非小细胞肺癌顺铂化疗耐药相关微RNA的筛选及鉴定

 目的　探讨肿瘤细胞微RNA（miRNA）对化疗药物敏感性的作用。方法　通过miRNA芯片技术检测顺铂（DDP）耐药细胞株A549／DDP与非耐药细胞株A549的miRNA表达的差异，利用荧光定量聚合酶链反应（PCR）技术验证相应miRNA的表达情况，通过在细胞株中抑制或过表达目标miRNA，研究其对细胞化疗药物敏感性的影响。结果　A549／DDP细胞对DDP的耐药为A549细胞的18倍。A549／DDP细胞与A549细胞存在51个表达水平差异在4倍以上的miRNA，其中24个表达上调，27个表达下调。PCR进一步证实miR-376c、miR-31、miR-29a、miR-221在A549／DDP细胞中显著上调，miR-196a、miR-20a、miR-20b、miR-17、miR-451在A549／DDP细胞中显著下调。在提高A549／DDP细胞中miR-17的表达后，细胞对DDP的敏感度增加了11.7 %，提高miR-451的表达或者抑制miR-29a的表达后，对DDP的敏感度分别下降了15.5 %、12.9 %，抑制miR-376c、miR-31、miR-221或过表达miR-196a、miR-20a、miR-20b均不影响A549／DDP细胞对DDP的敏感度。结论　非小细胞肺癌DDP耐药细胞与非耐药细胞的miRNA表达谱有差异，miRNA参与肺癌化疗耐药，miR-17具有逆转非小细胞肺癌DDP耐药的潜力。

Screening and identification of microRNA associated with cisplatin resistance in non-small cell lung cancer

Objective　To analyze the differences in microRNA (miRNA) expression between A549 and A549/DDP cells and explore the association between miRNA expression and drug resistance in non-small cell lung cancer (NSCLC). Methods　The drug resistance of A549/DDP cells was evaluated using CCK-8 assay and flow cytometry. Microarray technique and RT-PCR were used to analyze the differential expression of the miRNA between A549 and A549/DDP cells. Enforced or inhibited target miRNA expression in cisplatin resistant cell was used to investigate whether miRNA involve in modulating the sensitivity of NSCLC cells to chemotherapeutic agent, exploiting the emerging knowledge of miRNA for the development of new human therapeutic applications for overcoming anticancer drug resistance and trying to discover biomarkers that were better able to predict the cancer chemotherapy sensitivity. Results　The drug resistance index of A549/DDP cells relative to the parental A549 cells was 18. Microarray analysis of A549 and A549/DDP cells identified 51 differentially expressed genes (≥4-fold), including 24 up-regulated and 27 down-regulated genes in A549/DDP cells. RT-PCR identified 9 miRNA that were differentially expressed between A549 and A549/DDP cells. Of these differentially expressed miRNA, miR-376c, miR-31, miR-29a, miR-221 showed significantly increased expression, and miR-196a, miR-20a, miR-20b, miR-17, miR-451 showed significantly lowered expression in A549/DDP cells as indicated by the results of microarray analysis and RT-PCR. DDP sensitivity was increased 11.7 % in A549/DDP cells transfected with miR-17,but the chemosensitivity was decreased when miR-451 was over-expressed or miR-29a was inhibited by selective inhibitor, the reduction was 15.5 %, 12.9 %, respectively, whereas chemosensitivity did not change when miR-376c, miR-31, miR-221 were inhibited or miR-196a, miR-20b, miR-20a were over-expressed. Conclusion　A549/DDP cells show a different miRNA expression profile from its parental A549 cells, suggesting the involvement of miRNA in tumor cell drug resistance. miR-17 has the potential to be an efficient agent for preventing and reversing DDP-resistance in NSCLC. These results provide a strong rationale for the development of miRNA-based therapeutic strategies aiming to overcome cancer cell resistance.