Figure 4.

Manipulating miR-200b and miR-200c expressions in Kyse170 and Kyse510 cells alter cell epithelial–mesenchymal feature and modulate cell sensitivity to CCRT treatment. (A) miR-200b and miR-200c levels were manipulated by anti-miR-200b and miR-200c oligonucleotides transfection in Kyse170 cells, the reduction of miR-200b and miR-200c expressions were confirmed by qRT-PCR analysis. (B) Examination of E-cadherin, N-cadherin, snail, and slug expressions in anti-miR-200b, miR-200c, and miR-200b/c oligonucleotides-transfected Kyse170 cells. Total cell lysates were collected and subjected to Western blot using indicated antibodies (anti-E-cadherin, N-cadherin, snail, and slug). β-actin was determined as loading control. (C) miR-200b and miR-200c suppression modulate Kyse170 cells resisting to CCRT treatment. Control and anti-miR-200b, anti-miR-200c, or anti-miR-200b/c oligonucleotides-transfected Kyse170 cells were plated into a 96-well plate, treated with 5 Gy radiation and at the indicated concentrations of cisplatin (0, 5, 10, 20, 30, 40, 50, and 100 μM). After 48h incubation, cell viability was determined by MTT assay. Relative percentage of viable cells compared to cells without CCRT treatment were shown. (D) Kyse510 parental cells were transfected with control, miR-200b, miR-200c and miR-200b/c precursors, respectively. qRT-PCR analysis was used to confirm ectopic miR-200b, miR-200c and miR-200b/c expressions in Kyse510 parental cells. (E) Total cell lysates of transfected Kyse510 parental cells from (D) were collected and subjected to Western blot using indicated antibodies (anti-E-cadherin, N-cadherin, snail, and slug). β-actin was used as loading control. (F) Control and miR-200b, miR-200c or miR-200b/c precursors-transfected Kyse510 were subjected to MTT assay to examine the effect of CCRT treatment on their cell viability. Treatment conditions were described as in (C). Relative percentage of viable cells compared to cells without CCRT treatment were shown. * p < 0.05, *** p < 0.001.