Mir-200c Improve Paclitaxel And Gefitinib Inhibition In The A549Human Lung Cancer Cell

Objective:1. To identifie the optimal transfected concentrations of miR-200c transfectedwithA549cells.2. To investigate after the upregulation of miR-200c, paclitaxel combined withgefitinib influence the proliferation inhibition and apoptosis capabilities of humanlung cancer cell A549.3. To discusse after the upregulation of miR-200c，whether is the paclitaxelcombination with gefitinib synergistic effect.Method:1. Transfection experiments and PCR detection： we first transfected withmiR-200c mimics as experimental groups,transfected miR-200c NC as negativecontrol group, the lung cancer A549cells was the mock group. Set three transfectedconcentration20nm,50nm and100nm, transfection with lipo2000after72hours,SYBR Green Real-time PCR detect expression change of miR-200c and itsdownstream target genes ZEB1after transfection, to determine the optimaltransfected concentration.2. Cell proliferation assay: then transfected cells with the optimal concentration,divided into three treatment-groups, paclitaxel monotherapy group（0-16nmol/L）,gefitinib monotherapy group（0-40umol/L）, the two-drug combination group. MTTassay cell proliferation and calculate the50%inhibition rate (IC50) of each group.3. Apoptosis experiment: the transfection group and negative control group weredivided into four groups, to treat with paclitaxel IC50, gefitinib IC50, combined withtwo drugs (paclitaxel IC50+gefitinib IC50) and no drugs. After72hours, it detectthe impact of miR-200c on apoptosis by flow cytometry.Resμlts:1. Compared with the mock group and negative control groups, real-time PCRresμlt showed that the miR-200c expression increased1365.29times in the20nmgroup; increased16,440.88times in the50nm group; increased4352.11times in the 100nm group, P <0.01. Compared with the mock group and negative control groups,The ZEB1expression decreased24%in the20nm group; decreased51%in the50nmgroup; decreased34%in the100nm group, P <0.05. Therefore,50nm is the optimaltransfection concentrations.2. Compared transfected miR-200c NC group with transfected miR-200c mimicsgroup, the IC50of paclitaxel monotherapy was8.40vs6.09nmol/L, P <0.05; theIC50of gefitinib monotherapy was20.76vs15.39umol/L, P <0.05; the IC50ofpaclitaxel and gefitinib combined was6.71vs5.83nmol/L (with paclitaxel as areference) or16.69vs14.59umol/L (with gefitinib as a reference), P>0.05.3. Compared transfected miR-200c NC group with transfected miR-200c mimicsgroup, the total apoptosis rate of paclitaxel monotherapy group was16.61±2.51vs26.66±1.78, P <0.05, the total apoptosis rate of gefitinib monotherapy was11.56±0.85vs15.82±1.95, P <0.05; the total apoptosis rate of two-drug combination groupwas30.05±0.97vs32.87±1.75, P>0.05.Conclusion:1. The50nm is the best transfection concentration of by detecting the expressionof miR-200c and ZEB1after transfection lung cancer cells A549with miR-200c.2. miR-200c can increase the proliferation inhibition and promote the apoptosisof gefitinib and paclitaxel on lung cancer cellsA549.3. After the upregulation of miR-200c，the paclitaxel combination with gefitinibisn’t synergistic effect on proliferation and apoptosis.