JNK酶活性下调对人前列腺癌、肝细胞癌和乳腺癌细胞生长的抑制作用

doi:10.13481/j.1671-587x.20190513

摘要/Abstract

摘要： 目的：通过c-Jun氨基末端激酶（JNK）抑制剂SP600125和脂质纳米粒子包裹的JNK-siRNA 2种方法在体内和体外分别对JNK基因的表达进行干扰，探讨下调JNK酶活性在肿瘤治疗中的作用。方法：体外实验，将实验分为siRNA组和抑制剂SP600125组。在siRNA组中，使用JNK-siRNA及其对照NC-siRNA分别转染人前列腺癌细胞（PC细胞）、人肝细胞癌细胞（SMMC-7721细胞）和人乳腺癌细胞（MCF-7细胞）；在抑制剂SP600125组中将SP600125导入肝细胞癌细胞中，采用Western blotting法检测转染JNK-siRNA和SP600125后人前列腺癌、肝细胞癌和乳腺癌细胞中JNK或磷酸化JNK（p-JNK）蛋白表达水平；采用WST-1增殖实验检测各组肿瘤细胞的细胞活力。体内实验，通过皮下注射SMMC-7721细胞建立小鼠皮下肝细胞癌移植瘤动物模型，将8只小鼠饲养至肿瘤生长至3 mm×3 mm，再将模型小鼠随机分为抑制剂SP600125组和阴性对照组，JNK-siRNA组和NC-siRNA对照组，共4组，各组小鼠分别瘤内注射5 nmol SP600125、阴性对照溶液、脂质纳米粒子包裹的JNK-siRNA和NC-siRNA阴性对照，观察各组小鼠肿瘤体积；采用免疫组织化学染色检测肿瘤组织中JNK或p-JNK蛋白表达。结果：体外实验，与NC-siRNA对照组比较，JNK-siRNA组人前列腺癌、肝细胞癌和乳腺癌细胞中JNK蛋白表达水平降低（P<0.01），抑制剂SP600125组肝细胞癌细胞中p-JNK蛋白表达水平较其阴性对照组明显降低（P<0.01）。体内实验，以肝细胞癌细胞为例，抑制剂SP600125组小鼠肿瘤体积较阴性对照组明显减小，而JNK-siRNA组与其阴性对照组比较肿瘤体积变化不明显。免疫组织化学染色，抑制剂SP600125组小鼠移植瘤组织中p-JNK蛋白表达量和JNK-siRNA组小鼠移植瘤组织中JNK蛋白表达量较各自阴性对照组降低（P<0.01）。结论：在体外和体内实验中下调JNK酶活性在体内和体外均能降低JNK基因的表达水平，进而抑制肿瘤细胞的生长。

关键词: 前列腺肿瘤, 肝细胞癌, 乳腺肿瘤, 脂质纳米粒子, c-Jun氨基末端激酶, RNA干扰, 酶活性

Abstract: Objective:To discuss the role for down-regulation of JNK enzymatic activity in the tumor treatments by interrupting JNK gene expression with JNKinhibitor SP600125 and JNK-siRNA encapsulated by lipid nanoparticles in vivo and in vitro. Methods:In vitro experiments, the experiment was divided into siRNA group and inhibitor SP600125 group. In siRNA group,JNK-siRNA and NC-siRNA were transfected into the human prostate cancer cells (PC cells), human hepatocellular carcinoma cells(SMMC-7721 cells) and human breast cancer cells(MCF cells), respectively. In inhibitor SP600125 group, SP600125 was delivered to human hepatocellular carcinoma cells. The expression levels of JNK or p-JNK proteins in human prostate cancer cells,human hepatocellular carcinoma cells,and human breast cancer cells after transfected with JNK-siRNA and inhibitor SP600125 were detected by Western blotting method. The cell viabilities of tumor cells in various groups were examined by WST-1 proliferation assay. In vivo experiments,the human hepatocellular carcinoma SMMC-7721 cells were used to establish the subcutaneous hepatocellular carcinoma xenograft tumor models by subcutaneous injection. The eight mice were fed until the tumors were generated to 3 mm×3 mm. They were randomly divided into inhibitor SP600125 group and negative control group,JNK-siRNA group and NC-siRNA control group. Each mouse in various groups was injected intratumorally with 5 nmol SP600125,negative control solution, lipid nanoparticles-mediated JNK-siRNA and NC-siRNA negative control. The volumes of tumors of the mice in various groups were observed. The expressions of JNK or p-JNK proteins in tumor tissue were examined by immunohistochemical staining. Results: In vitro experiments, compared with NC-siRNA control group, the expression level of JNK protein in human prostate cancer cells, human hepatocellular carcinoma cells and human breast cancer cells in JNK-siRNA group were decreased(P<0.01); the expression level of p-JNK protein in human hepatocellular carcinoma cells in inhibitor SP600125 group was significantly increased compared with its negative control group(P<0.01).In vivo experiment, the human hepatocellular carcinoma cells were taken as an example, the volume of tumor of the mice in inhibitor SP600125 group was significantly reduced compared with negative control group, whereas the change of tumor volume in JNK-siRNAs group was not significant compared with NC-siRNA control group. The immunohistochemical staining results showed that compared with their negative control groups, the expression amount of p-JNK protein in tumor tissue of the mice in SP600125 group and the expression amount of JNK protein in tumor tissue of the mice in JNK-siRNA group were decreased (P<0.01). Conclusion:Down-regulation of enzymatic activity of JNK can decrease the expression level of JNK gene and then inhibit the tumor growth both in vivo and in vitro.

Key words: prostate neoplasms, hepatocellular carcinoma, breast neoplasms, lipid nanoparticle, c-Jun N-terminal kinase, RNA interference, enzymatic activity

中图分类号:

R965

岳圆, 徐瀛濂, 王晶晶, 孙敏英, 张泽南, 赵馨越, 李宗璞, 台桂香. JNK酶活性下调对人前列腺癌、肝细胞癌和乳腺癌细胞生长的抑制作用[J]. 吉林大学学报(医学版), 2019, 45(05): 1046-1051.

YUE Yuan, XU Yinglian, WANG Jingjing, SUN Minying, ZHANG Zenan, ZHAO Xinyue, LI Zongpu, TAI Guixiang. Inhibitory effects of down-regulation of JNK enzymatic activity on growth of human prostate cancer cells, hepatocellular carcinoma cells and breast cancer cells[J]. Journal of Jilin University(Medicine Edition), 2019, 45(05): 1046-1051.

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