【摘要】：目的:探討LKB1在人肝癌組織中的表達情況,并分析其與肝癌臨床病理特征之間的關(guān)系,明確LKB1在肝癌中的臨床意義。方法:收集廣西醫(yī)科大學附屬腫瘤醫(yī)院2014年1月至2014年12月經(jīng)外科手術(shù)切除并經(jīng)病理診斷為肝細胞肝癌癌組織及對應癌旁組織蠟塊標本79例。以及人肝癌細胞株MHCC-97H、QGY-7703、SSMC-7721、HepG2、Huh7和人正常肝細胞系HL7702作為實驗材料。利用q RT-PCR分析LKB1 m RNA在肝癌細胞株的表達情況,Western Blotting檢測肝癌細胞株中LKB1蛋白的表達情況,比較肝癌細胞株與正常肝細胞株中LKB1的表達。采用免疫組化檢測79例肝癌及癌旁組織標本中LKB1蛋白的表達情況,并將其分為LKB1低表達組(0-5分)和LKB1高表達組(6-12分),分析LKB1不同表達情況下與肝癌臨床病理特征和遠期預后之間的關(guān)系。選取Huh7肝癌細胞株,利用RNAi技術(shù)沉默LKB1的表達,研究LKB1基因沉默后對肝癌細胞的增殖、侵襲、遷移能力的影響并初步探討其可能的調(diào)控機制。結(jié)果:⑴、LKB1在不同的肝癌細胞中表達水平不同,除Huh7細胞株以外,在MHCC-97H、QGY-7703、SSMC-7721、Hep-G2細胞株中,LKB1的表達水平均低于HL7702(P0.05)。⑵、LKB1低表達與腫瘤最大直徑(P=0.005)和組織學分期(P=0.024)有關(guān),和患者年齡、性別、乙肝、BCLC分期、肝功能分期、腫瘤數(shù)目、癌栓、血清AFP濃度等無關(guān)。生存分析結(jié)果提示,低表達LKB1的患者術(shù)后總生存時間更短。COX單因素統(tǒng)計分析提示腫瘤最大直徑(P=0.038),腫瘤數(shù)目(P=0.012),組織學分期(P=0.032),LKB1低表達(P=0.018)與肝癌的預后密切相關(guān)。COX多因素模型分析表明,腫瘤數(shù)目(P=0.015),LKB1低表達(P=0.021)是肝癌患者預后的獨立因素。⑶、RNAi沉默LKB1表達后,肝癌細胞株Huh7中LKB1 m RNA及蛋白表達量均明顯下調(diào)。LKB1基因沉默后肝癌細胞的增殖、侵襲、遷移能力均明顯增強。同時發(fā)現(xiàn)當肝癌細胞中LKB1表達減少時,磷酸化p38的表達量明顯增加,E-鈣黏蛋白表達下降,波形蛋白表達上調(diào)。結(jié)論:在肝癌患者中,LKB1呈現(xiàn)普遍低表達水平,且其低表達與肝癌腫瘤最大直徑、組織學分期有關(guān),低表達LKB1患者預后較差。肝癌細胞株中Huh7細胞中LKB1呈現(xiàn)高表達水平,利用RNAi技術(shù)沉默基因表達后肝癌細胞增殖、侵襲、遷移能力均明顯增強。LKB1作為一個抑癌基因,正常情況下有可能通過抑制p38的磷酸化來抑制細胞的EMT進程,在LKB1表達下降后則對p38磷酸化作用加強并調(diào)控下游分子的表達,從而導致腫瘤細胞惡性程度增高。因此,深入研究其調(diào)控機制有望為肝癌的治療尋找新的藥物靶點及判斷預后的生物標記物。
[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.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.7

【參考文獻】

相關(guān)期刊論文 前5條

1 Jaleh Varshosaz;Maryam Farzan;;Nanoparticles for targeted delivery of therapeutics and small interfering RNAs in hepatocellular carcinoma[J];World Journal of Gastroenterology;2015年42期

2 祝普利;尹超;馮建龍;;原發(fā)性肝癌綜合治療進展[J];臨床肝膽病雜志;2015年06期

3 李航宇;李巖;劉丹;孫宏治;劉金鋼;;LPS通過p38/MAPK調(diào)控膽管癌細胞系ICBD的上皮間質(zhì)轉(zhuǎn)化[J];世界華人消化雜志;2013年21期

4 ;原發(fā)性肝癌診療規(guī)范(2011年版)[J];臨床腫瘤學雜志;2011年10期

5 梁璇;南克俊;徐勤枝;;絲氨酸/蘇氨酸蛋白激酶1基因沉默對肺癌細胞生物學行為的影響[J];南方醫(yī)科大學學報;2007年09期

，

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