LKB1在肝癌组织中的表达、功能及其机制的研究

发布时间：2018-10-17 21:14

【摘要】：目的:探讨LKB1在人肝癌组织中的表达情况,并分析其与肝癌临床病理特征之间的关系,明确LKB1在肝癌中的临床意义。方法:收集广西医科大学附属肿瘤医院2014年1月至2014年12月经外科手术切除并经病理诊断为肝细胞肝癌癌组织及对应癌旁组织蜡块标本79例。以及人肝癌细胞株MHCC-97H、QGY-7703、SSMC-7721、HepG2、Huh7和人正常肝细胞系HL7702作为实验材料。利用q RT-PCR分析LKB1 m RNA在肝癌细胞株的表达情况,Western Blotting检测肝癌细胞株中LKB1蛋白的表达情况,比较肝癌细胞株与正常肝细胞株中LKB1的表达。采用免疫组化检测79例肝癌及癌旁组织标本中LKB1蛋白的表达情况,并将其分为LKB1低表达组(0-5分)和LKB1高表达组(6-12分),分析LKB1不同表达情况下与肝癌临床病理特征和远期预后之间的关系。选取Huh7肝癌细胞株,利用RNAi技术沉默LKB1的表达,研究LKB1基因沉默后对肝癌细胞的增殖、侵袭、迁移能力的影响并初步探讨其可能的调控机制。结果:⑴、LKB1在不同的肝癌细胞中表达水平不同,除Huh7细胞株以外,在MHCC-97H、QGY-7703、SSMC-7721、Hep-G2细胞株中,LKB1的表达水平均低于HL7702(P0.05)。⑵、LKB1低表达与肿瘤最大直径(P=0.005)和组织学分期(P=0.024)有关,和患者年龄、性别、乙肝、BCLC分期、肝功能分期、肿瘤数目、癌栓、血清AFP浓度等无关。生存分析结果提示,低表达LKB1的患者术后总生存时间更短。COX单因素统计分析提示肿瘤最大直径(P=0.038),肿瘤数目(P=0.012),组织学分期(P=0.032),LKB1低表达(P=0.018)与肝癌的预后密切相关。COX多因素模型分析表明,肿瘤数目(P=0.015),LKB1低表达(P=0.021)是肝癌患者预后的独立因素。⑶、RNAi沉默LKB1表达后,肝癌细胞株Huh7中LKB1 m RNA及蛋白表达量均明显下调。LKB1基因沉默后肝癌细胞的增殖、侵袭、迁移能力均明显增强。同时发现当肝癌细胞中LKB1表达减少时,磷酸化p38的表达量明显增加,E-钙黏蛋白表达下降,波形蛋白表达上调。结论:在肝癌患者中,LKB1呈现普遍低表达水平,且其低表达与肝癌肿瘤最大直径、组织学分期有关,低表达LKB1患者预后较差。肝癌细胞株中Huh7细胞中LKB1呈现高表达水平,利用RNAi技术沉默基因表达后肝癌细胞增殖、侵袭、迁移能力均明显增强。LKB1作为一个抑癌基因,正常情况下有可能通过抑制p38的磷酸化来抑制细胞的EMT进程,在LKB1表达下降后则对p38磷酸化作用加强并调控下游分子的表达,从而导致肿瘤细胞恶性程度增高。因此,深入研究其调控机制有望为肝癌的治疗寻找新的药物靶点及判断预后的生物标记物。
[Abstract]:Objective: to investigate the expression of LKB1 in human hepatocellular carcinoma (HCC) and its relationship with the clinicopathological features of HCC, and to determine the clinical significance of LKB1 in HCC. Methods: from January 2014 to December 2014, 79 specimens of hepatocellular carcinoma tissues and corresponding adjacent tissues were collected from the affiliated Cancer Hospital of Guangxi Medical University, which were surgically resected and pathologically diagnosed. Human hepatoma cell line MHCC-97H,QGY-7703,SSMC-7721,HepG2,Huh7 and human normal liver cell line HL7702 were used as experimental materials. The expression of LKB1 m RNA in hepatocellular carcinoma cell line was analyzed by Q RT-PCR. The expression of LKB1 protein in hepatoma cell line was detected by, Western Blotting, and the expression of LKB1 in hepatoma cell line was compared with that in normal liver cell line. The expression of LKB1 protein in 79 cases of liver cancer and adjacent tissues was detected by immunohistochemistry. They were divided into two groups: low expression group (0-5 points) of LKB1 and high expression group of LKB1 (6-12 points). The relationship between LKB1 expression and clinicopathological features and long-term prognosis of HCC was analyzed. Huh7 hepatoma cell lines were selected to silence the expression of LKB1 by RNAi technique. The effects of LKB1 gene silencing on the proliferation, invasion and migration of HCC cells were studied and the possible regulatory mechanisms were discussed. Results: 1the expression level of LKB1 was different in different hepatoma cells, except for Huh7 cell line, the expression level of LKB1 in MHCC-97H,QGY-7703,SSMC-7721,Hep-G2 cell line was lower than that in HL7702 cell line (P0.05). 2 the low expression of LKB1 was related to the maximum diameter of tumor (P0. 005) and histological stage (P0. 024), and to the age and sex of the patients. Hepatitis B, BCLC stage, liver function stage, tumor number, tumor thrombus, serum AFP concentration were not related. The results of survival analysis suggest that, The total survival time of the patients with low expression of LKB1 was shorter. COX univariate statistical analysis showed that the maximum diameter of tumor (P0. 038), tumor number (P0. 012), histological stage (P0. 032) and low expression of LKB1 (P0. 018) were closely related to the prognosis of HCC. The number of tumor (Pn0. 015) and the low expression of LKB1 (Pn0. 021) were independent factors of prognosis of HCC patients. After silencing the expression of LKB1, the expression of LKB1 m RNA and protein in hepatoma cell line Huh7 were significantly down-regulated. LKB1 gene silenced the proliferation and invasion of HCC cells. The migration ability was obviously enhanced. At the same time, the expression of phosphorylated p38 was increased, the expression of E-cadherin was decreased, and the expression of vimentin was up-regulated when the expression of LKB1 was decreased. Conclusion: in HCC patients, the expression of LKB1 is generally low, and its low expression is related to the maximum diameter and histological stage of HCC, and the prognosis of patients with low expression of LKB1 is poor. The expression of LKB1 was high in Huh7 cells. The ability of proliferation, invasion and migration of hepatoma cells was significantly enhanced by silencing gene expression with RNAi technique. LKB1 was a tumor suppressor gene. Under normal conditions, it is possible to inhibit the EMT process by inhibiting the phosphorylation of p38. After the decrease of LKB1 expression, the phosphorylation of p38 is enhanced and the downstream expression is regulated, which leads to the increase of malignant degree of tumor cells. Therefore, further study of its regulatory mechanism is expected to find new drug targets and biomarkers for the treatment of liver cancer.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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