Screening And Identification Of MiRNA Associated With DDP Resistance In Non-small Cell Lung Cancer

Chemotherapy is an important therapeutic approach for the therapy of advanced NSCLC cancer, but a successful long-term treatment is prevented by the development of drug resistance. Recent works have underlined the involvement of non-coding RNAs,MicroRNAs (miRNAs)are a small noncoding family of～22 nt RNAs that play an important role in the negative regulation of gene expression at the post-transcriptional level by base pairing to the 3'untranslated region of target messenger RNAs.miRNAs are involved in a variety of basic processes, such as cell proliferation,differentiation, apoptosis, metabolism and signaling pathways.A lot of studies have indeed described the aberrant expression of miRNAs in human tumors and have investigated their potential role as oncogenes or tumor suppressor genes, depending on the targets they regulate.Furthermore,more and more studies have demonstrated that an important role of miRNAs in anticancer drug resistance and miRNA expression profiling can be correlated with the development of anticancer drug resistance,miRNA effecting the expression of genes involved in pathways of drug absorption,metabolism and disposition,and target receptors that participate in the overall clinical effect of a drug may contribute to drug resistance.To explore whether miRNAs are involved in the regulation of the sensitivity of tumor cells to chemotherapeutic agents,we analyzed miRNA expression levels in DDP-resistant NSCLC cell line A549/DDP, in comparison with its parent cell line, A549, using a miRNA microarray. Some miRNAs were further validated by real-time reverse transcription-PCR. Enforced or inhitited taget miRNAs expression in cisplatin resistant cell was used to investigate whether miRNAs involve in modulating the sensitivity of NSCLC cells to chemotherapeutic agent, exploiting the emerging knowledge of miRNAs for the development of new human therapeutic applications for overcoming anticancer drug resistance and hopes future research will discover biomarkers that are better able to predict the cancer chemotherapy sensitivity.Methods:1. Cell lines and cell cultureHuman non–small-cell lung cancer A549 (parental) and A549/DDP (cisplatin- selected) cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere with 10% CO2/90% air. Cisplatin (1ug/ml) was also added for the culture of A549/DDP cells.2. Detection the IC50 of DDP to A549 and A549/DDP cell linesA549 and A549/DDP cells was tested by increasing concentrations of DDP and measuring the surviving cells with the CCK8 cell proliferation assay at 48h after the cells were treated by DDP.Absorbance at 490 nm was directly proportional to the number of living cells in culture.3. Microarrays detection of miRNA expressionFor miRNA microarray analysis, total RNA was extracted from 70 to 85% con?uence of A549 and A549/DDP cells with Trizol according to manufacturer's instructions. Microarray assay was performed using a service provider (LC Sciences).4. quantitative RT-PCRTotal RNA was extracted using Trizol (Invitrogen) and reverse-transcribed to cDNA with the TaKaRa RT-PCR kit. Primers of miRNA and U6 were offered by GuangZhou RIBOBIO. miRNA quantification was performed using SYBR? Green Realtime PCR Master Mix, and theΔCt values were normalized using endogenous U6 snRNA as control. The real-time reactions were performed using ABI PRISM 7500 Sequence Detection System (Applied Biosystems).5. Regulation of miRNA expression in A549/DDP cellsIn order to determine whether we can enforce or inhitite taget miRNAs expression in A 549/DDP cells, the mimics or inhibtors was transfected into drug-resistant A549/DDP cells using TurboFectTMsiRNA Transfection Reagent (Fermentas), the final concentration of mimics and inhibtors were 50nM mimics and 100nM,respectively.At 24h post-transfection,the expression of miRNA was determined by quantitative RT-PCR.6. Alteration of miRNA expression in A549/DDP cells and measurement of drug sensitivityThe miRNA mimics and inhibtor were purchased from GuangZhou RIBOBIO. The mimics and inhibtor were transfected into drug-resistant A549/DDP cells at a final concentration of 50 nM and100 nM,using TurboFectTMsiRNA Transfection Reagent (Fermentas).At 48h post- transfection,the sensitivity of the mimic-transfected or inhibitor-transfected cells to DDP, was determined by examing the viability of cell using Cell Counting Kit-8 , following the manufacturer's instructions. Absorbance at 450 nm was directly proportional to the number of living cells in culture.7. Statistical analysisAll values were expressed as mean±standard deviation (SD). (qPCR) and cytotoxicity were analyzed by one-way ANOVA (SPSS 15.0). IC50 value was assessed by probit regression analysis using SPSS15.0 statistical software.Diffe- rence was considered as significant if the probability was less than 0.05 (P < 0.05).Results:1. The IC50 of DDP to A549 and A549/DDP cell lines were 0.48ug/ml and 8.64μg/ml, respectively. The IC50 of resistant A549/DDP cells to DDP was18 times higher compared with their parental cells,A549.2. we show that DDP-resistant A549/DDP cells exhibit a considerable dysregulation of the miRNAome profile as compared with their parental lines A549 among, the higest and the lowest levels of miRNA was miR-376c and miR-451,respectively.3. As consistent with the microarray data, quantitative RT-PCR analysis demonstrated the expression of miR-376c,miR-31,miR-29a,miR-221 was increased by 3.5-fold,2.5-fold,2.2-fold,and 3.1-fold respectively,the lever of miR-451,miR-196a,miR-20a ,miR-20b,miR-17 was decreased by 3.2-fold, 4.9-fold, 3.0-fold, 7.7-fold,71.4-fold respectively,as comparison with A549 cells.4. The anti-miR-376c and miR-451 were transfected into drug-resistant A549/DDP cells respectively, the lever of miR-376c expression was down-regulated 37 times, whereas the lever of miR-451 expression was up-regulated more than 1500 times.5. 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 inhibtor,the reduction was 15.5%,12.9%, respectively, whereas chemosensitivity did not change when miR-376c, miR-31, miR-221 was inhibited or miR-196a,miR-20b,miR-20a was over-expressed.Conclusions:1. MicroRNA expression profiling revealed a limited set of microRNAs with altered expression in DDP-resistant lung cancer cell line A549/DDP compared to its parental A549 cell line,it indicate that miRNA could play an important role in the development of drug resistanc in lung cancer cells. These findings may be beneficial for further research of predicting drug-resistanc in patients and designing personalized therapy for lung cancer patients.2. The result of miRNA microarray was confirmed by quantitative RT-PCR, showing high throughput miRNA microarray can be an effective tool detected deregulation of miRNA expression in diseases.3. we can regulated the expression of target miRNA effectively by over-expressing or suppressing the expression of miRNA in vitro ,which shows that miRNA has the potential as a therapeutic target for cancers.4. In our study,the elevated level of miR-17 by transfection of miR-17 mimic sensitizes A549/DDP cells to DDP,on the contrary ,the enforced increase of miR-451 levels in the A549/DDP cells or down-regulates expression of miR-29a and miR-222 decreases sensitivity of the A549-resistant cancer cells to DDP. These results provide a strong rationale for the development of miRNA-based therapeutic strategies aiming to overcome cancer cell resistance. miR-17has the potential to be an efficient agent for preventing and reversing DDP-resistance in non-small cell lung cancer cells.