miR-200c增加H2228细胞对克唑替尼和紫杉醇及顺铂敏感性的研究

Effect of Increased H2228 Cells through miR- 200c on Sensitivity of Crizotinib,Paclitaxel and Cisplatin

背景miR-200c可以增加肿瘤细胞对紫杉醇、顺铂的敏感性及表皮生长因子受体(EGFR)阳性肺癌细胞对分子靶向药物厄洛替尼、吉非替尼及阿法替尼的敏感性,但其是否可以增加棘皮动物微管相关样蛋白4(EML4)-间变性淋巴瘤激酶(ALK)融合基因阳性肺腺癌细胞(ALK阳性肺癌细胞)对克唑替尼、紫杉醇和顺铂的敏感性尚无相关研究。目的观察miR-200c是否可以增加H2228细胞对克唑替尼、紫杉醇及顺铂的敏感性,并探讨其机制。方法 2014年2月—2015年5月,培养H2228细胞并进行转染,根据转染物的不同,将细胞分为增强转染组(转染miR-200c mimics)、增强对照组(转染miR-200c mimics阴性对照)、抑制转染组(转染miR-200c inhibitor)、抑制对照组(转染miR-200c inhibitor阴性对照)。采用实时荧光定量PCR法(Real-time PCR法)检测H2228细胞中miR-200c基因表达水平,MTT法检测不同浓度抗肿瘤药物〔克唑替尼(50、100、200 nmol/L)、紫杉醇(6.25、12.50、25.00 nmol/L)、顺铂(25、50、100 nmol/L)〕作用下H2228细胞增殖水平,Transwell法检测H2228细胞迁移率,Real-time PCR法检测增强转染组、增强对照组H2228细胞中E-钙黏蛋白、N-钙黏蛋白、波状蛋白、CD24mRNA表达水平,Western blotting法检测增强转染组、增强对照组H2228细胞中E-钙黏蛋白、N-钙黏蛋白、波状蛋白、CD24表达水平。结果增强转染组H2228细胞中miR-200c基因表达水平高于增强对照组(P<0.05)。抑制转染组H2228细胞中miR-200c基因表达水平低于抑制对照组(P<0.05)。增强转染组各浓度抗肿瘤药物作用下H2228细胞增殖水平均低于增强对照组(P<0.05)。抑制转染组各浓度抗肿瘤药物作用下H2228细胞增殖水平均高于抑制对照组(P<0.05)。增强转染组H2228细胞迁移率小于增强对照组(P<0.05)。抑制转染组H2228细胞迁移率大于抑制对照组(P<0.05)。增强转染组H2228细胞中E-钙黏蛋白及其mRNA表达水平高于增强对照组,N-钙黏蛋白、波状蛋白、CD24及其mRNA表达水平低于增强对照组(P<0.05)。结论 miR-200c可以通过使肺癌细胞发生间质-上皮转变(MET)而增加H2228细胞对克唑替尼、紫杉醇及顺铂的敏感性。

Background The miR- 200 c can increase the sensitivity of tumor cells on paclitaxel and cisplatin,and the sensitivity of positive lung carcinoma cells in epidermal growth factor receptor（ EGFR） on molecular targeted drug such as erlotinib hydrochloride tablets, gefitinib and afatinib. However, there are no related studies on whether it can increase the sensitivity of echinodermata microtubule- associated protein- like 4（ EML4）- anaplastic lymphoma kinase（ ALK） fusion gene positive lung adenocarcinoma cells on crizotinib,paclitaxel and cisplatin. Objective To observe whether miR- 200 c can increase the sensitivity of H2228 cells on crizotinib,paclitaxel and cisplatin,and to explore its mechanism. Methods From February 2014 to May 2015,H2228 cells were cultured and transfected. According to the different transfectants,the cells were divided into enhanced transfection group（ transfected with miR- 200 c mimics）, enhanced control group（ transfected miR- 200 c mimics negative control）, inhibitory transfection group（ transfected with miR- 200 c inhibitor） and inhibitory control group（ transfected with miR- 200 c inhibitor negative control）. The real- time fluorescence quantification PCR method was used to detect the expression level of miR- 200 c gene in H2228 cells,MTT assay was performed to measure the proliferation level of H2228 cells under antineoplastic drugs 〔ketazidine（ 50,100,200 mmol / L）, paclitaxel（ 6. 25,12. 50,25. 00 mmol / L） and cisplatin（ 25,50,100 nmol / L） 〕of different concentrations,Transwell assay was used to measure the migration rate of H2228 cells, Real- time PCR method was applied to detect the expression level of E- cadherin, N- cadherin,corrugated protein and CD24 mRNA in H2228 cells in transfection group and enhanced control group,Western blotting was used to measure the expression levels of E- cadherin, N- cadherin, corrugated protein and CD24 in H2228 cells in enhanced transfection group and enhanced control group. Results The gene expression level of miR- 200 c in H2228 cells in enhanced transfection group was significantly higher than that in the enhanced control group（ P〈 0. 05）. The gene expression level of miR- 200 c in H2228 cells in inhibitory transfection group was significantly lower than that in inhibitory control group（ P 〈0. 05）. The cell proliferation level of H2228 under the effect of antineoplastic drugs of different concentrations in enhanced transfection group was lower than that in enhanced control group（ P〈 0. 05）. The proliferation level of H2228 cells under the effect of the effect of antineoplastic drugs of different concentrations in inhibitory transfection group was higher than that in inhibitory control group（ P 〈0. 05）. The migration rate of H2228 cells in enhanced transfection group was greater than that in enhanced control group（ P〈 0. 05）. The migration rate of H2228 cells in inhibitory transfection group was greater than that in the inhibitory control group（ P〈 0. 05）. The expression level of E- cadherin and its mRNA in H2228 cells in enhanced transfection group was higher than that in enhancedcontrol group,and the expression level of N- cadherin,corrugated protein,CD24 and their mRNA was lower than that in enhanced control group（ P 〈0. 05）. Conclusion Through the mesenchymal epithelial transiton in lung carcinoma cells,miR- 200 c can enhance the sensitivity of H2228 cells on crizotinib,paclitaxel and cisplatin.