The Expression And Mechanism Of Micrornas In Drug-resistant Nsclc Cell Line A549/ddp

Objective and background: MicroRNAs (miRNAs) are 21-25 nucleotide ( nt) non-coding RNA molecules that regulate gene expression negatively at post transcriptional level. MiRNAs may extensively take part in initiation and development of tumor, and possess the function similar to oncogenes or tumor suppressors. Recent studies show that miRNAs could have intimate relationship with multidrug resistance of cancer. Cisplatin (CDDP) is a commonly used chemotherapeutic agent for non-small-cell lung cancer (NSCLC). However, treatment with this agent is characterized by resistance, both acquired and intrinsic. Our study is to analyze the difference in miRNAs expression between A549 and A549/DDP cells and explore the association between miRNA and cisplatin resistanc of NSCLC. Furthermore, we investigated the functional roles of miRNA which contributes to the mechanisms of resistance with cisplatin in NSCLC. Theses studies provide a strong rationale for the development of miRNA based therapeutic strategies aiming to overcome NSCLC cell drug resistance.Methods: (1) The drug resistance of A549/DDP cells was evaluated using MTT assay. (2) The total RNA of the two cell lines was isolated and examined. miRNA expression profiles of A549/DDP and A549 were analyzed using microarray. (3) The results were confirmed by real time PCR. (4) MTT assay was used to evaluate the sensitivity of NSCLC cells transfected with miR-21 precursor or inhibitor. (5) Total cell RNA was extracted after 24 hours of transfecation. The relative miR-21 levels were determined by real time PCR. (6) Potential mRNA targets of miR-21 were predicted by MiRanda, TargetScan and PicTar software. (7) Total cell protein extracted after 48 hours of transfecation. The expression of Bcl-2 and PTEN protein in NSCLC cells transfected with the negative control or miRNAs was detected by western blot.Results: (1) IC50 of cisplatin to A549 cells and A549 /DDP cells were 7.47μmol /L and 137.32μmol /L (P<0.01). The drug resistance index of A549/DDP cells relative to the parental A549 cells was 18.38. (2) The purification of total RNA from A549 and A549/DDP was relatively high. Microarray analysis showed that 34 human miRNAs were differentially expressed between two cell lines (Fold Change>1.5 or<0.67 and p-value<0.05). Compared to A549 cells, there were 19 miRNAs up-regulated and 15 miRNAs down-regulated in A549/DDP. (3) Real time PCR identified 7 miRNAs that were differentially expressed between A549 and A549/DPP cells, including 3 up-regulated and 4 down-regulated ones in A549/DPP cells. Of these differentially expressed miRNAs, mir-21, mir-31, mir-374b showed significantly increased expression, and mir-378, mir-886-5p, mir-886-3p, mir-520c-3p showed significantly lowered expression in A549/DPP cells as indicated by the results of miarray analysis and real time PCR. (4) In vitro drug sensitivity assay demonstrated that overexpression of miR-21 conferred A549 cells cisplatin resistance whereas inhibition of miR-21 using antisense oligonucleotides sensitized A549/DDP cells to the anticancer drug. (5) Real time PCR revealed that anti-miR-21 significantly reduced miR-21 level and pre-miR-21 significantly increased miR-21 level. (6) The downregulation of miR-21 in A549/DDP cells was concurrent with the downregulation of Bcl-2 protein and upregulation of PTEN protein. While the upregulation of miR-21 in A549 cells was concurrent with the upregulation of Bcl-2 protein and downregulation of PTEN protein.Conclusion: (1) These results indicated that NSCLC resistant to cisplatin was associated with a group of miRNAs. This finding provides a experimental basis for further study of mechanism underlying the cisplatin resistance of NSCLC. (2) In vitro drug sensitivity assay demonstrated that overexpression of miR-21 conferred A549 cells cisplatin resistance whereas inhibition of miR-21 using antisense oligonucleotides sensitized A549/DDP cells to anticancer drugs. (3) miR-21 could modulate the sensitivity of NSCLC cells to cisplatin, at least in part, through regulating Bcl-2 and PTEN expression. Our data provide a novel insight into the mechanisms of cisplatin resistance in NSCLC and maybe useful for the future development of chemo-sensitizing strategy through manipulating miRNA expression.