miR-591靶向ABCC3对肺癌细胞吉西他滨耐药性的影响

Effect of miR-591 targeting ABCC3 on resistance to gemcitabine in lung cancer cells

目的:探究miRNA-591靶向ABCC3对肺癌细胞吉西他滨耐药性的影响。方法:采用低浓度递增法构建肺癌吉西他滨耐药细胞A549/GR,CCK-8检测细胞增殖,qRT-PCR和Western blot检测miR-591和ABCC3 mRNA、蛋白表达。TargetScan预测和双荧光素酶报告实验分析miR-591与ABCC3的靶向关系。A549/GR细胞中转染miR-591或si-ABCC3,并给予吉西他滨处理,或共转染miR-591和pcDNA-ABCC3,并给予吉西他滨处理。CCK-8和流式细胞术检测细胞增殖与凋亡,Western blot检测ABCC3、细胞周期蛋白D1(CyclinD1)、p21、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)蛋白表达。结果:与A549细胞相比,0.01、0.1、1、2、5、10μmol/L吉西他滨显著降低A549/GR细胞增殖抑制率。A549/GR细胞中miR-591表达明显减少,ABCC3 mRNA和蛋白表达显著增加(P<0.05)。miR-591靶向调控ABCC3表达。miR-591过表达联合吉西他滨显著提高A549/GR细胞抑制率、凋亡率、p21、Bax蛋白表达,显著降低CyclinD1、Bcl-2蛋白水平(P<0.05),与干扰ABCC3表达联合吉西他滨作用相同。ABCC3过表达逆转miR-591过表达对吉西他滨作用的A549/GR细胞增殖、凋亡、CyclinD1、p21、Bcl-2、Bax蛋白表达的影响。结论:miR-591过表达通过靶向调控ABCC3表达,降低肺癌细胞对吉西他滨的耐药性。

Objective:To investigate effect of miR-591 targeting ABCC3 on resistance of lung cancer cells to gemcitabine.Methods:Lung cancer gemcitabine-resistant cells A549/GR were constructed by low-concentration increasing method.Cell proliferation was measured by CCK-8,miR-591,ABCC3 mRNA and protein expressions were detected by qRT-PCR and Western blot.TargetScan prediction and dual luciferase reporting experiments to analyze targeting relationship between miR-591 and ABCC3.A549/GR cells were transfected with miR-591 or si-ABCC3,and treated with gemcitabine,or A549/GR cells were co-transfected with miR-591 and pcDNA-ABCC3,and treated with gemcitabine.CCK-8 and flow cytometry to detect cell proliferation and apoptosis.Western blot to detect ABCC3,CyclinD1,p21,Bcl-2,Bax protein expressions.Results:Compared with A549 cells,gemcitabine at concentrations of 0.01,0.1,1,2,5 and 10μmo/l L significantly decreased proliferation inhibition rate of A549/GR cells.Expression of miR-591 in A549/GR cells was dramatically reduced,and expressions of ABCC3 mRNA and protein were evidently increased(P<0.05).miR-591 targeting regulated ABCC3 expression.miR-591 overexpression combined with gemcitabine obviously increased inhibition rate,apoptosis rate,p21,and Bax protein expressions of A549/GR cells,apparently decreased CyclinD1,Bcl-2 protein levels(P<0.05),and influenced of interfered with ABCC3 expression combined with gemcitabine were the same.ABCC3 overexpression reversed effects of miR-591 overexpression on proliferation,apoptosis,and CyclinD1,p21,Bcl-2 and Bax protein expressions of gemcitabine-treated A549/GR cells.Conclusion:Overexpression of miR-591 decreased resistance of lung cancer cells to gemcitabine by targeting regulation of ABCC3 expression.