Abstract
Objective To compare the reversal effects of short hairpin RNA (shRNA) interference and tetrandrine on multidrug resistance (MDR) of human colorectal cancer cell line LoVo/5-fluorouracil(5-FU ).Methods An eukaryotic expression plasmid of shRNA targeting MDR1 was constructed and transfected into human colorectal cancer cell line LoVo/5-FU (transfection group).LoVo/5-FU was also pretreated with tetrandrine (tetrandrine group).Drug sensitivity was detected by methyl thiazolyltetrazolium colorimetric method.Cell cycle,apoptosis of cells and positive expression rate of P-glycoprotein (P-gp) were determined by flow cytometry assay.The expressions of MDR1 mRNA and P-gp were detected by real-time polymerase chain reaction and Western blot,respectively.All data were analyzed by analysis of variance and SNK-q test.Results (1)Drug sensitivity:the 50% concentration of inhibition(IC50)of the control group,tetrandrine group and transfection group were (7.3 ± 0.3),(4.4 ±0.7) and (2.4 ±0.4) mmol/L,respectively,a significant difference between the 3 groups was found(F =65.27,P < 0.05 ).There was a significant difference in the IC50 between the tetrandrine group and the transfection group (q =6.67,P < 0.05 ).(2) Changes of cell cycle:the proportion of cells in the G1 phase and S phase of the control group,tetrandrine group and transfection group were 38.13% ± 3.75%,51.36% ± 2.76%,59.24%±4.31% and 20.46%±2.23%,14.32%± 1.91%,9.40%± 1.65%,respectively,a significant difference between the 3 groups was found(F =25.23,24.37,P < 0.05 ).There were significant differences in the proportion of cells in the G1 phase and S phase between the tetrandrine group and the transfection group(q =3.67,4.35,P < 0.05 ).(3) Cell apoptosis:the cell apoptotic rates of the control group,tetrandrine group and transfection group were 1.32% ± 0.47%,3.24% ± 0.26%,5.88% ±- 0.44%,respectively,a significant difference between the 3 groups was found(F =99.26,P < 0.05 ).There was a significant difference in the cell apoptotic rate between the tetrandrine group and transfection group(q =11.48,P < 0.05 ).(4)The expression of P-gp:the positive expression rates of P-gp of the control group,tetrandrine group and transfection group were 96.9% ± 2.3%,61.6% ± 4.9%,76.6% ± 3.6%,respectively,a significant difference between the 3 groups was found(F =67.83,P < 0.05 ).There was a significant difference in the positive expression rate of P-gp between the tetrandrine group and transfection group (q =6.97,P < 0.05 ).(5)The mRNA expression of MDR1:the mRNA expressions of MDR1 of the control group,tetrandrine group and transfection group were 1462 ±161,570 ±85,233 ± 81,respectively,a significant difference between the 3 groups was found(F =90.59,P < 0.05 ).There was a significant difference in the mRNA expression of MDR1 between the tetrandrine group and transfection group (q =5.12,P < 0.05 ).Conclusions MDR1 shRNA and tetrandrine could reverse M DR1 gene-mediated m.ultidrug resistance in human colon cancer cell line LoVo/5-FU,but the effect of MDR1 shRNA is better than that of tetrandrine.MDR1 shRNA and tetrandrine might take effect by inhibiting P-gp expression and down-regulating mRNA expression of MDR1. Key words: Colorectal neoplasms; Short hairpin RNA; Tetrandrine; Multidrug resistance gene; P-glycoprotein
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