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纸质出版:2014
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张丹,郝宁波,唐波,吕沐瀚,谢睿,胡长江,汪苏敏,杨仕明.miR-1182调控胃癌细胞人端粒酶反转录酶的分子机制及其对迁移能力的影响[J].陆军军医大学学报(原第三军医大学学报),2014,36(10):1074-1077.
Zhang Dan,Hao Ningbo,Tang Bo,Lyu Muhan,Xie rui,Hu ChangJiang,Wang Sumin,Yang Shiming.Mechanism of miR-1182 regulating human telomerase reverse transcriptase in gastric cancer cells and its effect on cell migration capability[J].J Amry Med Univ (J Third Mil Med Univ),2014,36(10):1074-1077.
张丹,郝宁波,唐波,吕沐瀚,谢睿,胡长江,汪苏敏,杨仕明.miR-1182调控胃癌细胞人端粒酶反转录酶的分子机制及其对迁移能力的影响[J].陆军军医大学学报(原第三军医大学学报),2014,36(10):1074-1077. DOI:
Zhang Dan,Hao Ningbo,Tang Bo,Lyu Muhan,Xie rui,Hu ChangJiang,Wang Sumin,Yang Shiming.Mechanism of miR-1182 regulating human telomerase reverse transcriptase in gastric cancer cells and its effect on cell migration capability[J].J Amry Med Univ (J Third Mil Med Univ),2014,36(10):1074-1077. DOI:
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(P0.01)。 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. Quantitative
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 indicated 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-expression 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.
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