The Screen Of MicroRNAs In Cisplatin-resistant Lung Cells(A549/DDP) And Mechanism Study Of Cisplatin-resistant Regulated By MiR-138

Objective:To screen drug-resistant related miRNAs in lung adenocarcinoma cell, miRNA microarray was used to analyze the miRNA expression profiles in A549cell line and its drug-resistant A549/DDP cell line, real-time RT-PCR assay was utilized to validate the microarray data.Methods:Total RNA of A549and A549/DDP cells were isolated by Trizol method. The small RNA samples which ensured the successflul microarry hybridization were labelized with Cy3and Cy5respectively. Then the miRNA was hybridized on miRNA microarray. Original image signals were collected and digitized. The differentially expressed miRNA were screened and identified through comparison between the two chips. The results of microarray were confirmed by Real time RT-PCR.Results:The results of miRNA microarray showed that12miRNAs expressed differentially between A549and A549/DDP cells. As compared to A549cells, expression of miR-1246、miR-1470、miR-638、 miR-663、miR-181a、miR-1228*、miR-149*、miR-1908and miR-34a was up-regulated in A549/DDP cells by more than two folds, while expression of miR-138、miR-224and miR-886was down-regulated by more than two folds. miR-138、miR-224and miR-638were further confirmed by Real time RT-PCR. The results of Real time RT-PCR were consistent with that of microarray. But the difference of miR-638expression was found to be less than miRNA microarray data according to crossing point.Conclusions:Several miRNAs were differentially expressed between A549and A549/DDP cells. Our results suggested that these differentially expressed rniRNAs particularly miR-138、miR-224may play an important role in the mechanisms of lung adenocarcinoma drug-resistant. Objective:To observe effect of cell proliferation、cell cycle and cell apoptosis after up-regulation of miR-138and miR-224, A549/DDP cells were transiently transfected with plasmid.Methods:By lipofecatamine2000, A549/DDP cells were transfected with recombinant plasmid (pCDNA3.1-pri-miRNA) which chemically synthesized precursors of miR-138and miR-224oligonucleotides structured. The experiment divided into3groups:blank plasmid control group、pri-miR-138transfection group and pri-miR-224transfection group. The cells were transfected for24h and then treated with cisplatin at different concentrations (3.12ug/ml,6.25ug/ml,12.5ug/ml,25ug/ml,50ug/ml,100ug/ml,200ug/ml). Then cells were cultured for an additional48h and cell survival was measured by MTT. At24h after transfection, cisplatin was added at25μg/ml in media and the cells were incubated for48h, flow cytometry was used to analyze the distribution of cell cycle with PI staining and cell apoptosis with Annexin V-FITC staining.Results:After transfection in A549/DDP cells, MTT results showed that cell survival in pri-miR-138transfection group was less than blank plasmid control group in same doses of cisplatin, the IC50value was respectively14.45and49.32, reversal fold was3.41(p<0.05); And the IC50value was40.93in pri-miR-224transfection group, there was no statistically significant difference compared to blank plasmid control group. Cell cycle analysis showed that pri-miR-138transfection group G2phase cell was12.02±1.4%, which lower than blank plasmid control group25.75±1.8%(p<0.05), resulted in shortening of G2/M phase; And pri-miR-224transfection group G2phase cell was26.29±2.4%, there was no statistically significant difference compared to blank plasmid control group. Cell apoptosis analysis showed that the rates of apoptosis in pri-miR-138transfection group was34.09±1.2%, which higher than blank plasmid control group13.47±0.7%(p<0.05), resulted in rising of apoptotic rate. And the rates of apoptosis in pri-miR-224transfection group was19.19±1.5%, there was no statistically significant difference compared to blank plasmid control group.Conclusions:Up-regulation of miR-138increased the sensitivity of A549/DDP cells to cisplatin by enhanced inhibition of cell proliferation shortened G2/M phase and increased apoptosis. But over-expressed of miR-224had no obvious chang in cell proliferation、cell cycle and cell apoptosis. Objective:To observe regulation of cisplatin-resistant related genes at the mRNA and protein level after up-regulation of miR-138, A549/DDP cells were transiently transfected with plasmid.Methods:At72h after transfection, total RNA was extracted from A549/DDP cells, and was reversely transcribed to cDNA. MDR1、LRP、P53、Bcl-2、 ERCC1genes were amplified by semi-quantitative PCR while β-actin and GAPDH were performed as inner control. The mRNA expression levels of these genes in A549/DDP cells were analyzed. Western blot was used to detect expression of cisplatin-resistant related genes MDR1、 LRP、P53、Bcl-2and ERCC1at the protein level.Results:After up-regulation of miR-138in A549/DDP cells, RT-PCR results showed the mRNA expression levels of MDR1、LRP、P53、Bcl-2、ERCC1gene in pri-miR-138transfection group were respectively0.767±0.053、0.477±0.102、0.739±0.084、0.978±0.071、0.822±0.068, there was no statistically significant difference compared to blank plasmid control group. Western blot showed that the expression of ERCC1protein in the pri-miR-138transfection group was0.431±0.025, and significantly higher than blank plasmid control group(0.918±0.061)(P<0.05). And the protein expression levels of MDR1、LRP、P53、Bcl-2were respectively0.881±0.066、0.381±0.087、0.756±0.044、0.893±0.033, there was no statistically significant difference compared to blank plasmid control group.Conclusions:The enforced increase of miR-138levels in the A549/DDP cells downregulates expression of ERCC1at the protein level but not mRNA. Our results suggested that miR-138may play an important role in the development of cisplatin resistance in lung adenocarcinoma.