本文選題：miR-181a + ATG5��； 參考：《南京醫(yī)科大學(xué)》2017年博士論文

【摘要】：背景和研究目的:肝細(xì)胞癌(hepatocellularcarcinoma,HCC)是最常見的原發(fā)性肝細(xì)胞癌,約占原發(fā)性肝癌的90%,是全球癌癥死亡的主要原因。在HCC的發(fā)展中,有缺陷的自噬反應(yīng)有助于這個(gè)過程。因此,它仍然需要闡明自噬在肝癌中的作用,以及如何調(diào)節(jié)細(xì)胞自噬。miR-181a被發(fā)現(xiàn)在多種腫瘤,如乳腺癌,胃癌,和神經(jīng)母細(xì)胞瘤可抑制腫瘤細(xì)胞自噬。然而,miR-181a在肝癌細(xì)胞自噬中作用尚不明確。最近的研究發(fā)現(xiàn),肝癌miR-181a表達(dá)上調(diào),這可能與HCC的發(fā)病機(jī)制相關(guān)。因此,我們將探討miR-181a在肝癌細(xì)胞自噬中的作用機(jī)制。方法:本課題通過臨床上收集人肝細(xì)胞性肝癌組織和肝血管瘤組織,采用QPCR檢測(cè)組織中miR1-181a的表達(dá)量。WB檢測(cè)肝癌組織中自噬相關(guān)基因的表達(dá)量。建立腺病毒 Ad-NC 以及 Ad-miR181a-sponge HCC/HepG2,WB 檢測(cè)抑制 mir181a對(duì)肝癌細(xì)胞中自噬水平的影響。雙熒光素酶報(bào)告分析系統(tǒng)分析miR-181a作用于HCC的靶基因;利用流式細(xì)胞術(shù)分析抑制mir181a對(duì)肝癌細(xì)胞早期凋亡的影響;通過裸鼠皮下成瘤實(shí)驗(yàn)進(jìn)一步驗(yàn)證mi R-181a對(duì)肝癌細(xì)胞增殖的影響。結(jié)果:我們研究發(fā)現(xiàn)miR-181a在人肝癌(HCC)組織中表達(dá)增加。通過檢測(cè)肝癌組織中LC3,p62和Atg5蛋白的表達(dá)水平,發(fā)現(xiàn)自噬相關(guān)基因LC3、Atg5在人肝癌(HCC)組織中表達(dá)降低。miR-181a抑制可促進(jìn)HepG2細(xì)胞自噬上調(diào)。miR-181a靶基因通過熒光素酶檢測(cè)進(jìn)行驗(yàn)證,發(fā)現(xiàn)結(jié)果表明miR-181a可以綁定3'-UTRAtg5,Atg5是miR-181a在HepG2細(xì)胞中的靶基因。利用流式細(xì)胞儀檢測(cè),miR-181a抑制可促進(jìn)HepG2細(xì)胞的凋亡。在注射腺病毒miR-181a轉(zhuǎn)染HepG2細(xì)胞的小鼠,腫瘤體積及重量明顯小于對(duì)照組,研究提示抑制miR-181a表達(dá)可抑制腫瘤生長(zhǎng)。結(jié)論:我們研究發(fā)現(xiàn)miR-181a可以通過靶向基因Atg5抑制肝癌細(xì)胞自噬,降低肝癌細(xì)胞的凋亡,促進(jìn)腫瘤的生長(zhǎng)。這些發(fā)現(xiàn)可能會(huì)為肝癌治療提供了一個(gè)新的目標(biāo)。
[Abstract]:Background and objective: hepatocellular carcinoma (HCC) is the most common primary hepatocellular carcinoma, accounting for about 90% of primary liver cancer, and is the main cause of cancer death in the world. In the development of HCC, defective autophagy contributes to this process. Therefore, it still needs to clarify the role of autophagy in liver cancer and how to regulate autophagy. MiR-181a has been found to inhibit autophagy in many tumors, such as breast cancer, gastric cancer, and neuroblastoma. However, the role of miR-181a in autophagy of hepatocellular carcinoma cells is unclear. Recent studies have found that miR-181a expression in HCC is up-regulated, which may be related to the pathogenesis of HCC. Therefore, we will explore the mechanism of miR-181a in autophagy of hepatoma cells. Methods: human hepatocellular carcinoma and hepatic hemangioma were collected clinically. The expression of miR1-181a was detected by QPCR. WB was used to detect the expression of autophagy related genes in HCC. Adenovirus Ad-NC and Ad-miR181a-sponge HCC / HepG2 WB assay were established to detect the effect of mir181a on autophagy in hepatoma cells. Double luciferase report system was used to analyze the target genes of miR-181a acting on HCC, flow cytometry was used to analyze the effect of inhibiting mir181a on early apoptosis of hepatoma cells, and the effect of mi R-181a on the proliferation of hepatoma cells was further verified by subcutaneous tumorigenesis in nude mice. Results: our study found that the expression of miR-181a was increased in human hepatocellular carcinoma (HCC) tissues. By detecting the expression level of LC3p62 and Atg5 protein in HCC, it was found that the decrease of LC3Ag5 expression in human hepatocellular carcinoma (HCC) could promote the upregulation of HepG2 cell autophagy. MiR-181a target gene was verified by luciferase assay. The results showed that miR-181a could bind to 3- UTRAtg5 Atg5 as a target gene of miR-181a in HepG2 cells. The inhibitory effect of miR-181a on apoptosis of HepG2 cells was detected by flow cytometry. The tumor volume and weight of HepG2 cells transfected with adenovirus miR-181a were significantly lower than that of control group. It was suggested that inhibition of miR-181a expression could inhibit tumor growth. Conclusion: we found that miR-181a can inhibit autophagy, decrease apoptosis and promote tumor growth through targeting gene Atg5. These findings may provide a new goal for the treatment of liver cancer.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7

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本文編號(hào)：1874062