ARID3A调控AKR1C3促进结肠癌细胞对5-FU敏感性的机制

Mechanism of ARID3A promoting chemosensitivity of colon cancer cells to 5-FU by regulating AKR1C3

目的 探讨AT富交互域3A (ARID3A)对结肠癌细胞化疗敏感性的影响及其作用机制。方法 在人结肠癌细胞系HCT116和SW1116中构建ARID3A过表达或敲低细胞系,设置HCT116-PLVX/SW1116-PLVX过表达对照组、HCT116-ARID3A/SW1116-ARID3A过表达组、HCT116-ARID3A-sh1/SW1116-ARID3A-sh1敲减组、HCT116-ARID3A-sh2/SW1116-ARID3A-sh2敲减组和HCT116-SH/SW1116-SH敲减对照组。应用MTT实验检测5-FU作用于各组后的抑制效率。蛋白质谱检测筛选下游调控基因醛酮还原酶家族1成员C3(AKR1C3),采用qRT-PCR和蛋白质印迹法检测ARID3A过表达或敲减对AKR1C3表达的影响。采用双荧光素酶报告基因和染色质免疫沉淀(ChIP)实验分析ARID3A对AKR1C3的调控作用。进一步构建AKR1C3过表达或敲低细胞系,再次应用MTT实验检测其对5-FU的敏感性。结果 与对照组相比,HCT116-ARID3A过表达组(F=151.300,P<0.001;F=11.980,P=0.003;F=5.250,P=0.005)和SW1116-ARID3A过表达组(F=404.902,P<0.001;F=215.901,P<0.001;F=3.185,P=0.023)细胞受5-FU的抑制效率增加,药物和ARID3A表达水平的作用效应存在交互作用。而敲减ARID3A后,相较于对照组细胞表现出了对5-FU敏感性的降低,均P<0.05。进一步筛选出ARID3A的下游靶基因AKR1C3,qRT-PCR实验结果显示,在过表达ARID3A的HCT116和SW1116细胞中,AKR1C3的mRNA水平均低于相应对照组细胞(HCT116:0.395±0.054 vs 1.000±0.162,t=6.147,P=0.004;SW1116:0.250±0.017 vs 1.000±0.332,t=3.911,P=0.017);而在ARID3A敲减细胞中则相反,即AKR1C3的mRNA水平高于相应对照组细胞(HCT116:2.886±0.421 vs 1.000±0.080,t=7.621,P=0.002;SW1116:2.990±0.851 vs 1.000±0.148,t=3.909,P=0.016)。在过表达ARID3A的HCT116和SW1116细胞中,AKR1C3的蛋白水平均低于相应对照组细胞(HCT116:0.780±0.010 vs 1.000±0.012,t=19.630,P=0.004;SW1116:0.130±0.012 vs 0.240±0.029,t=4.880,P=0.032)。而在ARID3A敲减细胞中则相反,即AKR1C3的蛋白水平均高于相应对照组细胞(HCT116:1.630±0.040 vs 1.030±0.026,t=18.140,P=0.005;SW1116:1.070±0.153 vs 0.450±0.033,t=5.590,P=0.031)。并进一步明确ARID3A与AKR1C3结合并发挥其对AKR1C3的转录抑制作用。在HCT116和SW1116细胞中,过表达AKR1C3后,相较于对照组细胞均表现出了对5-FU敏感性的减弱,药物和AKR1C3表达水平的作用效应存在交互作用,均P<0.05。而敲减AKR1C3后,相较于对照组细胞表现出了对5-FU敏感性的增强,均P<0.05。结论 ARID3A通过抑制结肠癌细胞中的耐药相关基因AKR1C3促进结肠癌细胞对化疗药物5-FU的敏感性。

Objective To investigate the effect of AT rich interacting domain 3 A(ARID3 A) on the chemosensitivity of colon cancer cells and its mechanism.Methods ARID3 A overexpression or knockdown cell lines were constructed in human colon cancer cell lines HCT116 and SW1116,overexpression control group HCT116-PLVX/SW1116-PLVX,overexpression group HCT116-ARID3 A/SW1116-ARID3 A,knockdown control group HCT116-SH/SW1116-SH,knockdown group HCT116-ARID3 A-sh1/SW1116-ARID3 A-sh1,HCT116-ARID3 A-sh2/SW1116-ARID3 A-sh2 were set.MTT assay was used to detect and compare the effect of 5-FU on those groups.The downstream regulatory gene aldo-ketone reductase family 1 member C3(AKR1C3) was screened out by protein mapping, and the effect of ARID3 A overexpression or knockdown on the expression of AKR1 C3 was detected by qRT-PCR and Western blot experiments.The dual-luciferase reporter gene and chromatin immunoprecipitation(ChIP)assay were used to analyze and evaluate the effect of ARID3Aon the sensitivity of colon cancer cells to chemotherapeutics and its mechanism.The AKR1C3overexpression or knockdown cell line was further constructed,and its sensitivity to 5-FU was tested again by MTT assay.Results In HCT116and SW1116cells,compared with the control group,the cells in the ARID3Aoverexpression group were more efficiently inhibited by 5-FU,and there was an interaction between the effect of the drug and the expression level of ARID3A(HCT116:Fdrug concentration=151.300,P<0.001,FARID3A=11.980,P=0.003,Fdrug concentration×ARID3A=5.250,P=0.005,SW1116:Fdrug concentration=404.902,P<0.001,FARID3A=215.901,P<0.001,Fdrug concentration×ARID3A=3.185,P=0.023).After knocking down ARID3A,the cells showed a decrease in sensitivity to 5-FU compared with the control group(all P<0.05).The downstream target gene AKR1C3of ARID3Awas further screened out,and the mRNA level of AKR1C3in HCT116and SW1116cells overexpressing ARID3Awas lower than that in control cells(HCT116:0.395±0.054 vs 1.000±0.162,t=6.147,P=0.004,SW1116:0.250±0.017 vs 1.000±0.332,t=3.911,P=0.017).While in the ARID3Aknockdown group,the opposite was true,that is,the mRNA level of AKR1C3was higher than in control cells(HCT116:2.886±0.421 vs 1.000±0.080,t=7.621,P=0.002,SW1116:2.990±0.851 vs 1.000±0.148,t=3.909,P=0.016).In HCT116and SW1116cells overexpressing ARID3A,the protein level of AKR1C3was lower than that in control cells(HCT116:0.780±0.010 vs1.000±0.012,t=19.630,P=0.004,SW1116:0.130±0.012 vs 0.240±0.029,t=4.880,P=0.032).While the opposite was true in the ARID3Aknockdown group,namely(HCT116:1.630±0.040 vs 1.030±0.026,t=18.140,P=0.005,SW1116:1.070±0.153 vs0.450±0.033,t=5.590,P=0.031).And further clarified that ARID3Abinds to AKR1C3 and exerts its transcriptional inhibitory effect on AKR1C3.In HCT116and SW1116cells,after overexpression of AKR1C3,compared with the control cells,the sensitivity to 5-FU was weakened,and the effect of drug and AKR1C3 expression level interacted(all P<0.05).After knocking down AKR1C3,the cells showed enhanced sensitivity to 5-FU compared with the control group(all P<0.05).Conclusion ARID3Apromotes the sensitivity of colon cancer cells to the chemotherapeutic drug 5-FU by inhibiting the resistance-related gene AKR1C3 in colon cancer cells.