摘要
目的研究miR-1182在胃癌细胞中调控人端粒酶反转录酶(human telomerase reverse transcriptase,hTERT)的机制及对其迁移能力的影响。方法胃癌细胞MKN28中转染miR-1182类似物,qRT-PCR检测转染后细胞miR-1182的相对表达量,Western blot检测转染后细胞hTERT的表达变化;进一步通过生物信息学预测miR-1182与hTERT mRNA的结合位点,双荧光素酶实验分析miR-1182对hTERT mRNA的作用机制;Transwell实验检测转染miR-1182类似物对MKN28细胞体外迁移能力的影响。结果 qRT-PCR表明miR-1182组的miR-1182的相对表达量(10.168±2.645)明显高于对照组(1.008±0.167)(P<0.01)。Western blot结果显示在胃癌细胞系中过表达miR-1182后,hTERT的蛋白水平下调。双荧光素酶实验表明miR-1182可与hTERT mRNA的ORF区结合,且其主要结合部位为hTERT mRNA的ORF-1;在Transwell迁移实验中,miR-1182类似物转染细胞后,miR-1182组穿膜细胞数为(23.333±4.509)/视野,对照组穿膜细胞数为(71.000±4.582)/视野,miR-1182组细胞迁移能力明显低于对照组(P<0.01)。结论 miR-1182通过与胃癌细胞hTERT的ORF区结合,从而在转录后水平抑制hTERT的表达,并发现其可抑制胃癌细胞的迁移能力。
Objective To investigate the mechanism of miR-1182 regulating human telomerase reverse transcriptase （hTERT） in gastric cancer cells and to study the effect of miR-1182 on the migration capability of gastric cancer cells. Methods MKN28 gastric cancer cells were transfected with miR-1182 mimics. Quantita- tive RT-PCR was used to test the level of miR-1182, and Western blot analysis to detect the expression of hTERT. Bioinformatic analysis was carried out to predict the possible binding sites of miR-1182 to hTERT mRNA. Dual-luciferase assay was adopted to analyze the mechanism of miR-1182 acting on hTERT. Transwell migration assay was used to analyze cell migration ability. Results The results of quantitative RT-PCR indica- ted that the relative expression of miR-1182 in the miR-1182 mimics treatment group was higher than that in the control group （10. 168 ±2. 645 vs 1. 008±0. 167, P 〈0. 01 ）. Western blot results suggested that over-expres- sion of miR-1182 could inhibit the expression of hTERT. Dual-luciferase assay results suggested that miR-1182 bond to the open reading frame（ORF） of hTERT, with ORF-1 as the main target site. In Transwell migration assay, the cells passing through the membrane were 23. 333 ± 4. 509 in visual field in the miR-1182 mimics treatment group and 71.000 ±4. 582 in the control group. The migration ability of the cells in the miR-1182 mimics treatment group was significantly lower than that in the control group （P 〈 0. 01 ）. Conclusion MiR- 1182 down-regulates hTERT expression by binding to its ORF, and also inhibits the migration capability of gastric cancer cells.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2014年第10期1074-1077,共4页
Journal of Third Military Medical University
