泛素特异性肽酶22经Wnt/β-catenin通路调控结直肠癌化疗耐药的机制研究

Mechanism study of ubiquitin specific protease 22 regulating the chemoresistance of colorectal cancer through Wnt/β-catenin signal pathway

目的探讨泛素特异性肽酶22(USP22)作用于Wnt/β-catenin信号通路参与结直肠癌的发生和化疗耐药的分子机制。方法采用氟尿嘧啶(5-Fu)处理结直肠癌细胞株HT-29,建立耐药细胞株HT-29/5-Fu;构建USP22 siRNA稳定表达细胞株(表示为HT-29-sh USP22、HT-29/5-Fu-sh USP22)。CCK-8法检测HT-29/5-Fu细胞对5-Fu的敏感性、抑制USP22表达后结直肠癌细胞对5-Fu敏感性的影响;采用Western blotting检测不同质量浓度5-Fu诱导下HT-29细胞中USP22蛋白表达,HT-29和HT-29/5-Fu细胞中USP22和β-catenin蛋白表达,以及抑制USP22表达对结直肠癌细胞USP22、β-catenin表达的影响。结果 (1)HT-29与HT-29/5-Fu细胞对5-Fu的IC_(50)值分别为(1.58±0.23)mg/L和(14.58±0.94)mg/L,其中HT-29/5-Fu细胞对5-Fu的敏感性显著下降(n=6,t=8.476,P<0.01)。(2)在0.5、5.0 mg/L的5-Fu诱导后,HT-29细胞内USP22蛋白表达水平(USP22/β-actin)显著升高(0.5 mg/L vs 0 mg/L:t=7.618,P<0.05;5.0 mg/L vs 0 mg/L:t=6.992,P<0.05)。HT-29/5-Fu组中USP22蛋白表达水平为0.92±0.11,显著高于HT-29组的0.18±0.06(t=7.618,P<0.05)。(3)HT-29-sh USP22组对5-Fu的IC_(50)为(0.25±0.23)mg/L,显著低于HT-29组(t=6.662,P<0.01)。HT-29/5-Fu-sh USP22组对5-Fu的IC_(50)为(1.36±0.14)mg/L,显著低于HT-29/5-Fu组(t=7.002,P<0.01),但与HT-29组相比,差异无统计学意义(t=1.586,P>0.05),抑制USP22表达可增强结直肠癌细胞HT-29对5-Fu的敏感性。(4)HT-29-sh USP22组的USP22蛋白表达水平为0.07±0.01,与HT-29组相比下调(t=7.105,P<0.01)。HT-29/5-Fu-sh USP22组的USP22蛋白表达水平为0.33±0.02,与HT-29/5-Fu组相比,USP22蛋白表达水平下调(t=6.153,P<0.01)。HT-29-sh USP22、HT-29/5-Fu-sh USP22中β-catenin蛋白表达水平与相应对照组HT-29、HT-29/5-Fu相比显著下调(HT-29-sh USP22 vs HT-29:t=8.823,P<0.01;HT-29/5-Fu-sh USP22vs HT-29/5-Fu:t=7.656,P<0.01)。结论 5-Fu可诱导结直肠癌细胞USP22表达增高。抑制USP22表达可增加结直肠癌细胞对5-Fu的药物敏感性,其机制可能是抑制Wnt/β-catenin信号通路,从而有效逆转结直肠癌细胞对5-Fu的耐药。

Objective To investigate the mechanism of ubiquitin specific protease 22 (USP22) regulating the occurrence and chemotherapy resistance of colorectal cancer through Wnt/β-catenin pathway. Methods 5-fluorouracil (5-Fu) was used for treating the colorectal cancer cell line HT-29 and establishing the drug-resistant cell line HT-29/5-Fu. The sensitivity of HT-29/5-Fu cells and HT-29 cells to 5-Fu was detected by CCK-8 assay. Western blot was used for detecting the effect of 5-Fu on the protein expression of USP22 in HT-29 colorectal cancer cell lines. Using HT-29 parental cell lines of colorectal cancer, a stable expression cell line of USP22 siRNA was constructed (expressed as HT-29-sh USP22, HT-29/5-Fu-sh USP22). The effect of inhibition of USP22 expression on 5-Fu sensitivity of colorectal cancer cells was investigated by CCK-8 test. Western blotting was used to detect the effects of inhibition of USP22 expression on the proteins expression of USP22,β-catenin through Wnt/β-catenin signaling pathway in HT-29 colorectal cancer cell lines. Results (1) The IC50 values of HT-29 and HT-29/5-Fu cells to 5-Fu were (1.58±0.23) mg/L and (14.58±0.94) mg/L, respectively. The sensitivity of HT-29/5-Fu cells to 5-Fu was significantly decreased (n=6, t=8.476, P<0.01).(2) After induction of 0.5, 5.0 mg/L 5-Fu, the expression of USP22 protein (USP22/beta-actin) in HT-29 cells increased significantly (0.5 mg/L vs 0 mg/L: t=7.618, P<0.05;5.0 mg/L vs 0 mg/L: t=6.992, P<0.05). The expression level of USP22 protein in HT-29/5-Fu group was 0.92±0.11, significantly higher than that in HT-29 group (0.18±0.06)(t=7.618, P<0.05).(3) TheIC50 of 5-Fu in HT-29-sh USP22 group was (0.25±0.23) mg/L, significantly lower than that in HT-29 group (t=6.662, P<0.01). The IC50 of 5-Fu in HT-29/5-Fu-sh USP22 group was (1.36±0.14) mg/L, significantly lower than that in HT-29/5-Fu group (t=7.002, P<0.01), but there was no significant difference between HT-29/5-Fu-sh USP22 group and HT-29 group (t=1.586, P>0.05). Inhibition of USP22 expression could enhance the sensitivity of colorectal cancer cell HT-29 to 5-Fu.(4) The expression of USP22 protein in HT-29-sh USP22 group was (0.07±0.01), which was down-regulated compared with HT-29 group (t=7.105, P<0.01). The expression level of USP22 protein in HT-29/5-Fu-sh USP22 group was 0.33±0.02. Compared with HT-29/5-Fu group, the expression level of USP22 protein was down-regulated (t=6.153, P<0.01). The expression of beta-catenin protein in USP22 siRNA stably expressed cell lines HT-29-sh USP22 and HT-29/5-Fu-sh USP22 was significantly lower than that in the corresponding control group HT-29, HT-29/5-Fu (HT-29-sh USP22 vs HT-29: t=8.823, P<0.01;HT-29/5-Fu-sh USP22 vs HT-29/5-Fu: t=7.656, P<0.01). Conclusions 5-Fu can increase the protein expression of USP22 in colorectal cancer cells. The inhibition of USP22 expression can increase the drug sensitivity of colorectal cancer cells to 5-Fu, and effectively reverse the resistance of colorectal cancer cells to 5-Fu by mediating the Wnt/β-catenin signaling pathway.