敲低长链非编码RNA AFAP1-AS1抑制TPC-1甲状腺乳头状癌细胞上皮间质转化及其侵袭和迁移

Knockdown of long non-coding RNA actin filament-associated protein 1 antisense RNA1 (AFAP1-AS1) inhibits epithelial-mesenchymal transition,invasion and migration of TPC-1 papillary thyroid cancer cells

目的检测甲状腺乳头状癌组织长链非编码RNA(lncRNA)肌动蛋白丝相关蛋白1反义RNA1(AFAP1-AS1)的表达,敲低TPC-1甲状腺乳头状癌细胞AFAP1-AS1,研究其对TPC-1细胞上皮间质转化(EMT)的影响及相关分子机制。方法采用实时定量PCR检测60例甲状腺乳头状癌组织lncRNA AFAP1-AS1表达;利用RNA干扰技术(RNAi)敲低TPC-1甲状腺乳头状癌细胞AFAP1-AS1,设置AFAP1-AS1敲低(shAFAP1-AS1)组和阴性对照RNA(shNC)组及未转染的TPC-1细胞为对照组。通过集落形成实验检测TPC-1细胞集落形成能力、 TranswellTM侵袭实验检测细胞侵袭能力,划痕愈合实验检测细胞的迁移能力;通过实时定量PCR检测敲低AFAP1-AS1后,细胞上皮钙黏素(E-cadherin)、波形蛋白(vimentin)、β联蛋白(β-catenin)、锌指转录因子snail2的mRNA水平, Western blot法检测其蛋白水平。结果与癌旁组比较,甲状腺乳头状癌组中AFAP1-AS1 mRNA表达水平显著增加;敲低AFAP1-AS1后, TPC-1细胞的集落形成能力、侵袭和迁移能力均显著降低;与shNC组和对照组相比,敲低AFAP1-AS1后,细胞snail2、 vimentin和β-catenin的mRNA和蛋白表达显著降低,而E-cadherin的mRNA和蛋白表达显著增加。结论 lncRNA AFAP1-AS1在甲状腺乳头状癌组织高表达,敲低TPC-1细胞AFAP1-AS1后,癌细胞的集落形成能力、侵袭和迁移能力显著下调,可能与抑制细胞EMT过程有关。

Objective To detect the expression of long non-coding RNA(lncRNA) actin filament-related protein 1 antisense RNA1(AFAP1-AS1) in papillary thyroid carcinoma tissue, and to investigate the effects of the knockdown of AFAP1-AS1 in TPC-1 papillary thyroid carcinoma cells on cell epithelial-mesenchymal transition(EMT) and related molecular mechanism in TPC-1 cells. Methods Real-time quantitative PCR was used to detect the expression of lncRNA AFAP1-AS1 in 60 cases of papillary thyroid carcinoma tissues. RNA interfering(RNAi) was used to knockdown AFAP1-AS1 in TPC-1 cells. TPC-1 cells were divided into AFAP1-AS1 knockdown(shAFAP1-AS1) group, negative control RNA(shNC) group and untransfected control group. The colony-formation assay, TranswellTM invasion and scratch healing assays were employed to detect the colony-forming ability, cell invasion ability and cell migration ability of TPC-1 cells, respectively. After knockdown of AFAP1-AS1, real-time quantitative PCR and Western blot analysis were used to detect the mRNA and protein levels of E-cadherin, vimentin, β-catenin and snail2, respectively. Results Compared with the paracancerous tissue, the expression level of AFAP1-AS1 mRNA in the papillary thyroid carcinoma tissue significantly increased. Knockdown of AFAP1-AS1 significantly reduced the colony-forming ability, invasion and migration ability of TPC-1 cells. Compared with shNC group andcontrol group, knockdown of AFAP1-AS1 significantly reduced the mRNA and protein expression of snail2, vimentin and β-catenin. In contrast, the mRNA and protein expression of E-cadherin increased considerably. Conclusion The lncRNA AFAP1-AS1 is highly expressed in papillary thyroid carcinoma tissue. After knockdown of AFAP1-AS1 in TPC-1 cells, the colony-forming ability, invasion and migration ability of cancer cells are significantly down-regulated, which may be related to the inhibition of EMT.