赖氨酸特异性组蛋白去甲基化酶1在肝细胞型肝癌组织的表达及对细胞增殖及侵袭力的影响

Expression of lysine-specific demethylase 1 in hepatocellular carcinoma tissues and its effect on cell proliferation and invasion

目的观察赖氨酸特异性组蛋白去甲基化酶1(LSD1)在肝细胞型肝癌组织中表达,以及下调LSD1基因表达对人肝癌HepG2细胞增殖及侵袭力的影响。方法选取我院及河南省肿瘤医院择期行手术治疗的肝细胞型肝癌患者91例,免疫组织化学法检测肝癌和癌旁组织中LSD1蛋白表达,培养人肝癌HepG2细胞,分为LSD1干扰序列组、阴性对照组和空白组,实时荧光定量聚合酶链反应(FQ-PCR)术检测细胞中LSD1基因表达,噻唑蓝(MTT)法检测细胞增殖能力,Transwell小室检测细胞侵袭力,Western blot法检测细胞中LSD1、Snail、上皮型钙黏蛋白(E-cadherin)和神经型钙黏蛋白(N-cadherin)表达。结果肝癌组织中LSD1蛋白阳性表达率为81.32%,高于癌旁组织的35.16%(P<0.01);肝癌组织中LSD1蛋白表达与分化程度、TNM分期和血管浸润明显相关(P<0.05);LSD1干扰序列组细胞中LSD1 mRNA相对表达量为1.19±0.10,低于阴性对照组和空白组(2.29±0.14、2.34±0.11,P<0.01),LSD1干扰序列组24、48、72、96 h时吸光度(A)值分别为0.23±0.05、0.32±0.08、0.38±0.08、0.52±0.03,低于阴性对照组(0.38±0.08、0.49±0.05、0.67±0.04、0.77±0.10)和空白组(0.33±0.03、0.52±0.09、0.70±0.08、0.78±0.07,P<0.05);LSD1干扰序列组侵袭细胞数(86.3±3.9)低于阴性对照组(110.6±9.4)和空白组(117.7±7.0,P<0.05);LSD1干扰序列组细胞中LSD1、Snail和N-cadherin蛋白相对表达量(0.25±0.03、0.30±0.02、0.32±0.03)低于阴性对照组(0.77±0.06、0.76±0.10、0.67±0.04)和空白组(0.84±0.14、0.74±0.07、0.69±0.06,P<0.05),而E-cadherin蛋白相对表达(0.71±0.05)高于阴性对照组(0.37±0.02)和空白组(0.34±0.04,P<0.05)。结论LSD1蛋白在肝细胞型肝癌组织中呈高表达,可能通过上皮-间充质转化(EMT)过程而在人肝癌HepG2细胞增殖和侵袭中发挥重要作用。

Objective To investigate the expression of lysine-specific demethylase 1 (LSD1) in hepatocellular carcinoma tissues, and to explore the effects of down-regulation of LSD1 gene expression on proliferation and invasion of human hepatoma HepG2 cells. Methods A total of 91 cases of hepatocellular carcinoma undergoing elective surgery in our hospital and Henan Cancer Hospital were selected. Immunohistochemistry was used to detect the expression of LSD1 protein in hepatocellular carcinoma tissues and adjacent tissues. Human hepatoma HepG2 cells were cultured and divided into LSD1 interference sequence group, negative control group and blank group. Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was used to detect the expression of LSD1 gene in cells. Methyl thiazol tetrazolium (MTT) assay was used to detect cell proliferation. Transwell chamber was used to detect cell invasion. Western blotting was used to detect the expression of LSD1, Snail, E-cadherin and N-cadherin proteins in cells. Results The positive expression rate of LSD1 protein in hepatocellular carcinoma tissues was 81.32%, which was significantly higher than that in adjacent tissues (35.16%, P<0.01). The expression of LSD1 protein in hepatocellular carcinoma tissues was related to the degree of differentiation, TNM staging and vascular invasion (P<0.05). The relative expression level of LSD1 mRNA in LSD1 interference sequence group was 1.19±0.10, which was significantly lower than that in the negative control group and blank group (2.29±0.14 and 2.34±0.11, P<0.05). The absorbance (A) values at 24, 48, 72 and 96 h in the LSD1 interference sequence group were 0.23±0.05, 0.32±0.08, 0.38±0.08 and 0.52±0.03, respectively, which were significantly lower than those in the negative control group (0.38±0.08, 0.49±0.05, 0.67±0.04 and 0.77±0.10) and blank group (0.33±0.03, 0.52±0.09, 0.70±0.08 and 0.78±0.07, P<0.05). The number of invasive cells in the LSD1 interference sequence group was (86.3±3.9), which was significantly less than that in the negative control group (110.6±9.4) and the blank group (117.7±7.0, P<0.05). The relative expression levels of LSD1, Snail, and N-cadherin proteins in the LSD1 interference sequence group were (0.25±0.03, 0.30±0.02 and 0.32±0.03), which were significantly lower than those in the negative control group (0.77±0.06, 0.76±0.10 and 0.67±0.04) and blank group (0.84±0.14, 0.74±0.07 and 0.69±0.06, P<0.05), and the relative expression level of E-cadherin protein (0.71±0.05) was significantly higher than in the negative control group (0.37±0.02) and blank group (0.34±0.04, P<0.05). Conclusion LSD1 protein was highly expressed in hepatocellular carcinoma tissues, and might play an important role in the proliferation and invasion of human hepatoma HepG2 cells through epithelial-mesenchymal transition.