MicroRNA-224Modulates Chemosensitivity Of NSCLC Cells To Cisplatin By Targeting P21^WAF1/CEP1

Background:Non-small cell lung cancer (NSCLC) accounts for more than80%of primary lung cancer, more than60%of patients with advanced, often unresectable. Chemotherapy is the main treatment for stage Ⅲb-Ⅳ NSCLC. Cisplatin (DDP), an important component of chemotherapy in NSCLC, is still first-line drug. However, its acquired resistance restricts the play of its efficacy.MicroRNAs (miRNAs) are small, endogenous noncoding RNAs that have been identified as post-transcriptional regulators of gene expression. MiRNAs exert their functions through imperfect base-pairing with the3’-untranslated region (3’-UTR) of target mRNAs. In human cancer, miRNAs can act as oncogenes or tumour suppressor genes during tumourigenesis. As the relationship between miRNA and cancer being revealed, there is evidence that tumor cell sensitivity to anticancer drugs by the miRNA. MiR-224is up-regulated in a vatiety of human malignancies, as an oncogene involved in the biological porcess of proliferation, apoptosis and metastasis. Recent researches have shown the expression of miR-224is relative to methotrexate resistance to colon cancer. To date, however, the relationship between the expression of miR-224and chemoresistance in NSCLC remains unexplored.Materials and Methods:(1) A microarray technique was used to determine the miRNA profile in resistant A549/DDP and parental A549cells. Real-time quantitative PCR (qRT-PCR) was used to confirm the array results.(2) miR-224inhibitors was synthesized and introduced to cells using LipofectamineTM2000. The expression of miR-224in transfected cells was detected by qRT-PCR. The half maximal inhibitory concentration (IC50) values of DDP in resistant A549/DDP, parental A549and transfection A549/DDP cells were determined by MTT assay. Flow cytometric analysis was used to analyze the apoptosis and cell cycle distribution of transfection A549/DDP cells treated with DDP.(3) Bioinformatis analysis showed that there were p21WAF1/CIP13’UTR binding sites in miR-224sequence. qRT-PCR and western blot were used to detecte the expression of p21WAF1/CIP1in resistant A549/DDP, parental A549, A549/DDP/miR-224-inhibitor, A549/DDP/miR-NC cells at the level of mRNA and protein. P21WAF1/CIP13’UTR luciferase reporter was constructed and a dual luciferase assay was used to detect the activity.(4) PcDNA3.1/p21WAF1/CIP1vector was constructed and transfected into A549/DDP cells. The expression of p21WAF1/CIP1was detected by qRT-PCR and wesitern blot in transfection A549/DDP cells. The half IC50value of DDP in transfection A549/DDP cells was determined by MTT assay. Flow cytometric analysis was used to analyze the apoptosis and cell cycle distribution of transfection A549/DDP cells treated with DDP.(5) In vivo experiments were used to observe the effect of decreased miR-224on A549/DDP cell sensitivity to DDP. The in vivo levels of miR-224and p21WAF1/CIP1were detected by qRT-PCR and Immunohistochemistry.(6) Western blotting was used to detecte P53signaling pathway and the downstream targets Bcl-2, Bax in A549/DDP/miR-224-inhibitor, A549/DDP/miR-NC cells treated with or without DDP.Results:(1) The level of miR-224was significantly higher in resistant A549/DDP cells than that in sensitive A549cells.(2) Inhibition of miR-224in A549/DDP cells sensitized the cells to DDP. The IC50values of DDP in A549/DDP/miR-224-inhibitor, A549/DDP/miR-NC cells were16.57±1.62μg/ml and29.47± 2.15μg/ml, respectively. Compared with the control group, inhibition of miR-224in A549/DDP cells introduced increased apoptosis and proportion of G1cell cycle.(3) The inverse correlation between miR-224and p21WAF1/CIP1expression was found in vitro.The level of p21WAF1/CIP1was significantly lower in resistant A549/DDP cells than that in sensitive A549cells. The expression of p21WAF1/CIP1was significantly up-regulated in A549/DDP cells transfected with miR-224-inhibitor. MiR-224directly targets p21WAF1/CIP1in A549/DDP cells.(4) Ectopic expression of p21WAF1/CIP1could mimic the effect of decreased miR-224.(5) In vivo experiments confimed that inhibiton of miR-224resensitized A549/DDP cells to DDP and the expression of miR-224reversed to that of P21WAF1/CIP1.(6) Inhibition of miR-224regulates P53signaling pathway by activiation P53, introduction downstream targets Bcl-2, Bax down-regulated and up-regulated respectively in A549/DDP cells treated with DDP.Conclusions:In the present study we provide the first evidence that miR-224reverses resistance to DDP in A549/DDP cells, at least in part, by targeting p21WAF1/CIP1and mediating P53signaling pathway in vitro and vivo.