【摘要】：目的:探討LKB1在人肝癌組織中的表達(dá)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(xué)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(xì)胞肝癌癌組織及對應(yīng)癌旁組織蠟塊標(biāo)本79例。以及人肝癌細(xì)胞株MHCC-97H、QGY-7703、SSMC-7721、HepG2、Huh7和人正常肝細(xì)胞系HL7702作為實(shí)驗(yàn)材料。利用q RT-PCR分析LKB1 m RNA在肝癌細(xì)胞株的表達(dá)情況,Western Blotting檢測肝癌細(xì)胞株中LKB1蛋白的表達(dá)情況,比較肝癌細(xì)胞株與正常肝細(xì)胞株中LKB1的表達(dá)。采用免疫組化檢測79例肝癌及癌旁組織標(biāo)本中LKB1蛋白的表達(dá)情況,并將其分為LKB1低表達(dá)組(0-5分)和LKB1高表達(dá)組(6-12分),分析LKB1不同表達(dá)情況下與肝癌臨床病理特征和遠(yuǎn)期預(yù)后之間的關(guān)系。選取Huh7肝癌細(xì)胞株,利用RNAi技術(shù)沉默LKB1的表達(dá),研究LKB1基因沉默后對肝癌細(xì)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(jī)制。結(jié)果:⑴、LKB1在不同的肝癌細(xì)胞中表達(dá)水平不同,除Huh7細(xì)胞株以外,在MHCC-97H、QGY-7703、SSMC-7721、Hep-G2細(xì)胞株中,LKB1的表達(dá)水平均低于HL7702(P0.05)。⑵、LKB1低表達(dá)與腫瘤最大直徑(P=0.005)和組織學(xué)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(dá)LKB1的患者術(shù)后總生存時間更短。COX單因素統(tǒng)計(jì)分析提示腫瘤最大直徑(P=0.038),腫瘤數(shù)目(P=0.012),組織學(xué)分期(P=0.032),LKB1低表達(dá)(P=0.018)與肝癌的預(yù)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(dá)(P=0.021)是肝癌患者預(yù)后的獨(dú)立因素。⑶、RNAi沉默LKB1表達(dá)后,肝癌細(xì)胞株Huh7中LKB1 m RNA及蛋白表達(dá)量均明顯下調(diào)。LKB1基因沉默后肝癌細(xì)胞的增殖、侵襲、遷移能力均明顯增強(qiáng)。同時發(fā)現(xiàn)當(dāng)肝癌細(xì)胞中LKB1表達(dá)減少時,磷酸化p38的表達(dá)量明顯增加,E-鈣黏蛋白表達(dá)下降,波形蛋白表達(dá)上調(diào)。結(jié)論:在肝癌患者中,LKB1呈現(xiàn)普遍低表達(dá)水平,且其低表達(dá)與肝癌腫瘤最大直徑、組織學(xué)分期有關(guān),低表達(dá)LKB1患者預(yù)后較差。肝癌細(xì)胞株中Huh7細(xì)胞中LKB1呈現(xiàn)高表達(dá)水平,利用RNAi技術(shù)沉默基因表達(dá)后肝癌細(xì)胞增殖、侵襲、遷移能力均明顯增強(qiáng)。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(xì)胞的EMT進(jìn)程,在LKB1表達(dá)下降后則對p38磷酸化作用加強(qiáng)并調(diào)控下游分子的表達(dá),從而導(dǎo)致腫瘤細(xì)胞惡性程度增高。因此,深入研究其調(diào)控機(jī)制有望為肝癌的治療尋找新的藥物靶點(diǎn)及判斷預(yù)后的生物標(biāo)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(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.7

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