| Objective:To investigate whether overexpression of Twist1 promotes the acquisition of multidrug resistance(MDR)in colorectal cancer sw620 cells,and its mechanism is studied preliminarily.Methords:The lentivirus vector Twist1-GFP-LV and the negative control virus NC-GFP-LV were used to transfect SW620 cells,which were called experimental group SW620/Twist1 and control group SW620-NC respectively.Two groups of cells were tested as follows:1)Determining the effect of expression:(1)RT-qPCR was performed to examine the mRNA expression levels of Twist1;(2)western-blot analysis was performed to investigate the expression of Twist1 proteins;2)Detection of drug resistance to oxaliplatin and 5-fu in vitro: Firstly,the half maximal inhibitory concentration(IC50)of two drugs on sw620 cells was detected by CCK8 assay,and the drug of this concentration was used to act on the two groups of cells respectively.The difference of cell inhibition ratio was detected at 24 h,48h and 72 h after administration.3)Detection of drug resistance associated indexes:(1)the mRNA expression levels of ABCB1 and ABCG2 were detected by RT-qPCR;(2)the protein expression levels of ABCB1(p-gp)and ABCG2(BCRP)were detected by western blot analysis;4)Detection of cancer stem cell markers: the expression levels of CD133 and CD44 were detected by flow direct-labeled antibody analysis.Results:1)The over-expression of effect determination:(1)detection of Twist1 mRNA expression by RT-qPCR:Twist1 mRNA relative expression value in experimental group and control group was(2.54 ± 0.31)and(0.97 ± 0.33)respectively,compared with the control group,Twist1 in the experimental group increased by about 2.6 times(P<0.01);(2)the protein expression levels of Twist1 was detected by Western blot analysis in the experimental group and the control group respectively,the results were(1.12±0.03)and(0.53±0.02),the experimental group was significantly higher(P<0.01);These resultssuggest that Twist1 overexpression is satisfactory and cell model has been established successfully.2)The IC50 of oxaliplatin and 5-FU on sw620 cells were 14.33ug/ml and41.62ug/ml respectively.The concentration(IC50)of oxaliplatin and 5-FU were separately acted on the two groups,at 24 h,48h and 72 h,the inhibition ratio were measured.The results showed that the inhibitory ratio of the two chemotherapeutic drugs in the experimental group was lower than that in the control group at different time points,but there was no significant difference at 24h(P>0.05)and the inhibition ratio was significantly decreased at 48h(P<0.05)and 72h(P<0.01).The results suggested that the inhibition ratio of proliferation of the two chemotherapeutic drugs on the experimental group was lower than that of the control group.The cells were resistant to the two chemotherapeutic drugs after overexpression of Twist1,showing the characteristics of MDR.3)Detection of drug resistance associated genes and proteins:(1)the mRNA relative expression levels of ABCB1(p-gp)detected by RT-qPCR in the experimental group and control group were(2.02±0.12)and(0.94±0.23);the results of ABCG2(BRCP)were(2.45±0.38)and(0.91± 0.22);compared with the control group,the m RNA relative expression levels of two genes in the experimental group increased significantly(P<0.01);(2)The expression of ABCB1(p-gp)protein in experimental and control groups was detected by western blot analysis,the results were(0.70 ± 0.03)and(0.35 ±0.05);the results of ABCG2(BRCP)were(0.75±0.12)and(0.29±0.08),compared with NC group,the changes of two proteins in experimental group increased significantly(P<0.01);4)Detection of CSCs markers: the positive ratio of CD133 in the two groups were(54.3 ± 2.6)% and(78.6 ± 3.8)%respectively and that were(5.6 ± 1.2)% and(12.5 ± 2.0)% about CD44;After over expression of Twist1,the expression of two CSCs markers increased significantly(P<0.01).Conclusion: Overexpression of Twist1 could promote the expression of ABCB1(p-gp)and ABCG2(BRCP)in colorectal cancer SW620 cells and obtain MDR in tumor cells.This phenomenon may be related to the overexpression of Twist1 to enhance the expression of CSCs markers of sw620 cells and promote the acquisition of stemness. |
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