本文選題：肝細(xì)胞肝癌　切入點(diǎn)：Hippo信號(hào)通路　出處：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的通過(guò)去甲基化藥物上調(diào)LATS1基因在人肝癌細(xì)胞中的表達(dá),研究LATS1基因?qū)θ烁伟┘?xì)胞增殖、凋亡、裸鼠成瘤及Hippo信號(hào)通路的影響。方法利用蛋白免疫印跡法(Western Blot)檢測(cè)人肝癌細(xì)胞Hep G2與SMMC-7721中大腫瘤抑制基因-1(Large tumor suppressor gene 1,LATS1)和下游癌基因Yes-相關(guān)蛋白(Yes-associated protein,YAP)的蛋白表達(dá)水平,使用甲基化特異性PCR(Methylation-specific PCR,MSP)與亞硫酸氫鹽測(cè)序法(Bisulfite sequencing PCR,BSP)檢測(cè)Hep G2與SMMC-7721細(xì)胞中LATS1基因的甲基化水平,利用5-氮雜-2脫氧胞苷(5-Aza-2’-deoxycytidine,DAC)去甲基化處理Hep G2與SMMC-7721細(xì)胞后,用MSP和Western Blot分別檢測(cè)LATS1的甲基化水平與LATS1和YAP的蛋白表達(dá)水平,流式細(xì)胞術(shù)(Flow cytometry,FCM)檢測(cè)各組細(xì)胞凋亡和周期情況,建立細(xì)胞裸鼠皮下移植瘤模型,檢測(cè)各組細(xì)胞成瘤情況。結(jié)果MSP顯示人肝癌細(xì)胞系Hep G2與SMMC-7721中LATS1存在高度甲基化;BSP定量顯示Hep G2細(xì)胞中LATS1甲基化程度(甲基化的CPG島位點(diǎn)占全部CPG島位點(diǎn)的比例)為92.4%,SMMC-7721細(xì)胞中LATS1甲基化程度為93.1%,LATS1基因啟動(dòng)子區(qū)域高度甲基化;WB提示LTAS1低表達(dá),YAP高表達(dá)(P0.05)。經(jīng)DAC去甲基化處理Hep G2與SMMC-7721,MSP顯示LATS1的甲基化水平顯著降低(P0.05),同時(shí)LAST1蛋白表達(dá)水平顯著升高(P0.05),但YAP蛋白表達(dá)水平顯著降低(P0.05),細(xì)胞周期停滯在G1期(P0.05)、細(xì)胞凋亡率顯著增加(P0.05)、裸鼠皮下移植瘤大小與體積顯著減小(P0.05)。結(jié)論LATS1基因在人肝癌細(xì)胞中甲基化程度較高,去甲基化后能夠上調(diào)LATS1基因的蛋白表達(dá)水平同時(shí)降低YAP蛋白表達(dá)水平,抑制并降低Hep G2與SMMC-7721細(xì)胞增殖能力、誘導(dǎo)其凋亡,遏制肝癌細(xì)胞的成瘤能力。
[Abstract]:Objective to study the effect of LATS1 gene on proliferation and apoptosis of human hepatoma cells by upregulating the expression of LATS1 gene in human hepatoma cells by demethylating drugs. Methods the expression of large tumor suppressor gene 1 (LATS1) and Yes-associated protein (Yes-associated protein) in human hepatoma cell line Hep G2 and SMMC-7721 were detected by Western blot. The methylation level of LATS1 gene in Hep G2 and SMMC-7721 cells was detected by methylation specific PCR(Methylation-specific PCR MSPs and bisulfite sequencing PCR BSPs. Hep G2 and SMMC-7721 cells were treated with 5-aza-2-deoxycytidine demethylation (5-Aza-2-deoxycytidine). The methylation level of LATS1 and the protein expression of LATS1 and YAP were detected by MSP and Western Blot, and apoptosis and cell cycle were detected by flow cytometry. Results MSP showed that there was hypermethylation of LATS1 in Hep G2 and LATS1 in SMMC-7721. The extent of LATS1 methylation in Hep G2 cells was determined quantitatively (the percentage of methylated CPG island loci to all CPG island sites) in Hep G2 cells. The degree of LATS1 methylation in SMMC-7721 cells was 93.1. The high methylation of LTAS1 in the promoter region of LATS1 gene suggested that the low expression of LTAS1 and the high expression of P0.050.The methylation level of Hep G2 and SMMC-7721 showed that the methylation level of LATS1 was significantly lower than that of LAST1 protein surface after DAC demethylation treatment in SMMC-7721 cells. The expression of YAP protein decreased significantly, the cell cycle stagnated in G1 phase, the apoptosis rate increased significantly, and the size and volume of subcutaneous transplanted tumor decreased significantly in nude mice. Conclusion the expression of LATS1 gene in human hepatoma cells is significantly lower than that in human hepatoma cells. Higher levels of methylation, Demethylation can up-regulate the expression of LATS1 gene and decrease the expression of YAP protein, inhibit and decrease the proliferation of Hep G2 and SMMC-7721 cells, induce apoptosis and inhibit the tumorigenic ability of hepatoma cells.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735.7

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相關(guān)期刊論文 前1條

1 Piotr M Wierzbicki;Krystian Adrych;Dorota Kartanowicz;Marcin Stanislawowski;Anna Kowalczyk;Janusz Godlewski;Iwona Skwierz-Bogdanska;Krzysztof Celinski;Tomasz Gach;Jan Kulig;Bartlomiej Korybalski;Zbigniew Kmiec;;Underexpression of LATS1 TSG in colorectal cancer is associated with promoter hypermethylation[J];World Journal of Gastroenterology;2013年27期

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本文編號(hào)：1654073