本文關(guān)鍵詞：肝癌中NFAT2基因啟動子區(qū)CpG島甲基化狀態(tài)的研究　出處：《江蘇大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 肝細(xì)胞性肝癌 活化T細(xì)胞核因子2 甲基化

【摘要】：目的肝細(xì)胞性肝癌(Hepatocellular carcinoma,HCC)的高患病率與低生存率嚴(yán)重危害著大眾健康。在探尋肝癌的有效早期診斷和分子靶向治療方面,對其致癌分子機(jī)制的了解不可或缺。有研究表明活化T細(xì)胞核因子(nuclear factor of activated T cells,NFAT)家族與腫瘤的發(fā)生與生長有關(guān),其中活化T細(xì)胞核因子2(NFAT2)基因在肝癌組織與癌旁組織中表達(dá)差異最大,較癌旁組織,在癌組織中表達(dá)下降。本文即是從表觀遺傳學(xué)角度,探究肝細(xì)胞性肝癌(HCC)中NFAT2基因表達(dá)下降的機(jī)制,探索該基因啟動子區(qū)CpG島甲基化的狀態(tài)。并進(jìn)一步驗(yàn)證甲基化對NFAT2基因表達(dá)的影響。方法通過實(shí)時熒光定量聚合酶鏈反應(yīng)(quantificational real-time PCR,qRT-PCR)檢測組織標(biāo)本NFAT2 mRNA的表達(dá),通過蛋白質(zhì)免疫印跡(Western Blot,WB)檢測組織標(biāo)本蛋白的表達(dá),驗(yàn)證癌組織與癌旁組織中NFAT2的表達(dá)改變情況。通過重硫酸鹽測序法(bisulfite sequencing PCR,BSP)研究肝癌組織與癌旁組織及不同肝細(xì)胞系NFAT2啟動子的甲基化狀態(tài)。通過甲基化酶抑制劑(5-氮雜-2’-脫氧胞苷,5-aza-dC)的細(xì)胞加藥試驗(yàn)進(jìn)一步探索NFAT2啟動子區(qū)的甲基化狀態(tài)及其對NFAT2表達(dá)的影響。結(jié)果肝癌組織中NFAT2 mRNA和蛋白水平低于癌旁組織(P0.05)。癌組織中NFAT2基因啟動子CpG島甲基化百分比高于癌旁組織(33.0%±13.9%VS.21.6%±8.3%)(P=0.003)。人肝癌細(xì)胞系(HuH7、HepG2、Hep3B)中NFAT2基因啟動子CpG島甲基化頻率高于人正常肝細(xì)胞系(L02)(34.8%±7.3%,40.4%±10.3%and 37.0%±10.1%VS.16.2%±6.9%)(P0.01)。Spearman相關(guān)分析顯示NFAT2 mRNA水平與其啟動子甲基化程度呈負(fù)相關(guān)(r=-0.661,P=0.027)。添加甲基化酶抑制劑(5-aza-dC)的PLC細(xì)胞(實(shí)驗(yàn)組)NFAT2 mRNA和蛋白的表達(dá)高于添加二甲基亞楓(dimethyl sulfoxide,DMSO)的PLC細(xì)胞(對照組)(P0.05)。結(jié)論肝癌中NFAT2基因啟動子區(qū)CpG島處于高甲基化狀態(tài),并且該甲基化可能參與NFAT2表達(dá)降低的調(diào)控。
[Abstract]:Objective Hepatocellular carcinoma was used in hepatocellular carcinoma (HCC). The high prevalence rate and low survival rate of HCC seriously endanger public health. In order to explore the effective early diagnosis and molecular targeted therapy of liver cancer. It is necessary to understand the molecular mechanism of its carcinogenesis. Some studies have shown that nuclear factor of activated T cells can be activated. The expression of activated T cell nuclear factor 2 (NF-2NFAT2) gene in HCC and paracancerous tissues was higher than that in paracancerous tissues. From the perspective of epigenetics, we explore the mechanism of the decrease of NFAT2 gene expression in hepatocellular carcinoma (HCC). To explore the status of CpG island methylation in the promoter region of the gene, and to further verify the effect of methylation on the expression of NFAT2 gene. Quantificational real-time PCR. The expression of NFAT2 mRNA was detected by qRT-PCR, and the expression of protein was detected by Western blot. To verify the change of NFAT2 expression in cancer tissues and adjacent tissues, bisulfite sequencing PCR was detected by bisulfate sequencing. The methylation status of NFAT2 promoter in hepatocellular carcinoma tissues, paracancerous tissues and different liver cell lines was studied by means of 5-aza-2-deoxycytidine (deoxycytidine), a methylase inhibitor. 5-aza-dc). The methylation status of NFAT2 promoter and its effect on the expression of NFAT2 in HCC were further explored by cell addition assay. Results the levels of NFAT2 mRNA and protein in HCC tissues were lower than those in paracancerous tissues (P < 0.05). The percentage of CpG island methylation of NFAT2 promoter in cancer tissue was higher than that in paracancerous tissue (33.0% 鹵13.9VS.21.6% 鹵8.3%). Human hepatoma cell line HuH7. The methylation frequency of CpG island of NFAT2 gene promoter was higher than that of human normal liver cell line L02H3B (34.8% 鹵7.3%). 40.4% 鹵10.3 and 37.0% 鹵10.1 vs 16.2% 鹵6.9%. Spearman correlation analysis showed that the level of NFAT2 mRNA was negatively correlated with its promoter methylation. The expression of NFAT2 mRNA and protein in PLC cells supplemented with methylase inhibitor 5 aza-dC was higher than that in the experimental group (P < 0. 027). Dimethyl sulfoxide. Conclusion the CpG island in the promoter region of NFAT2 gene in HCC is hypermethylated. And this methylation may be involved in the regulation of NFAT2 expression reduction.
【學(xué)位授予單位】：江蘇大學(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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本文編號：1379557