微小RNA-1243对人甲状腺乳头状癌TPC-1细胞增殖和迁移的作用及其机制

Mechanism of microRNA-1243 on proliferation and migration of human papillary thyroid carcinoma TPC-1 cells

目的观察微小RNA(miRNA,miR)-1243过表达对人甲状腺乳头状癌细胞TPC-1增殖和迁移的生物学影响。方法利用实时定量反转录聚合酶链反应(RT-qPCR)技术检测甲状腺乳头状癌2×105个TPC-1细胞不同转染组miR-1243的表达,水溶性甲臢化合物(MTS)法检测2×10^4个细胞的增殖能力及迁移小室(Transwell)检测1×10^5对数生长期的TPC-1细胞的迁移能力。利用生物信息学软件预测miR-1243的靶基因。采用双荧光素酶报告基因验证miR-1243对靶基因的直接调控。结果空白对照和阴性对照组中miR-1243的表达量分别为0.97±0.31,1.208±0.40,显著低于miR-1243模拟物转染组932.84±52.34,差异有统计学意义(P=0.000);细胞转染48h后,空白对照及阴性对照组细胞相对存活率分别为4.82±0.10和4.91±0.09,显著高于miR-1243模拟物转染组4.41±0.06,细胞转染miR-1243的增殖能力被显著抑制(P=0.032)。miR-1243模拟物转染组迁移细胞数为15.5±6.5,均少于空白对照组33.5±9.5和阴性对照组31.75±10.75,差异均有统计学意义(P=0.016)。经过生物信息学网站miRanda和RNAhybrid共同分析,可见AKT丝氨酸/苏氨酸激酶1(AKT1)mRNA是miR-1243的潜在靶基因。双荧光素酶报告基因结果显示,与空载体组(98.97±9.96),突变组(85.02±11.04)及阴性对照模拟物组(97.38±10.29)比较,共转染miR-1243模拟物的野生型组荧光素酶活性显著下降(45.92±10.91)(P=0.005),证实miR-1243直接调控制AKT1表达。结论过表达miR-1243可以抑制TPC-1细胞的增殖和迁移能力。miR-1243可能通过直接靶向AKT1参与调控甲状腺乳头状癌的进展。

Objective To observe the biological effects of microRNA (miRNA, miR)-1243 overexpression on proliferation and migration of human papillary thyroid carcinoma cell line TPC-1. Methods The expression of miR-1243 in 2×10^5 TPC-1 cells was detected by real-time quantitative polymerase chain reaction (RT-qPCR). The capacities of migration and the migration in TPC-1 cells were tested by water soluble methacon compounds (MTS) or Transwell assays, respectively. The candidate targets for miR-1243 were investigated using predication algorithm provided by miRanda and RNAhybrid. Luciferase activity was measured by dual luciferase reporter assay. Results The expression levels of miR-1243 in blank control and negative control groups were 0.97±0.31 and 1.208±0.40 respectively, significantly lower than 932.84±52.34 in miR-1243 mimic transfected group (P=0.000). The relative survival rates of the blank control group and the negative control group were 4.82±0.10 and 4.91±0.09, respectively, which were significantly higher than that of 4.41±0.06 in miR-1243 mimic transfected group (P=0.032). The proliferation ability of TPC-1 cells transfected with miR-1243 was significantly inhibited. The number of migratory cells in the miR-1243 mimic group was 15.5±6.5, which was significantly less than that in the blank control group (33.5±9.5) and the negative control group (31.75±10.75) (P=0.016). The bioinformatics analysis revealed that the AKT serine/threonine kinase 1 (AKT1) mRNA may be the target of miR-1243. The results of dual luciferase assay showed that WT 3’ untranslated region (UTR) of AKT1 cotransfected with miR-1243 mimics significantly decreased luciferase activity (45.92±10.91) as compared with empty vector group (98.97±9.96), mutant group (85.02±11.04) and negative control mimic group (97.38±10.29) (P=0.005). Conclusion Overexpression of miR-1243 can inhibit the proliferation and migration of TPC-1 cells. The miR-1243 may be involved in the regulation of thyroid papillary carcinoma by direct targeting of AKT1.