上调微小RNA-200a表达对非小细胞肺癌紫杉醇化疗敏感度的影响及其机制
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摘要
目的探讨微小RNA-200a(miRNA-200a)对非小细胞肺癌紫杉醇化疗敏感度的影响及其机制。方法采用荧光定量逆转录聚合酶链反应(qRT-PCR)检测非小细胞肺癌A549细胞中miRNA-200a的表达情况;采用脂质体法将miRNA-200a模拟物及miRNA-200a阴性对照转染至非小细胞肺癌A549细胞,采用qRT-PCR检测转染效果;采用噻唑蓝(MTT)法检测上调miRNA-200a表达对紫杉醇处理的非小细胞肺癌A549细胞增殖抑制情况的影响;采用蛋白质印迹法(Western blot)检测上调miRNA-200a表达及紫杉醇处理后非小细胞肺癌A549细胞中Wnt/β-catenin信号通路相关β-catenin蛋白的表达情况。结果非小细胞肺癌A549细胞中miRNA-200a的相对表达量为(0.26±0.08),明显低于正常人肺上皮16HBE细胞的(1.02±0.03)(P﹤0.01)。转染48 h后,空白对照组、mimics NC组、miRNA-200a mimics组细胞中miRNA-200a的相对表达量分别为(1.06±0.12)、(1.18±0.25)和(5.02±0.16),3组细胞中miRNA-200a的相对表达量比较,差异有统计学意义(P﹤0.01)。miRNA-200a mimics组细胞中miRNA-200a的相对表达量明显高于空白对照组(P﹤0.01)。MTT法检测结果显示,随着紫杉醇浓度的增加,A549细胞受到的增殖抑制作用明显增强,且呈一定的浓度依赖性;与紫杉醇组相比,转染miRNA-200a mimics后,随着紫杉醇浓度的增加,A549细胞受到的增殖抑制作用增强(P﹤0.05)。Western blot检测结果显示,空白对照组、紫杉醇组、miRNA-200a mimics组和miRNA-200a+紫杉醇组细胞中β-catenin蛋白的相对表达量分别(0.78±0.03)、(0.62±0.05)、(0.48±0.05)和(0.25±0.04),4组细胞中β-catenin蛋白的相对表达量比较,差异有统计学意义(P﹤0.01)。与空白对照组相比,紫杉醇组、miRNA-200a mimics组和miRNA-200a+紫杉醇组细胞中β-catenin蛋白的相对表达量均明显降低(P﹤0.01);与紫杉醇组相比,miRNA-200a+紫杉醇组细胞中β-catenin蛋白的相对表达量明显降低(P﹤0.01)。结论上调miRNA-200a表达能够增强非小细胞肺癌A549细胞对紫杉醇化疗药物的敏感度,其作用机制与阻断Wnt/β-catenin信号通路有关。
Objective To investigate the effect of microRNA-200 a(miRNA-200 a) on paclitaxel sensitivity in nonsmall cell lung cancer and its mechanism. Method The expression of miRNA-200 a in NSCLC A549 cells was detected by qRT-PCR; the miRNA-200 a mimics and miRNA-200 a negative control were transfected into A549 cells by liposome,and the transfection effects were measured by qRT-PCR. The inhibitive effect of up-regulation of the expression of miRNA-200 a on the proliferation of paclitaxel-treated NSCLC A549 cells was determined by MTT assay. The expression of Wnt/β-catenin signaling pathway related β-catenin protein in NSCLC A549 cells after up-regulation of miRNA-200 a and paclitaxel treatment were examined by Western blot. Result The relative expression of miRNA-200 a in NSCLC cell line A549 was(0.26±0.08), and was significantly lower than that in normal human lung epithelial 16 HBE cell line at(1.02±0.03)(P<0.01). In 48 h after transfection, the relative expression levels of miRNA-200 a in blank control group, mimics NC group and miRNA-200 a mimics group were(1.06±0.12),(1.18±0.25) and(5.02±0.16), and the relative expression levels of miRNA-200 a in the three groups were significantly different(P<0.01). The relative expression of miRNA-200 a in miRNA-200 a mimics group was significantly higher than that in blank control group(P<0.01). The results of MTT showed that the inhibitive effect on A549 cells was markedly increased and was dose-dependent; compared with the paclitaxel group, after transfection of miRNA-200 a mimics, the inhibition observed in A549 cells was significantly elevated as the concentration of paclitaxel increased(P<0.05). Western blot results indicated that the relative expression of β-catenin protein in blank control group, paclitaxel group, miRNA-200 a mimics group and miRNA-200 a + paclitaxel group were(0.78±0.03),(0.62±0.05),(0.48±0.05) and(0.25±0.04), respectively, and the relative expression of β-catenin protein in the four groups differedstatistically(P<0.01). Compared with the blank control group, the relative expressions of β-catenin protein in paclitaxel group, miRNA-200 a mimics group and miRNA-200 a+ paclitaxel group were decreased notably(P<0.01). Conclusion Up-regulation of miRNA-200 a expression can enhance the sensitivity of A549 cells to paclitaxel chemotherapeutic drugs, and its mechanism may be related to the inhibition of Wnt/β-catenin signaling pathway.
