miR-181a通过调控靶基因ATG5抑制肝细胞性肝癌的自噬

发布时间：2018-05-11 13:11

本文选题：miR-181a + ATG5　；参考：《南京医科大学》2017年博士论文

【摘要】：背景和研究目的:肝细胞癌(hepatocellularcarcinoma,HCC)是最常见的原发性肝细胞癌,约占原发性肝癌的90%,是全球癌症死亡的主要原因。在HCC的发展中,有缺陷的自噬反应有助于这个过程。因此,它仍然需要阐明自噬在肝癌中的作用,以及如何调节细胞自噬。miR-181a被发现在多种肿瘤,如乳腺癌,胃癌,和神经母细胞瘤可抑制肿瘤细胞自噬。然而,miR-181a在肝癌细胞自噬中作用尚不明确。最近的研究发现,肝癌miR-181a表达上调,这可能与HCC的发病机制相关。因此,我们将探讨miR-181a在肝癌细胞自噬中的作用机制。方法:本课题通过临床上收集人肝细胞性肝癌组织和肝血管瘤组织,采用QPCR检测组织中miR1-181a的表达量。WB检测肝癌组织中自噬相关基因的表达量。建立腺病毒 Ad-NC 以及 Ad-miR181a-sponge HCC/HepG2,WB 检测抑制 mir181a对肝癌细胞中自噬水平的影响。双荧光素酶报告分析系统分析miR-181a作用于HCC的靶基因;利用流式细胞术分析抑制mir181a对肝癌细胞早期凋亡的影响;通过裸鼠皮下成瘤实验进一步验证mi R-181a对肝癌细胞增殖的影响。结果:我们研究发现miR-181a在人肝癌(HCC)组织中表达增加。通过检测肝癌组织中LC3,p62和Atg5蛋白的表达水平,发现自噬相关基因LC3、Atg5在人肝癌(HCC)组织中表达降低。miR-181a抑制可促进HepG2细胞自噬上调。miR-181a靶基因通过荧光素酶检测进行验证,发现结果表明miR-181a可以绑定3'-UTRAtg5,Atg5是miR-181a在HepG2细胞中的靶基因。利用流式细胞仪检测,miR-181a抑制可促进HepG2细胞的凋亡。在注射腺病毒miR-181a转染HepG2细胞的小鼠,肿瘤体积及重量明显小于对照组,研究提示抑制miR-181a表达可抑制肿瘤生长。结论:我们研究发现miR-181a可以通过靶向基因Atg5抑制肝癌细胞自噬,降低肝癌细胞的凋亡,促进肿瘤的生长。这些发现可能会为肝癌治疗提供了一个新的目标。
[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.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7

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