lncRNA MIR31HG 对甲状腺乳头状癌细胞 增殖、迁移、侵袭的影响及作用机制研究

Effect and mechanism of lncRNA MIR31HG on proliferation, migration and invasion of TPC-1 cell line

摘要:

目的 探究长链非编码RNA（lncRNA）MIR31HG对甲状腺乳头状癌（PTC）细胞增殖、迁 移、侵袭的影响及可能的作用机制。方法 选取2018 年8 月—2019 年4 月在湖南省中医药大学第一附属 医院行甲状腺癌根治术的48 例患者的新鲜癌组织及癌旁组织标本，男女各24 例。利用TCGA 数据库验证 lncRNA MIR31HG 在甲状腺癌组织与癌旁组织中的表达，患者术后病理报告确诊为PTC，提取样本RNA 行 qRT-PCR 验证lncRNA MIR31HG 在PTC 组织与癌旁组织中表达情况及与患者临床病理特征的相关性；利 用lncRNA MIR31HG 的小干扰RNA 转染PTC 细胞系TPC-1 并验证抑制率，保证抑制率>60%，MTT 检测 TPC-1 细胞增殖能力，细胞划痕实验和Transwell 小室实验检测细胞迁移及侵袭能力，Western blotting 检测 Akt 信号通路关键蛋白分子Akt、磷酸化Akt（p-Akt）、PTEN 的表达。结果 宫颈鳞状细胞癌、乳头状肾细 胞癌、头颈部鳞癌、胰腺癌及甲状腺癌组织lncRNA MIR31HG 相对表达量较癌旁组织高（P <0.05），膀胱癌、 前列腺癌组织较癌旁组织低（P <0.05）。PTC 组织lncRNA MIR31HG 相对表达量较癌旁组织高（P <0.05）。 TPC-1、K1 及BCPAP 的lncRNA MIR31HG 相对表达量较Nthy-ori 3-1 高（P <0.05），且TPC-1 相对表 达量最高。实验组lncRNA MIR31HG 相对表达量均较siRNA-NC 组下降（P <0.05），其中siRNA-1 组与 siRNA-3 组沉默效率均>60%。各组OD 值比较，差异有统计学意义（P <0.05）。siRNA-1 组与siRNA-3 组 划痕愈合率较siRNA-NC 组低（P <0.05）。siRNA-1 组、siRNA-3 组侵袭至Transwell 小室下室的细胞数较 siRNA-NC 组低（P <0.05）。siRNA-1 组、siRNA-3 组PTEN mRNA 和蛋白较siRNA-NC 组升高（P <0.05）， 且siRNA-3 组最高；siRNA-1 组、siRNA-3 组p-Akt 蛋白较siRNA-NC 组低，且siRNA-3 组最低（P <0.05）。 结论 lncRNA MIR31HG 在PTC 组织中高表达并与PTC 患者肿瘤分期有关；下调lncRNA MIR31HG 的表 达可以抑制TPC-1 细胞增殖、迁移及侵袭能力，其机制可能与lncRNA MIR31HG 抑制PTEN 表达从而激 活Akt 通路有关。

关键词:癌，乳头状;甲状腺;长链非编码RNA/ 核糖核酸酶类

Abstract:

Objective To explore the effects of long-chain non-coding MIR31HG on cell proliferation, migration and invasion of papillary thyroid carcinoma (PTC) cells and its possible mechanisms. Methods Fresh specimens were collected from 48 patients (male: 24 females, females: 24 patients), all of them were confirmed as PTC by postoperative pathological report. TCGA database was used to verify the expression of lncRNA MIR31HG in thyroid carcinoma and paracanceroustissues; the specimens RNA was extracted and qRT-PCR was used to verify the expression of lncRNA MIR31HG in PTC tissues and paracancerous tissues. Then we transfected PTC cell line TPC- 1 with small interfering RNAs of lncRNA MIR31HG and verified transfection efficiency, ensured the inhibition rate of lncRNA MIR31HG was above 60%. The proliferation ability of TPC-1 cells was detected by MTT colorimetric assay. Cell migration and invasion ability were detected by cell scratch assay and transwell chamber assay. Western blotting was used to detect the expression of key protein molecules Akt, phosphorylated Akt (p-Akt) and PTEN in Akt signaling pathway. Results The relative expression of lncRNA MIR31HG in cervical squamous cell carcinoma, papillary renal cell carcinoma, head and neck squamous cell carcinoma, pancreatic carcinoma and thyroid carcinoma was higher than that in paracancerous tissues (P < 0.05), while that in bladder cancer and prostate cancer was lower than that in paracancerous tissues (P < 0.05). The relative expression of lncRNA MIR31HG in PTC tissue was higher than that in paracancerous tissue (P < 0.05). The relative expression of MIR31HG in TPC-1, K1 and BCPAP was higher than that in Nthy-ori 3-1 (P < 0.05), and the relative expression of TPC-1 was the highest. The relative expression of lncRNA MIR31HG in experimental group was lower than that in siRNA-NC group (P < 0.05), and the silencing efficiency of siRNA-1 group and siRNA-3 group was more than 60%. There was statistical significance in OD value of each group (P < 0.05). The scratch healing rate of siRNA-1 group and siRNA-3 group was lower than that of siRNA-NC group (P < 0.05). The number of cells invading the subventricular space of Transwell in siRNA-1 and siRNA-3 groups was lower than that in siRNA-nc group (P < 0.05). PTEN mRNA and protein in siRNA-1 group and siRNA-3 group were higher than those in siRNA-NC group (P < 0.05), and the highest in siRNA-3 group; p-Akt protein in siRNA-1 group and siRNA-3 group was lower than that in siRNA-NC group, and the lowest in siRNA-3 group (P < 0.05). Conclusions lncRNA MIR31HG is highly expressed in PTC tissues and positively correlated with tumor stage in PTC patients; inhibition of lncRNA MIR31HG expression can inhibit the proliferation ability, migration ability and invasion ability of TPC-1 cell. Its mechanism may be related to the inhibition of PTEN expression by lncRNA MIR31HG to activate the Akt pathway.

Key words:cancer, papillary; thyroid; long-chain non-coding rna / ribonucleases