长链非编码RNA GAS5调控乳腺癌细胞阿霉素耐药的分子机制

Molecular mechanism of long non-coding RNA GAS5 regulating adriamycin resistance in breast cancer cells

目的探讨长链非编码RNA(lncRNA) GAS5对耐阿霉素乳腺癌细胞的影响及其分子机制。方法采用浓度梯度(0.50、1.00和4.00 μg/ml) μm阿霉素长期处理MCF-7乳腺癌细胞(河南中医药大学第五临床医学院的), 建立阿霉素耐药的细胞株MCF-7/ADR, 采用荧光定量聚合酶链反应(PCR)检测MCF-7细胞和MCF-7/ADR细胞lncRNA GAS5表达水平;采用对照lncRNA和lncRNA GAS5慢病毒感染MCF-7/ADR细胞株, 构建lncRNA对照和lncRNA GAS5过表达细胞系(lncRNA对照组和lncRNA GAS5组)。采用细胞计数试剂盒(CCK-8)测定两组细胞对阿霉素的敏感性;采用平板克隆形成实验分析两组细胞细胞克隆形成率;采用流式细胞术分析两组细胞的凋亡;采用生物信息学和双荧光素酶报告基因分析lncRNA GAS5的靶基因;采用蛋白质免疫印迹(Western blot)分析lncRNA GAS5靶蛋白的表达水平。计量数据比较采用t检验。结果采用阿霉素浓度递增法成功构建对阿霉素耐药的乳腺癌细胞株MCF-7/ADR。MCF-7乳腺癌细胞lncRNA GAS5表达水平(1.09±0.11)明显高于MCF-7/ADR细胞lncRNA GAS5表达水平(0.51±0.09), 差异有统计学意义(t=9.127, P<0.05)。lncRNA对照组细胞吸光值(1.96±0.09)明显高于lncRNA GAS5组(1.42±0.09), 差异有统计学意义(t=9.258, P<0.05)。lncRNA对照组细胞克隆数[(218.81±23.51)个]明显高于lncRNA GAS5组细胞克隆数[(114.23±16.27)个], 差异有统计学意义(t=8.181, P<0.05)。lncRNA对照组细胞凋亡率[(4.70±0.89)%]明显高于lncRNA GAS5组细胞凋亡率[(18.53±2.99)%], 差异有统计学意义(t=9.913, P<0.05)。lncRNA对照组细胞MRP1、ABCB1、ABCG1、P-gp蛋白表达水平(1.25±0.10、0.97±0.08、1.80±0.15、1.50±0.12)明显高于lncRNA GAS5组细胞(0.75±0.07、0.43±0.06), 0.83±0.11、0.72±0.08), 差异有统计学意义(t=9.315、11.710、11.780、11.971, P<0.05)。结论 lncRNA GAS5在阿霉素耐药细胞株中表达水平显著下调, 通过调节肿瘤细胞耐药相关蛋白的表达参与乳腺癌耐药过程。

Objective To investigate the effect of long non-coding RNA(lncRNA)GAS5 on adriamycin resistant breast cancer cells and its molecular mechanism.Methods MCF-7 breast cancer cells from the Fifth Clinical Medical College of Henan University of Traditional Chinese Medicine were treated with adriamycin by a concentration gradient manner(0.50,1.00 and 4.00μg/ml).The adriamycin resistant cell line MCF-7/ADR was established.The expression level of lncRNA GAS5 in MCF-7 cells and MCF-7/ADR cells was detected by fluorescence quantitative polymerase chain reaction(PCR).MCF-7/ADR cell lines were infected with control lncRNA and lncRNA GAS5 lentivirus to construct lncRNA control and lncRNA GAS5 overexpression cell lines(lncRNA control group and lncRNA GAS5 group).Cell counting kit-8(CCK-8)assay was used to determine the sensitivity of the two groups of cells to adriamycin.The cell clone formation rate of the two groups was analyzed by plate clone formation experiment.The apoptosis of the two groups was analyzed by flow cytometry.Western blotting was used to analyze the expression level of resistance associated protein.The measurement data were compared by t-test.Results Adriamycin resistant breast cancer cell line MCF-7/ADR was successfully constructed by doxorubicin concentration increasing method.The expression level of lncRNA GAS5 in MCF-7 breast cancer cells(1.09±0.11)was significantly higher than that in MCF-7/ADR cells(0.51±0.09,t=9.127,P<0.05).The absorption value of cells in lncRNA control group(1.96±0.09)was significantly higher than that in lncRNA GAS5 group(1.42±0.09)(t=9.258,P<0.05).The number of cell clones in lncRNA control group[(218.81±23.51)]was significantly higher than that in lncRNA GAS5 group[(114.23±16.27),t=8.181,P<0.05].The apoptosis rate of lncRNA control group[(4.70±0.89)%]was significantly higher than that of lncRNA GAS5 group[(18.53±2.99)%,t=9.913,P<0.05].The expression levels of MRP1,ABCB1,ABCG1 and P-gp proteins in lncRNA control group(1.25±0.10,0.97±0.08,1.80±0.15,1.50±0.12)were significantly higher than those in lncRNA GAS5 group(0.75±0.07,0.43±0.06,0.83±0.11,0.72±0.08,t=9.315,11.710,11.780,11.971,P<0.05).Conclusion LncRNA GAS5 expression in adriamycin resistant cell lines was significantly reduced,which was involved in the drug resistance process of breast cancer by regulating the expression of drug-resistant related proteins in tumor cells.