[关键词]
[摘要]
目的 探究GP73是否通过上皮间质转化(EMT)影响肝癌细胞对奥沙利铂耐药。方法 采用实时荧光定量PCR(qRT-PCR)和Western blotting实验检测GP73在肝癌患者癌旁和癌组织中的表达变化;Western blotting检测GP73在SMMC7721、Bel-7404和HepG2细胞株中表达差异;采用浓度梯度递增方法建立HepG2奥沙利铂获得性耐药细胞株,通过MTT法检测奥沙利铂(2、4、8、16、32 μmol/L)对亲本细胞和耐药细胞活性的影响;采用Western blotting实验检测HepG2亲本细胞和耐药细胞中GP73及上皮间质转化相关分子N-cadherin、Vimentin、E-cadherin的表达;利用siRNA/质粒分别干扰或过表达GP73,采用MTT法检测奥沙利铂(2、4、8、16、32 μmol/L)对干扰GP73前后耐药细胞活性的影响;通过Westernblotting实验检测Vimentin、N-cadherin和E-cadherin的表达。结果 与癌旁组织比较,GP73 mRNA和蛋白水平在肝癌组织中均显著上调(P<0.05);与SMMC7721和Bel-7404GP73细胞比较,GP73在HepG2细胞中显著高表达(P<0.05)。奥沙利铂对亲本和耐药细胞的半数抑制浓度(IC50)分别为(12.03±0.06)、(19.26±0.04)μmol/L;干扰GP73后奥沙利铂对耐药细胞的抑制率升高,8、16、32 μmol/L浓度组差异显著(P<0.05)。与亲本细胞比较,耐药细胞中GP73、N-cadherin及Vimentin蛋白表达显著上调(P<0.05),而E-cadherin蛋白表达显著下调(P<0.05)。干扰GP73后,Vimentin、N-cadherin表达显著下调,E-cadherin的表达显著上调(P<0.05);过表达GP73后,Vimentin、N-cadherin表达显著上调,E-cadherin的表达显著下调(P<0.05)。结论 GP73对人肝癌细胞奥沙利铂耐药具有促进作用,其作用机制可能与调节EMT相关。
[Key word]
[Abstract]
Objective To investigate whether GP73 affects oxaliplatin resistance of hepatocellular carcinoma through EMT. Methods qPCR and Western blot were used to detect expression of GP73 in adjacent normal tissues and HCC tissues. Western blotting was used to detect the expression difference of GP73 in SMMC7721, BEL-7404 and HepG2 cell lines. OXA resistant cell was established by long term and low dose OXA incubation. MTT assay was used to detect the effect of OXA (2, 4, 8, 16, 32 μmol/L) on the activity of non-resistant and resistant cells and evaluate the establishment of OXA acquired resistant cell lines. Western blotting was used to detect the expression of GP73, EMT markers N-cadherin, Vimentin and E-cadherin in non-resistant and OXA resistant cells. GP73 were knocked down and overexpressed by siRNA/plasmid transfection in OXA resistant HepG2 cell. MTT assay was used to detect the effects of oxaliplatin (2, 4, 8, 16, 32 μmol/L) on the activity of drug-resistant cells before and after GP73 interference. Western blotting was used to detect the expression of GP73, EMT markers N-cadherin、Vimentin and E-cadherin. Results Compared with adjacent tissues, the mRNA and protein levels of GP73 were significantly up-regulated in liver cancer tissues (P<0.05). Compared with SMMC7721 and Bel-7404GP73 cells, the expression of GP73 was significantly higher in HepG2 cells (P<0.05). The IC50 values of oxaliplatin against parents and drug-resistant cells were (12.03±0.06), (19.26±0.04) μmol/L, respectively, respectively. After interfering with GP73, the inhibition rate of oxaliplatin on drug-resistant cells was increased, and there were significant differences in 8, 16 and 32 μmol/L concentration groups (P<0.05). Compared with parental cells, the protein expressions of GP73, N-cadherin and Vimentin in drug-resistant cells were significantly up-regulated (P<0.05), while the protein expression of E-cadherin was significantly down-regulated (P<0.05). After interference with GP73, the expression of Vimentin and N-cadherin was significantly down-regulated, while the expression of E-cadherin was significantly up-regulated (P<0.05). After overexpression of GP73, the expression of Vimentin and N-cadherin were significantly up-regulated, while the expression of E-cadherin was significantly down-regulated (P<0.05). Conclusion GP73 can promote oxaliplatin resistance in human hepatoma cells, and its mechanism may be related to regulating EMT.
[中图分类号]
R965
[基金项目]
黑龙江省省属高等学校基本科研业务费(2019-KYYWF-0968)