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

doi:10.3877/cma.j.issn.1674-0793.2019.02.005

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IC₅₀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IC₅₀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IC₅₀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 USP22 vs 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。

关键词: 结直肠肿瘤, 泛素特异性肽酶22, Wnt/β-catenin信号通路, 抗药性，肿瘤

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.

Key words: Colorectal neoplasms, Ubiquitin specific protease 22, Wnt/β-catenin signal pathway, Drug resistance, neoplasm

表1 结直肠癌细胞对5-Fu的敏感性和USP22表达情况（n=6）

组别	IC₅₀(mg/L)	USP22蛋白表达(USP22/β-actin)
HT-29组	1.58±0.23	0.18±0.06
HT-29/5-Fu组	14.58±0.94	0.92±0.11
HT-29-sh	0.25±0.23	0.07±0.01
HT-29/5-Fu-sh	1.36±0.14	0.33±0.02
t值^a,b,c	7.905，10.112，9.239	7.618，9.682，8.476
P值^a,b,c	<0.01，<0.01，<0.01	<0.01，<0.01，<0.01

表1 结直肠癌细胞对5-Fu的敏感性和USP22表达情况（n=6）

组别	IC₅₀(mg/L)	USP22蛋白表达(USP22/β-actin)
HT-29组	1.58±0.23	0.18±0.06
HT-29/5-Fu组	14.58±0.94	0.92±0.11
HT-29-sh	0.25±0.23	0.07±0.01
HT-29/5-Fu-sh	1.36±0.14	0.33±0.02
t值^a,b,c	7.905，10.112，9.239	7.618，9.682，8.476
P值^a,b,c	<0.01，<0.01，<0.01	<0.01，<0.01，<0.01

图1 Western blotting检测5-Fu对HT-29细胞USP22蛋白表达的影响

图2 Western blotting检测HT-29和HT-29/5-Fu细胞USP22蛋白表达的差异

图3 抑制USP22表达对结直肠癌细胞USP22表达的影响

表2 抑制USP22表达对结直肠癌细胞β-catenin表达的影响（n=6）

组别	β-catenin蛋白表达(β-catenin/β-actin)
HT-29组	0.25±0.017
HT-29/5-Fu组	0.99±0.059
HT-29-sh USP22组	0.11±0.009
HT-29/5-Fu-sh USP22组	0.47±0.035
t值^a,b,c	8.823，6.656，5.997
P值^a,b,c	<0.01，<0.01，<0.01

表2 抑制USP22表达对结直肠癌细胞β-catenin表达的影响（n=6）

组别	β-catenin蛋白表达(β-catenin/β-actin)
HT-29组	0.25±0.017
HT-29/5-Fu组	0.99±0.059
HT-29-sh USP22组	0.11±0.009
HT-29/5-Fu-sh USP22组	0.47±0.035
t值^a,b,c	8.823，6.656，5.997
P值^a,b,c	<0.01，<0.01，<0.01

图4 抑制USP22表达对结直肠癌细胞β-catenin表达的影响

[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 22 contributes 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 non-pharmacological 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 meta-analysis[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]. J Gastrointest 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 p53 transcriptional 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.
[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.

[1]	康夏, 田浩, 钱进, 高源, 缪洪明, 齐晓伟. 骨织素抑制破骨细胞分化改善肿瘤骨转移中骨溶解的机制研究[J]. 中华乳腺病杂志(电子版), 2023, 17(06): 329-339.
[2]	代莉, 邓恢伟, 郭华静, 黄芙蓉. 术中持续输注艾司氯胺酮对腹腔镜结直肠癌手术患者术后睡眠质量的影响[J]. 中华普通外科学文献(电子版), 2023, 17(06): 408-412.
[3]	王得晨, 杨康, 杨自杰, 归明彬, 屈莲平, 张小凤, 高峰. 结直肠癌微卫星稳定状态和程序性死亡、吲哚胺2,3-双加氧酶关系的研究进展[J]. 中华普通外科学文献(电子版), 2023, 17(06): 462-465.
[4]	付佳, 肖海敏, 武曦, 冯涛, 师帅. 年龄校正查尔森合并症指数对腹腔镜结直肠癌围手术期并发症的预测价值[J]. 中华普通外科学文献(电子版), 2023, 17(05): 336-341.
[5]	薛永婷, 高峰, 王雅楠, 屈莲平. 溶瘤病毒治疗在结直肠癌中的应用[J]. 中华普通外科学文献(电子版), 2023, 17(05): 380-384.
[6]	唐旭, 韩冰, 刘威, 陈茹星. 结直肠癌根治术后隐匿性肝转移危险因素分析及预测模型构建[J]. 中华普外科手术学杂志(电子版), 2024, 18(01): 16-20.
[7]	张生军, 赵阿静, 李守博, 郝祥宏, 刘敏丽. 高糖通过HGF/c-met通路促进结直肠癌侵袭和迁移的实验研究[J]. 中华普外科手术学杂志(电子版), 2024, 18(01): 21-24.
[8]	李婷, 张琳. 血清脂肪酸代谢物及维生素D水平与结直肠癌发生的关系研究[J]. 中华普外科手术学杂志(电子版), 2023, 17(06): 661-665.
[9]	常剑, 邱峰, 毛郁琪. 摄食抑制因子-1与腹腔镜结直肠癌根治术后肝转移的关系分析[J]. 中华普外科手术学杂志(电子版), 2023, 17(05): 502-505.
[10]	王晓燕, 肖佑, 肖戈, 王真权. 老年结直肠癌肺转移CT特征及高危因素研究[J]. 中华普外科手术学杂志(电子版), 2023, 17(05): 506-509.
[11]	倪文凯, 齐翀, 许小丹, 周燮程, 殷庆章, 蔡元坤. 结直肠癌患者术后发生延迟性肠麻痹的影响因素分析[J]. 中华结直肠疾病电子杂志, 2023, 12(06): 484-489.
[12]	范小彧, 孙司正, 鄂一民, 喻春钊. 梗阻性左半结肠癌不同手术治疗方案的选择应用[J]. 中华结直肠疾病电子杂志, 2023, 12(06): 500-504.
[13]	关旭, 王锡山. 基于外科与免疫视角思考结直肠癌区域淋巴结处理的功与过[J]. 中华结直肠疾病电子杂志, 2023, 12(06): 448-452.
[14]	顾睿祈, 方洪生, 蔡国响. 循环肿瘤DNA检测在结直肠癌诊治中的应用与进展[J]. 中华结直肠疾病电子杂志, 2023, 12(06): 453-459.
[15]	孙昕, 程海波, 沈卫星. 基于全转录组学探讨仙连解毒方治疗Ⅲ期结直肠癌患者的疗效机制[J]. 中华消化病与影像杂志(电子版), 2023, 13(05): 277-283.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

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

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

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