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

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

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