Objective To investigate the effect of microRNA-200 a(miRNA-200 a) on paclitaxel sensitivity in nonsmall cell lung cancer and its mechanism. Method The expression of miRNA-200 a in NSCLC A549 cells was detected by qRT-PCR; the miRNA-200 a mimics and miRNA-200 a negative control were transfected into A549 cells by liposome,and the transfection effects were measured by qRT-PCR. The inhibitive effect of up-regulation of the expression of miRNA-200 a on the proliferation of paclitaxel-treated NSCLC A549 cells was determined by MTT assay. The expression of Wnt/β-catenin signaling pathway related β-catenin protein in NSCLC A549 cells after up-regulation of miRNA-200 a and paclitaxel treatment were examined by Western blot. Result The relative expression of miRNA-200 a in NSCLC cell line A549 was(0.26±0.08), and was significantly lower than that in normal human lung epithelial 16 HBE cell line at(1.02±0.03)(P<0.01). In 48 h after transfection, the relative expression levels of miRNA-200 a in blank control group, mimics NC group and miRNA-200 a mimics group were(1.06±0.12),(1.18±0.25) and(5.02±0.16), and the relative expression levels of miRNA-200 a in the three groups were significantly different(P<0.01). The relative expression of miRNA-200 a in miRNA-200 a mimics group was significantly higher than that in blank control group(P<0.01). The results of MTT showed that the inhibitive effect on A549 cells was markedly increased and was dose-dependent; compared with the paclitaxel group, after transfection of miRNA-200 a mimics, the inhibition observed in A549 cells was significantly elevated as the concentration of paclitaxel increased(P<0.05). Western blot results indicated that the relative expression of β-catenin protein in blank control group, paclitaxel group, miRNA-200 a mimics group and miRNA-200 a + paclitaxel group were(0.78±0.03),(0.62±0.05),(0.48±0.05) and(0.25±0.04), respectively, and the relative expression of β-catenin protein in the four groups differedstatistically(P<0.01). Compared with the blank control group, the relative expressions of β-catenin protein in paclitaxel group, miRNA-200 a mimics group and miRNA-200 a+ paclitaxel group were decreased notably(P<0.01). Conclusion Up-regulation of miRNA-200 a expression can enhance the sensitivity of A549 cells to paclitaxel chemotherapeutic drugs, and its mechanism may be related to the inhibition of Wnt/β-catenin signaling pathway.
引文
[1]中华医学会呼吸病学分会肺癌学组,中国肺癌防治联盟.晚期非小细胞肺癌分子靶向治疗专家共识(2013版)[J].中华结核和呼吸杂志,2014,37(3):177-183.
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[13]倪明立,谢玲,秦贝贝.Wnt/β-catenin信号通路在非小细胞肺癌组织的表达及意义[J].广东医学,2016,37(15):2299-2301.
[14]Fu Q,Chen Z,Gong X,et al.β-Catenin expression is regulated by an IRES-dependent mechanism and stimulated by paclitaxel in human ovarian cancer cells[J].Biochem Biophys Res Commun,2015,461(1):21-27.
