MiR-200c Enhancing The Sensitivity To Human Lung Cancer A549Cell Treated With Paclitaxel And Gefitinib

Objectives：To observe the influence of drug sensitivity on treating with paclitaxel andgefitinib and effect of the combination of two drugs, to investigate the possiblemechanism from molecular levels when up-regulation miR-200c in A549cell.Methods：1. Cell transfection and transfection efficiency of detection: Lipofectamine2000was transfected into A549cell lines and transfect miR-200c mimic(transfection group,mimic NC(negative control group), only add medium for blank control group, todetect the expression of miR-200c and ZEB1by Real-time PCR and to compare theexpression difference among the groups.2. The experimental groups and drug treatment: transfect group and negativecontrol group were treating with paclitaxel(the group of miR-200c-paclitaxel and thegroup of Control-paclitaxel), gefitinib(the group of miR-200c-gefitinib and the groupof Control-gefitinib), combined with two drugs(the group of miR-200c-two drugs andthe group of Control-two drugs) respectively, without medice is named the group ofmiR-200c and the group of Control.3. Determined by MTT method to detect the influence of sensitivity treating withpaclitaxel, gefitinib and the combination of two drugs after transfection.4. Western Blot detect the expression of TUBB3protein, EGFR, Akt, MAPKphosphorylation protein and total protein after drug treatment among all groups.Results：1. The results of he expression of miR-200c and ZEB1detected by Real TimePCR, the comparison among the three groups, we find that the expression ofmiR-200c was up-regulation apparently in the transfect group, P＜0.01; theexpression of ZEB1was down-regulation apparently in the transfect group, P＜0.01.2. Determined by MTT method to detect the influence of sensitivity treating withdifferent drugs after transfection: the cell proliferationl inhibition rate was improvedin the group of miR-200c-gefitinib and miR-200c-paclitaxel compare to the group of Control-gefitinib,Control-paclitaxel and miR-200c, P＜0.05; There was no differencebetween the group of miR-200c-two drugs and Control-two drugs,the group ofmiR-200c-two drugs and miR-200c-paclitaxel, miR-200c-gefitinib, P＞0.05.3. Western Blot show that the comparison among the group of miR-200c-paclitaxel, Control-paclitaxel, miR-200c after given paclitaxel, the expression ofTUBB3protein was down-regulation in the group of miR-200c-paclitaxel, P＜0.01.The comparison among the group of miR-200c-gefitinib, Control-gefitinib, miR-200cafter given gefitinib, we find that the expression of EGFR, Akt phosphorylationprotein was down-regulation in the group of miR-200c-gefitinib, P＜0.05, there wasno obvious difference between the MAPK phosphorylation protein and total protein,EGFR, Akt total protein, P＞0.05. The comparison between the group ofmiR-200c-two drugs and Control-two drugs after given two drugs, there was noobvious difference between the EGFR, Akt, MAPK phosphorylation protein and totalprotein, P＞0.05.The comparison between the group of miR-200c-two drugs andmiR-200c-gefitinib, there was no obvious difference between the EGFR, Aktphosphorylation protein, P＞0.05; compare to the group of miR-200c-paclitaxel,EGFR, Akt phosphorylation protein has no difference, P＜0.05.Conclusion：1. MiR-200c could inhibit the expression of TUBB3protein to improve thesensitivity of A549cell treated with paclitaxe.2. MiR-200c could inhibit the expression of EGFR, Akt phosphorylation protein,to enhance the sensitivity of A549cell treating with gefitinib.3. MiR-200c has no synergistic antitumor effect treated with paclitaxel andgefitinib in lung cancer A549cell.