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
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摘要
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 IC_(50) 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) The IC_(50) 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 IC_(50) 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.
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 IC_(50) 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) The IC_(50) 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 IC_(50) 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.
引文
[1]Yuan L,Yuan P,Yuan H,et al.miR-542-3p inhibits colorectal cancer cell proliferation,migration and invasion by targeting OTUB1[J].Am J Cancer Res,2017,7(1):159-172.
[2]王宛明,董贾中,管淑敏.泛素特异性肽酶22诱导胰腺癌细胞对吉西他滨耐药的观察[J].中国临床药理学与治疗学,2017,22(4):412-417.
[3]Nusse R,Clevers H.Wnt/β-Catenin signaling,disease,and emerging therapeutic modalities[J].Cell,2017,169(6):985-999.
[4]Zhang JJ,Chen JT,Hua L,et al.miR-98 inhibits hepatocellular carcinoma cell proliferation via targeting EZH2 and suppressing Wnt/β-catenin signaling pathway[J].Biomed Pharmacother,2017,85:472-478.
[5]Gong Z,Liu J,Xie X,et al.Identification of potential target genes of USP22 via ChIP-seq and RNA-seq analysis in HeLa cells[J].Genet Mol Biol,2018,41(2):488-495.
[6]Melo-Cardenas J,Xu Y,Wei J,et al.USP22 deficiency leads to myeloid leukemia upon oncogenic Kras activation through a PU.1-dependent mechanism[J].Blood,2018,132(4):423-434.
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[8]陆周一,陈晓峰.WNT/β-catenin信号通路与miRNA在原发性肺癌中的研究进展[J].中国癌症杂志,2017,27(2):151-155.
[9]Jiang S,Song C,Gu X,et al.Ubiquitin-specific peptidase 22contributes to colorectal cancer stemness and chemoresistance via Wnt/β-catenin pathway[J].Cell Physiol Biochem,2018,46(4):1412-1422.
[10]He Z,Song A,Zhang Z,et al.Comparative efficacy of nonpharmacological adjuvant therapies for quality of life in the patients with breast cancer receiving chemo-or radio-therapy:A protocol for systematic review and Bayesian network metaanalysis[J].Medicine(Baltimore),2018,97(35):e12096.
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[12]Zhang Y,Li H,Cao R,et al.Suppression of miR-708 inhibits the Wnt/β-catenin signaling pathway by activating DKK3 in adult B-all[J].Oncotarget,2017,8(38):64114-64128.
[13]马勇,王礼宁,郭杨,等.姜黄素通过调节Wnt/β-catenin信号通路促进软骨细胞增殖的研究[J].广州中医药大学学报,2017,34(1):90-95.
[14]Lv L,Xiao XY,Gu ZH,et al.Silencing USP22 by asymmetric structure of interfering RNA inhibits proliferation and induces cell cycle arrest in bladder cancer cells[J].Mol Cell Biochem,2011,346(1-2):11-21.
[15]张洪印.术前放化疗在局部晚期结直肠癌患者中的应用及敏感性预测研究进展[J].中国普通外科杂志,2017,26(3):380-385.
[16]Lin Z,Yang H,Kong Q,et al.USP22 antagonizes p53transcriptional activation by deubiquitinating Sirt1 to suppress cell apoptosis and is required for mouse embryonic development[J].Molecular Cell,2012,46(4):484.
[17]刘英丽.泛素特异性蛋白酶USP22及其SNPs对Hela细胞凋亡及细胞周期调控作用的研究[D].石家庄:河北医科大学,2015.
[2]王宛明,董贾中,管淑敏.泛素特异性肽酶22诱导胰腺癌细胞对吉西他滨耐药的观察[J].中国临床药理学与治疗学,2017,22(4):412-417.
[3]Nusse R,Clevers H.Wnt/β-Catenin signaling,disease,and emerging therapeutic modalities[J].Cell,2017,169(6):985-999.
[4]Zhang JJ,Chen JT,Hua L,et al.miR-98 inhibits hepatocellular carcinoma cell proliferation via targeting EZH2 and suppressing Wnt/β-catenin signaling pathway[J].Biomed Pharmacother,2017,85:472-478.
[5]Gong Z,Liu J,Xie X,et al.Identification of potential target genes of USP22 via ChIP-seq and RNA-seq analysis in HeLa cells[J].Genet Mol Biol,2018,41(2):488-495.
[6]Melo-Cardenas J,Xu Y,Wei J,et al.USP22 deficiency leads to myeloid leukemia upon oncogenic Kras activation through a PU.1-dependent mechanism[J].Blood,2018,132(4):423-434.
[7]McCracken KW,Aihara E,Martin B,et al.Wnt/β-catenin promotes gastric fundus specification in mice and humans[J].Nature,2017,541(7636):182-187.
[8]陆周一,陈晓峰.WNT/β-catenin信号通路与miRNA在原发性肺癌中的研究进展[J].中国癌症杂志,2017,27(2):151-155.
[9]Jiang S,Song C,Gu X,et al.Ubiquitin-specific peptidase 22contributes to colorectal cancer stemness and chemoresistance via Wnt/β-catenin pathway[J].Cell Physiol Biochem,2018,46(4):1412-1422.
[10]He Z,Song A,Zhang Z,et al.Comparative efficacy of nonpharmacological adjuvant therapies for quality of life in the patients with breast cancer receiving chemo-or radio-therapy:A protocol for systematic review and Bayesian network metaanalysis[J].Medicine(Baltimore),2018,97(35):e12096.
[11]Cohen R,Sroussi M,Pilati C,et al.Unresectable metastatic colorectal cancer patient cured with cetuximab-based chemotherapy:a case report with new molecular insights[J].JGastrointest Oncol,2018,9(4):E23-E27.
[12]Zhang Y,Li H,Cao R,et al.Suppression of miR-708 inhibits the Wnt/β-catenin signaling pathway by activating DKK3 in adult B-all[J].Oncotarget,2017,8(38):64114-64128.
[13]马勇,王礼宁,郭杨,等.姜黄素通过调节Wnt/β-catenin信号通路促进软骨细胞增殖的研究[J].广州中医药大学学报,2017,34(1):90-95.
[14]Lv L,Xiao XY,Gu ZH,et al.Silencing USP22 by asymmetric structure of interfering RNA inhibits proliferation and induces cell cycle arrest in bladder cancer cells[J].Mol Cell Biochem,2011,346(1-2):11-21.
[15]张洪印.术前放化疗在局部晚期结直肠癌患者中的应用及敏感性预测研究进展[J].中国普通外科杂志,2017,26(3):380-385.
[16]Lin Z,Yang H,Kong Q,et al.USP22 antagonizes p53transcriptional activation by deubiquitinating Sirt1 to suppress cell apoptosis and is required for mouse embryonic development[J].Molecular Cell,2012,46(4):484.
[17]刘英丽.泛素特异性蛋白酶USP22及其SNPs对Hela细胞凋亡及细胞周期调控作用的研究[D].石家庄:河北医科大学,2015.