Down-regulation of nuclear MRP1 motivated the growth of MEC cells.

The expression of nuclear MRP1 is down-regulated by short-hairpin RNA (shRNA). The multidrug-resistant MC3/5FU cells were transfected with plasmids containing an MRP1 specific shRNA and a non-specific control shRNA, the resulting clones were MC3/5FU—S (short for S) and MC3/5FU-NS (short for NS). (A) Flow cytometry was used to determine the altered cell cycle of MEC cells as nuclear MRP1 decreased. Compared with MC3/5FU cells (49.9±1.01%) and MC3/5FU-NS cells (48.76±0.63%), the percentage of MC3/5FU-S cells in G0/G1 phase significantly decreased to 44.03±1.04% (P<0.01, n = 4). Compared with MC3/5FU (15.25±1.25%) and MC3/5FU-NS (16.61±1.04%), the percentage of MC3/5FU-S cells in G2/M phase was also significantly decreased to 11.20±0.77% (P<0.05, n = 4). Also compared with MC3/5FU (34.70±1.19%) and MC3/5FU-NS (34.26±0.63%), the cell number of MC3/5FU-S cells in S phase significantly increased to 43.63±1.49% (P<0.01, n = 4). (B) The growth curves showed that the viability of MC3/5FU-S cells was obviously higher than that of MC3/5FU cells and MC3/5FU-NS cells during the exponential growth phase between day 2 and day 7. (C) Compared with MC3/5FU (78.67±5.49) and MC3/5FU-NS cells (72.67±2.91), plate colony formation assay showed that colony number of MC3/5FU-S (165.0±9.07) cells substantially increased (P<0.01, n = 3). (D) After 14 days of culturing, the colonies of MEC cells were fixed then stained. The staining showed that not only the number of MC3/5FU-S colonies was obviously more substantial than other groups but also the size of the MC3/5FU-S colonies was larger in relation to other groups. **P<0.01, ***P<0.001.