d AFAP1-AS1-siRNA组、NT-siRNA组两组比较,差异均有统计学意义(P=0.043;P=0.013)。3组侵袭细胞数分别为(72.8±5.7)个、(145.6±8.9)个、(148.4±7.3)个,差异具有统计学意义(F=37.273,P=0.034),AFAP1-AS1-siRNA组侵袭细胞数少于NT-siRNA组(P=0.021)。3组细胞Rho A蛋白表达量分别为0.34±0.03、1.02±0.04、1.04±0.03,差异具有统计学意义(F=9.667,P=0.013),AFAP1-AS1-siRNA组Rho A蛋白表达量显著低于NT-siRNA组(P=0.018);3组Cyclin D1蛋白表达量分别为0.52±0.04、1.03±0.02、1.05±0.04,差异具有统计学意义(F=15.464,P=0.010),AFAP1-AS1-siRNA组Cyclin D1蛋白表达量显著低于NT-siRNA组(P=0.023);3组MMP-9蛋白表达量分别为0.42±0.04、1.05±0.03、1.02±0.04,差异有统计学意义(F=10.328,P=0.032),AFAP1-AS1-siRNA组MMP-9蛋白表达量显著低于NT-siRNA组(P=0.035)。结论 AFAP1-AS1沉默可抑制甲状腺癌细胞增殖、侵袭,其机制可能与下调Rho A、Cyclin D1、MMP-9蛋白表达相关。

关键词: 甲状腺肿瘤, RNA,长链非编码, 细胞增殖, 肿瘤侵润, 肌动蛋白纤维相关蛋白1-反义RNA1

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^TM 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.

Key words: Thyroid neoplasms, RNA,long noncoding, Cell proliferation, Neoplasm invasiveness, Actinfilament-associated protein 1-antisense RNA1

程虎, 刘名奎, 陈天平. 长非编码RNA AFAP1-AS1对甲状腺癌细胞增殖和侵袭的影响及其机制[J]. 国际肿瘤学杂志, 2020, 47(6): 327-332.

Cheng Hu, Liu Mingkui, Chen Tianping. Effects of lncRNA AFAP1-AS1 on proliferation and invasion of thyroid cancer cells and its mechanisms[J]. Journal of International Oncology, 2020, 47(6): 327-332.

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组别	A₄₅₀值
组别	0 d	1 d	2 d	3 d	4 d
AFAP1-AS1-siRNA组	0.26±0.02	0.37±0.04	0.51±0.05	0.68±0.06^a	0.96±0.08^b
NT-siRNA组	0.25±0.02	0.43±0.05	0.75±0.07	1.17±0.09	1.69±0.11
Blank组	0.26±0.02	0.44±0.04	0.75±0.07	1.22±0.09	1.72±0.12
F值	5.476	3.654	2.478	7.318	10.351
P值	0.076	0.082	0.276	0.016	0.004