| Setting and objective:Now the incidence and mortality rate of lung cancer has ranked first of all the malignant tumors in the worldwide. Chemotherapy is an important means of treatment for advanced NSCLC cancer, however, drug resistance due to long-term treatment ofen lead to the failure of chemotherapy. Recent studies have shown that non- protein encoding small molecule RNA(miRNA) can influence the occurrence and development of lung cancer and drug resistance through gene mutation, regulate target genes and proliferation and apoptosis related genes. miR-155, as one of the miRNA family, has been involved in the occurrence and development of a variety of malignant tumors, but the role of miR-155 in lung cancer and the mechanism of how it affect lung cancer is still controversial.. This study aims to observe the differential expression of miR-155 between the cell lines A549 and its drug-resistant cell lines A549/DDP, and to explore its effect on lung cancer cells and cisplatin sensitivity, providing new ideas for the individualized treatment of lung adenocarcinoma. Methods:1.Cell culture : Human cell lines of A549 and A549/DDP were cultured in RPMI-1640 medium(containing 10% fetal bovine serum and 1% of penicillin and streptomycin mixture) and put put in a suitable condition of cell culture box(37°C in a humidified atmosphere with 10% CO2/90% air). The culture medium of A549/DDP cells was added by cisplatin with the final concentration of 1?g / ml.2.Identification of the drug-resistant strains A549/DDP and the differential expression detection of miR-155 and TP53INP1 mRNA: MTT method was used for detection the value IC50 of A549/DDP and A549 cell lines of cisplatin by increasing concentrations of DDP and calculate the resistance ratio. Total RNA was extracted and examined from two kinds of cells.Real-time quantitative PCR(qRT-PCR) was used to detect the expression of miR-155 and TP53INP1 mRNA in A549/DDP resistant cells and A549 cells.3.Cell transfection: The experiment was divided into three groups: experimental group(miR155-inhibitor), negative control group(FAM-NC-inhibitor) and control group. The lipofectamine ?2000 and FAM-miR155-inhibitor or FAM-NC-inhibitor were mixed to transfect into A549 / DDP cell lines.4.Result detection:qRT-PCR was used to measured the expression of miR-155 and TP53INP1 mRNA and Western-blot was used to detect the protein expression of TP53INP1 ? Bax ? Bcl-2 in A549/DDP cells before and after transfecting the miR-155-inhibitor. MMT assay,flow-cytometric analysis were performed to detect drug sensitivity,cell proliferation ability,and apoptosis on treatment with DDP.5.Statistical analysis: SPSS 19.0 statistical package was used. Quantitative data was present as mean ± standard deviation( x ±S). Multiple groups were analyzed by one-way ANOVA. IC50 value was calculated by logistic regression analysis. Taking ? =0.05 as the standard value. Results:1.The value of IC50 of cisplatin on A549 and A549/DDP cells were 16.20±2.27 ?mol/L and 69.72±4.83 ?mol/L, IC50 value of A549/DDP is 4.3 times more than the A549 cells, the difference was statistically significant(t = 17.36, P = 0.001).2. Real time fluorescence quantitative PCR(qRT-PCR) showed that the expression of miR155 in A549/DDP cells was significantly up-regulated, which was about 8.3 times that of A549 cells, TP53INP1 mRNA expression was significantly down-regulated, the difference was statistically significant(t = 37.30, P = 0.001; t = 5.418, P = 0.001).3.After miR-155 inhibitor transfect A549/DDP cells, qRT-PCR showed that the expression of miR-155 in A549/DDP cell line was down-regulated by 57.37%.4.qRT-PCR and Western blot show that the expression of TP53INP1 mRNA and its protein were up-regulated compared with negative control group after miR-155 inhibitor transfect A549/DDP cells and the difference was statistically significant(P = 0.007 and P =0.001). the expression of miR-155 and TP53INP1 in lung adenocarcinoma cells were negatively correlated.5.MTT results show that after miR-155 inhibitors were transfected into A549/DDP cells, and the value of IC50 of cisplatin on A549/DDP cells were(29.35±3.69 ?mol/L),which was significantly higher than that of non transfected group(70.59±3.47 ?mol/L), and empty transfected group(74.18±6.56 ?mol/L), the difference was statistically significant( all P = 0.001).6.After miR-155 inhibitor transfected into A549/DDP cells, the expression of the apoptosis related protein Bcl-2 was down regulated, and Bax protein was up-regulated compared with the non transfected group and empty transfection group,the difference was statistically significant( all P = 0.001).7.Flow cytometry results show that early apoptosis rate, late apoptosis rate and total apoptosis rate in untransfected group were(15.77±2.89)%,(15.58±3.19)% and(32.16±3.59)%,respectively. Empty transfected group were(17.33±2.12)%,(18.37±1.73)% and(29.24±2.28)%, respectively. Inhibitor group were(33.1±2.38)%,(33.95±2.31)% and(61.4±2.65)%, respectively. Early apoptosis rate, late apoptosis rate and total apoptosis rate in transfected group were higher than that of untransfected group and empty transfected group, the difference was statistically significant(all P = 0.001). Conclusion:miR-155 is highly expressed in lung adenocarcinoma A549/DDP cisplatinresistant strains, and it has been involved in the production of cisplatin resistance in lung cancer. Down-regulation of miR-155 in A549/DDP cells can reverse cisplatin-resisitance and promote the apoptosis of A549/DDP cell line. The mechanism of which is probably through targete TP53INP1 gene or/and through other ways to cause the down-expression of Bcl–2 and up-expression of Bax. |
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