NEK6在HepG2细胞中的定位及其在肝癌中的表达和临床意义

The Localization of NEK6 in HepG2 Cell Line and Its Expression and Clinical Significance in HCC

摘要: 目的：检测NEK6在HepG2细胞中的定位，肝细胞肝癌组织与正常肝脏组织中的表达水平，分析其与临床病理学资料间的关系。方法：在HepG2细胞中转染pMCV-Nek6-Myc和pMCV-Nek6-Flag，应用间接免疫荧光法测定NEK6在HepG2细胞中的定位；应用RT-qPCR法检测47例肝细胞肝癌(HCC)组织与相应远癌旁组织(>1 cm)中NEK6 mRNA的表达水平；应用Western blot法检测该47例肝细胞肝癌组织与相应正常肝组织中NEK6蛋白的表达水平。收集并统计该组患者的临床病理学资料，包括性别、年龄、肿瘤大小、肿瘤数量(单个/多个)、血清AFP水平(≥400 ng/ml或<400 ng/ml)、Edmondson分级、纤维包膜、微血管侵犯(microvascular invasion, MVI)等，分析NEK6 mRNA表达水平与各临床病理特点的关系。配对样本应用配对t检验，单因素分析用卡方检验，多因素分析用二元Logistic回归分析，以P < 0.05认为结果有统计学意义。结果：免疫荧光实验显示，NEK6主要定位于HepG2细胞的细胞核中，在细胞质中也有少量存在；RT-qPCR结果显示，NEK6 mRNA在HCC中的表达量为(1.405 ± 0.472)，显著高于正常肝组织中的表达量(1.133 ± 0.474)，差异有统计学意义(t = 3.143, P = 0.0029)；Western blot结果显示，NEK6蛋白在HCC中的表达量为(1.031 ± 0.193)，显著高于正常肝组织中的表达量(0.906 ± 0.182)，差异有统计学意义(t = 3.464, P = 0.0012)。结论：NEK6主要存在于HepG2细胞的细胞核中，在肝细胞肝癌中表达上调，微血管侵犯是Nek6表达水平的独立相关因素，可能作为肝癌预后不良的判断指标之一。

Abstract: Objective: To detect the localization of NEK6 in HepG2 cells, the expression level of hepatocellular carcinoma tissues and normal liver tissues, and to analyze the relationship between NEK6 and clinicopathological data. Methods: HepG2 cells were transfected with pMCV-Nek6-Myc and pMCV-Nek6-Flag, and the localization of NEK6 in HepG2 cells was determined by indirect immunofluorescence method. RT-qPCR was used to detect the expression level of NEK6 mRNA in 47 cases of hepatocellular carcinoma (HCC) tissues and corresponding distant adjacent tissues (>1 cm). Western blot was used to detect the NEK6 protein expression levels in the 47 cases of hepatocellular carcinoma tissues and corresponding normal liver tissues. The clinical pathological data of this group of patients were collected and counted, including gender, age, tumor size, tumor number (single/multiple), serum AFP level (≥400 ng/ml or <400 ng/ml), Edmondson grade, fiber envelope , Microvascular invasion (microvascular invasion MVI), etc., to analyze the relationship between NEK6 mRNA expression level and various clinicopathological characteristics. Paired samples were tested by paired t test, chi-square test was used for univariate analysis, and binary logistic regression analysis was used for multivariate analysis. The results were considered statistically significant at P < 0.05. Results: Immunofluorescence experiments showed that NEK6 was mainly located in the nucleus of HepG2 cells, and a small amount in the cytoplasm; RT-qPCR results showed that the expression of NEK6 mRNA in HCC was (1.405 ± 0.472), which was significantly higher than that of normal liver tissue: the expression level in HCC (1.133 ± 0.474); the difference was statistically significant (t = 3.143, P = 0.0029); Western blot results showed that the expression level of NEK6 protein in HCC was (1.031 ± 0.193), which was significantly higher than that of normal liver. The expression in tissues (0.906 ± 0.182) has a statistically significant difference (t = 3.464, P = 0.0012). Conclusion: NEK6 mainly exists in the nucleus of HepG2 cells, and its expression is up-regulated in hepatocellular carcinoma. Microvascular invasion is an independent factor related to the expression of Nek6 and may be used as one of the indicators of poor prognosis of liver cancer.