MiR-126对肝细胞肝癌的转移和血管生成的作用及机制研究

发布时间：2018-04-20 01:41

本文选题：肝细胞肝癌 + miR-126　；参考：《浙江大学》2016年博士论文

【摘要】：背景肝细胞肝癌(Hepatocellular carcinoma, HCC)是世界上最常见的癌症之一,特别是在中国。尽管以手术为主的综合治疗的进步,但是HCC的死亡率依然很高。活跃的转移和血管生成是HCC的两大特征,严重影响肝癌患者的预后。然而,其机制仍然不甚清楚。MicroRNAs (miRNA)是一类高度保守的小RNA。它们通过结合于靶基因的3'UTR区域来参与调节细胞的各种生物学行为。越来越多的文献报道,miRNAs在肝癌的发展过程中发挥着重要的作用,包括凋亡,增殖,自噬和EMT等。到目前为止,在肝癌中同时调节转移和血管生成的miRNAs报道尚少。MiR-126已经被报道在肿瘤发展过程中有重要的作用。但是其对于肝癌转移和血管生成的作用仍然不清楚。所以探索miR-126和肝癌转移和血管生成的关系可能是肝癌治疗研究的一个不错的策略。目的本次研究的目的在于探索miR-126在肝癌组织和细胞中的表达水平,并统计其与肝癌标本临床病理资料之间的相关性。我们进一步研究了miR-126与肝癌转移和血管生成之间的关系,寻找其发挥作用的靶基因及相关机制。方法1. 通过实时荧光定量PCR的办法来研究miR-126在肝癌组织和细胞系中的表达。2. 用miR-126 mimics和inhibitors来过表达或抑制其在肝癌细胞中的水平,3. 运用Transwell实验和成血管实验来研究转移和血管生成能力。4. 利用裸鼠皮下成瘤实验,进行miR-126的体内功能研究。5. 用生物信息学网站和荧光双报告实验来预测和验证miR-126的靶基因。6. 用免疫组化检测CD34的表达水平,来反应体内血管生成的水平。结果1. 在70对肝癌病例中,我们发现肝癌组织中miR-126的表达量明显降低。但是没有发现miR-126表达量与临床病理资料有相关性。相对于正常肝永生化细胞L02和Changliver细胞,7株肝癌细胞系中,有6株的miR-126的表达降低。2. 在体外功能研究中,我们发现miR-126过表达之后,可以抑制肝癌细胞的转移和血管生成的能力,而miR-126表达抑制之后,肝癌细胞的转移和血管生成能力明显上升了。3. 裸鼠皮下成瘤实验发现,miR-126稳定过表达组的瘤体体积明显小于对照组。4. 我们应用生物信息学网站(miRanda, TargetScan)预测出LRP6和PIK3R2是miR-126的靶基因,并且通过荧光双报告实验和Western blot实验得到了验证。并且,在70例肝癌病例中LRP6和PIK3R2与miR-126的表达水平呈负相关的关系。5.在肝癌细胞中分别抑制了LRP6和PIK3R2的表达之后,肝癌细胞的转移能力和血管生成能力分别下降了。靶基因“补救实验”证明,LRP6和miR-126的共转染可以减弱miR.-126对转移的作用,而PIK3R2和miR-126共转染可以减弱miR-126对血管生成能力的作用。6. 在70例临床标本中CD34的表达量与miR-126的水平呈负相关,并且miR-126稳定高表达组的裸鼠瘤体中的CD34表达量比对照组低。结论1. 相比较于肝癌旁组织和正常肝细胞,miR-126在肝癌组织和肝癌细胞中的表达量明显降低。2. MiR-126抑制肝癌细胞的转移和血管生成,并且抑制裸鼠瘤体的生长速度。3.LRP6和PIK3R2被证明是miR-126的靶基因,并且在肝癌中介导miR-126对肝癌转移和血管生成的作用。
[Abstract]:Hepatocellular carcinoma (HCC) is one of the most common cancers in the world, especially in China. Despite the progress of surgery based comprehensive treatment, the death rate of HCC is still high. Active metastasis and angiogenesis are the two major features of HCC. Severe weight affects the prognosis of liver cancer patients. However, the mechanism is still not. It is clear that.MicroRNAs (miRNA) is a highly conserved small RNA. that participates in the regulation of various biological behavior of cells by combining the 3'UTR region of the target gene. More and more literature reports that miRNAs plays an important role in the development of liver cancer, including apoptosis, proliferation, autophagy and EMT. So far, in liver cancer MiRNAs reports that regulate metastasis and angiogenesis have been reported to play an important role in the development of cancer. However, the role of.MiR-126 is still unclear on the metastasis and angiogenesis of liver cancer. Therefore, the exploration of the relationship between miR-126 and the metastasis of liver cancer and angiogenesis may be a good strategy for the treatment of liver cancer. Objective the purpose of this study was to explore the expression level of miR-126 in the tissues and cells of liver cancer and to count the correlation between the clinical and pathological data of the liver cancer. We further studied the relationship between miR-126 and the metastasis of liver cancer and angiogenesis, and find the target gene and the related mechanism. Method 1. through the reality To study the expression of miR-126 in liver cancer tissue and cell lines by fluorescence quantitative PCR,.2. used miR-126 mimics and inhibitors to express or inhibit its level in the hepatoma cells. 3. the Transwell experiment and vascular experiment were used to study the metastasis and angiogenesis ability.4. using the nude mouse subcutaneous tumorigenesis experiment to perform miR-126 In vivo functional study.5. uses bioinformatics website and fluorescence double report experiment to predict and verify the target gene.6. of miR-126 to detect the level of CD34 expression by immunohistochemistry to reflect the level of angiogenesis in the body. Results 1. in 70 cases of liver cancer, we found that the expression of miR-126 in the liver cancer tissues was significantly reduced. The expression of miR-126 was correlated with the clinicopathological data. Compared with normal liver immortalized L02 and Changliver cells, 6 of the 7 hepatoma cell lines decreased the expression of.2. in the function study in vitro. We found that after miR-126 overexpression, we could inhibit the metastasis and angiogenesis of hepatoma cells and the expression of miR-126. After inhibition, the metastasis and angiogenesis of hepatoma cells increased obviously in.3. nude mice. The tumor volume of the miR-126 stable overexpression group was significantly smaller than that of the control group.4.. We used the bioinformatics website (miRanda, TargetScan) to predict that LRP6 and PIK3R2 were the target genes of miR-126, and the fluorescence double report was used. The test and Western blot test were verified. And the expression level of LRP6 and PIK3R2 was negatively correlated with the expression level of miR-126 in 70 cases of liver cancer. After the expression of LRP6 and PIK3R2 in the hepatoma cells, the metastasis ability and angiogenesis of hepatoma cells decreased respectively. The target gene "remedial experiment" proved that LRP6 and Co transfection of miR-126 could weaken the effect of miR.-126 on metastasis, while PIK3R2 and miR-126 co transfection could weaken the effect of miR-126 on angiogenesis..6. in 70 clinical specimens, the expression of CD34 was negatively correlated with the level of miR-126, and CD34 expression in the tumor body of the group of miR-126 stable high expression group was lower than that of the control group. Conclusion 1. Compared with para hepatoma and normal hepatocytes, the expression of miR-126 in HCC and HCC cells significantly reduced.2. MiR-126 to inhibit the metastasis and angiogenesis of hepatoma cells, and to inhibit the growth rate of nude mice,.3.LRP6 and PIK3R2 were proved to be the target genes of miR-126, and the metastasis of the liver cancer mediated miR-126 on the metastasis of liver cancer. And the role of angiogenesis.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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