LncRNA-PVT 1/miR-15 a/Bmi-1通路调控HGC-27胃癌细胞的体外增殖

LncRNA-PVT 1/miR-15 a/Bmi-1 Path way Regulates the Proliferation of HGC-27 Gastric Cancer Cells in Vitro

【目的】探讨lncRNA-PVT1/miR-15a/Bmi-1通路在胃癌细胞体外增殖中的调控作用。【方法】分别研究PVT1,miR-15a及Bmi-1在HGC-27胃癌细胞体外增殖中的作用。体外培养的人胃癌细胞株HGC-27分为如下组别:①空白对照组、si-PVT1(转染PVT1 siRNA)和si-PVT1 NC(转染PVT1 siRNAscramble阴性对照);②空白对照组、miR-15a(转染miR-15a模拟物)和miR-15a NC(转染miR-15a模拟物的阴性对照)。③空白对照组、si-Bmi-1(转染Bmi-1的siRNA)和si-Bmi-1 NC(转染Bmi-1的siRNAscramble阴性对照)。采用MTS法检测上述组别的细胞增殖情况。在抑制PVT1后检测不同组别PVT1、miR-15a和Bmi-1的表达情况。为验证miR-15a与Bmi-1之间的调节关系,将HGC-27胃癌细胞株分为3组:空白对照、miR-15a(转染miR-15a模拟物)和miR-15a NC(转染模拟物阴性对照),检测各组miR-15a和Bmi-1的表达情况。通过生物信息学网站预测PVT1与miR-15a以及miR-15a与Bmi-1的潜在结合位点。采用双荧光素酶报告基因技术验证PVT1与miR-15a以及miR-15a和Bmi-1之间的靶向调节关系。在前述基础上,逆向验证PVT1、miR-15a和Bmi-1三者之间的调控关系。【结果】抑制PVT1、Bmi-1或者过表达miR-15a后,HGC-27胃癌细胞的OD490值以及增殖率在0 h后的不同时间点均出现显著降低(P<0.01);抑制PVT1后,Bmi-1的表达出现降低,而miR-15a表达增高(P<0.01);转染miR-15a后,miR-15a表达升高,而Bmi-1表达出现降低(P<0.01)。与空白对照和miR-15a模拟物阴性对照组相比,PVT1以及Bmi-1野生型报告基因的萤光素酶活性在miR-15a模拟物组呈现显著降低,两者分别下降约56%和32%左右(P<0.01)。与空白对照组和si-PVT1 NC组相比,si-PVT1组PVT1和Bmi-1表达明显下降,miR-15a表达升高,在si-PVT1组加入miR-15a inhibitor之后,miR-15a表达下降,PVT1和Bmi-1的表达降低均出现了逆转;而si-PVT1组加入miR-15a后,miR-15a表达升高,在si-PVT1组加入miR-15a之后,miR-15a表达升高,PVT1和Bmi-1的表达出现进一步下降。【结论】LncRNA-PVT1能够通过靶向抑制miR-15a上调Bmi-1(lncRNA-PVT1/miR-15a/Bmi-1通路),进而促进胃癌细胞的体外增殖。

【Objective】To investigate the role of lncRNA-PVT 1/miR-15 a/Bmi-1 pathway in the proliferation of HGC-27 gastric cancer cells in vitro.【Methods】Gastric cancer cell line HGC-27 was cultured in vitro and divided into the following groups:(1)blank control,si-PVT 1 and si-PVT 1 NC.The si-PVT 1 and si-PVT 1 NC groups were transferred with PVT 1 siRNA and PVT 1 siRNA scramble,respectively.(2)blank control,miR-15 a and miR-15 a NC.(3)blank control,si-Bmi-1 and si-Bmi-1 NC.Cell proliferation was evaluated by using MTS.The expression of PVT 1,miR-15 a and Bmi-1 was detected in the condition of PVT 1 inhibition.In order to validate the relationship between miR-15 a and Bmi 1,miR-15 a mimic was transfected into HGC-27 gastric cancer cell line,and the expression of miR-15 a and Bmi-1 was examined.The potential complementary binding sites of PVT 1 and miR-15 a,and miR-15 a and Bmi-1 were predicted by bioinformatics.Dual-luciferase reporter assay was performed to detect luciferase activity of the different groups of cells in order to verify the relationship between PVT 1 and miR-15 a and miR-15 a and Bmi-1.Relevant rescue experiment was conducted in order to further validate the mutual relationship of lncRNA-PVT 1,miR-15 a and Bmi-1.【Results】OD 490 value and cell proliferation rate were significantly decreased in the condition of PVT 1 or Bmi-1 inhibition,or miR-15 a overexpression(P<0.01).The expression of Bmi-1 was decreased in si-PVT 1 group,whereas miR-15 a was increased(P<0.01).miR-15 a was increased after transfection,whereas the expression of Bmi-1 was decreased(P<0.01).The dual luciferase reporter assay indicated that both the PVT 1 and Bmi-1 reporter gene luciferase activity were decreased significantly in miR-15 a mimics group,down-regulating 56%and 32%,respectively(P<0.01).The expression of PVT 1 and Bmi-1 was elevated in the condition of PVT 1 knockdown and miR-15 a inhibition,whereas both of their expression were further decreased in the condition of PVT 1 knockdown and miR-15 a transfection.【Conclusion】LncRNA-PVT 1 could upregulate Bmi-1(lncRNA-PVT 1/miR-15 a/Bmi-1 pathway)to promote the proliferation of gastric cancer cells in vitro by inhibiting miR-15 a.