[15]涂雪松,胡利霞,瞿广桥,等.紫杉醇通过抑制Wnt/β-catenin信号通路抑制鼻咽癌细胞增殖的实验研究[J].癌症进展,2016,14(12):1216-1220.
[16]侯峰强,董山潮,雷喜锋,等.紫杉醇对人胆管癌QBC939细胞wnt/β-catenin信号通路基因和蛋白表达的影响[J].昆明医科大学学报,2017,38(4):10-13.
[17]Su J,Zhang A,Shi Z,et al.Micro RNA-200a suppresses the Wnt/β-catenin signaling pathway by interacting withβ-catenin[J].Int J Oncol,2012,40(4):1162-1170.
[2]Wang J,Song W,Shen W,et al.Micro RNA-200a suppresses cell invasion and migration by directly targeting gab1 in hepatocellular carcinoma[J].Oncol Res,2017,25(1):1-10.
[3]Yao J,Zhou E,Wang Y,et al.micro RNA-200a inhibits cell proliferation by targeting mitochondrial transcription factor A in breast cancer[J].DNA Cell Biol,2014,33(5):291-300.
[4]Li R,He JL,Chen XM,et al.Mi R-200a is involved in proliferation and apoptosis in the human endometrial adenocarcinoma cell line HEC-1B by targeting the tumor suppressor PTEN[J].Mol Biol Rep,2014,41(4):1977-1984.
[5]王勇,李宇男,于晓松.肺癌细胞及组织中mi R-181a、mi R-200a、mi R-200c表达变化及意义[J].山东医药,2014,54(48):16-18.
[6]许璐,方玉松,王丹云,等.mi R-200a抑制YAP1基因表达对肺癌细胞增殖的影响[J].中国肿瘤临床,2017,44(7):311-315.
[7]Zhen Q,Liu J,Gao L,et al.Micro RNA-200a targets EGFR and c-Met to inhibit migration,invasion,and gefitinib resistance in non-small cell lung cancer[J].Cytogenet Genome Res,2015,146(1):1-8.
[8]汪建林,杨西胜,李小磊,等.mi R-200a与肿瘤关系[J].现代肿瘤医学,2013,21(12):2853-2856.
[9]Liu N,Zhong L,Zeng J,et al.Upregulation of micro RNA-200a associates with tumor proliferation,CSCs phenotype and chemosensitivity in ovarian cancer[J].Neoplasma,2015,62(4):550-559.
[10]沈阿灵,刘丽雅,齐飞,等.片仔癀上调mi R-200a抑制大肠癌耐药细胞转移的作用机制[J].中华中医药杂志,2016,31(9):3682-3686.
[11]Gao Y,Song C,Hui L,et al.Overexpression of RNF146 in non-small cell lung cancer enhances proliferation and invasion of tumors through the Wnt/β-catenin signaling pathway[J].PLo S One,2014,9(1):e85377.
[12]Zhang XX,Zhang LL,Yang HL,et al.Mechanism of Wnt/β-catenin signaling pathway in enhanced malignant phenotype of non-small cell lung cancer induced by anti-angiogenesis therapy[J].Asian Pac J Trop Med,2016,9(1):58-62.
[13]倪明立,谢玲,秦贝贝.Wnt/β-catenin信号通路在非小细胞肺癌组织的表达及意义[J].广东医学,2016,37(15):2299-2301.
[14]Fu Q,Chen Z,Gong X,et al.β-Catenin expression is regulated by an IRES-dependent mechanism and stimulated by paclitaxel in human ovarian cancer cells[J].Biochem Biophys Res Commun,2015,461(1):21-27.
[15]涂雪松,胡利霞,瞿广桥,等.紫杉醇通过抑制Wnt/β-catenin信号通路抑制鼻咽癌细胞增殖的实验研究[J].癌症进展,2016,14(12):1216-1220.
[16]侯峰强,董山潮,雷喜锋,等.紫杉醇对人胆管癌QBC939细胞wnt/β-catenin信号通路基因和蛋白表达的影响[J].昆明医科大学学报,2017,38(4):10-13.
[17]Su J,Zhang A,Shi Z,et al.Micro RNA-200a suppresses the Wnt/β-catenin signaling pathway by interacting withβ-catenin[J].Int J Oncol,2012,40(4):1162-1170.