MiR-709下调GPC5表达促进肝癌细胞增殖、侵袭和转移的机制研究

发布时间：2018-05-19 15:48

本文选题：肝癌 + microRNA　；参考：《天津医科大学》2017年博士论文

【摘要】：肝细胞肝癌(hepatocellular carcinoma,HCC,以下简称肝癌)是世界上最常见的恶性肿瘤之一,其发病率和病死率分居全球恶性肿瘤的第5位和第3位。肝癌发病隐匿,早期诊断困难,预后差且极易转移复发,这也严重影响了肝癌的治疗效果。肝癌发生发展及转移复发的分子机制复杂且尚未完全清楚。MiRNA是近年来在真核生物体内发现的一种非编码的单链小分子RNA,miRNA高度保守,在体内主要起转录后负调控作用。研究已证实miRNA在肝癌的发生发展过程中起着重要作用。MiR-709在多种器官组织如脑、淋巴结、心脏、肺、肝脏和肾脏中含量丰富。MiR-709在肝癌中的作用还未见报道。因此,miR-709在肝癌的发生发展过程中的功能作用及其机制值得我们深入去研究。目的:探究miR-709在HCC发生发展过程中的作用。方法:运用qRT-PCR检测肝细胞、肝癌细胞、肝癌组织及癌旁正常组织检测miR-709水平,分析miR-709与肝癌临床病理特征间关系。将miR-709 mimic和miR-709 inhibitor转染到肝癌细胞中,检测miR-709的表达,分别采用CCK-8增殖实验、Transwell迁移实验和western blot实验检测转染后肝癌细胞株的增殖、周期、迁移及侵袭能力的变化情况。然后通过运用生物信息学软件进一步检测miR-709的靶基因,然后通过荧光素酶报告基因检测系统、qRT-PCR以及western Blot等方法验证miR-709的作用靶点。结果:(1)①MiR-709在肝癌细胞中表达上调:通过应采用qRT-PCR检测miR-709水平在肝癌细胞(HepG2,、SMMC7721、Hep3B和Bel7402)和肝细胞HL-7702间表达水平发现,miR-709在肝癌细胞中表达明显上调;(2)MiR-709在肝癌组织中表达上调:对6对研究的肝癌组织和其癌旁正常组织中的miR-709进行初步筛查发现肝癌组织中miR-709水平上调表达。然后对纳入的50对肝癌组织及其癌旁正常组织中的miR-709进行检测发现,miR-709在肝癌组织中表达明显上调(P0.001)。在50例肝癌病人中,有40例(80%)肝癌病人的miR-709在肝癌组织中的水平明显高于其癌旁正常组织。我们研究发现,高表达的miR-709和肝癌的pTNM分期、侵袭及转移呈正相关。(2)(1)过表达miR-709可促进肝癌细胞增殖:将miR-709 mimic and inhibitor成功转染到肝癌细胞内,运用CCK-8实验检测肝癌细胞增殖发现下调miR-709可抑制肝癌细胞增殖,转染miR-709 mimic可促进肝癌细胞增殖;对Ki-67蛋白及其mRNA水平检测发现转染miR-709 mimic可促使细胞增殖,而转染miR-709 inhibitor则抑制增殖。(2)过表达miR-709可促进肝癌细胞的转移及侵袭:与转染miR-709 inhibitor肝癌细胞相比,转染miR-709 mimic过表达miR-709可促进肝癌细胞的转移及侵袭。(3)GPC5是miR-709作用的靶基因:通过PicTar和TargetScan生物信息学软件预测发现,miR-709可和GPC5 3'-UTR互补配对。将野生型和突变型GPC5 3,UTR克隆植入荧光素酶报告质粒中,分别构建野生型(WT)和突变型(Mut)的荧光素酶报告质粒并与miR-709 mimic和Scramble转染到肝癌细胞中对萤光素酶活性进行检测,以明确miR-709与其靶基因3'UTR间的相互作用。在肝癌细胞HepG2细胞,转染miR-709 mimic对基因GPC5的野生型3'UTR报告基因质粒的萤光素酶的活性有明显的抑制作用(P0.001);但对基因GPC5的突变型3'UTR报告基因质粒的萤光素酶活性没有明显的影响。基因GPC5是miR-709的直接作用的靶基因,miR-709能直接与GPC5的3'UTR靶向结合,发挥抑制其表达的作用。转染miR-709 mimic可以明显抑制HepG2细胞的GPC5蛋白和mRNA的表达。将pcDNA质粒、pcDNA-GPC5质粒、pcDNA-GPC5+miR-709转染HepG2细胞检测对肝癌细胞的增殖和侵袭能力的影响,转染pcDNA-GPC5质粒后的肝癌细胞的增殖和侵袭能力明显减弱,转染pcDNA-GPC5+miR-709质粒后的肝癌细胞的增殖和侵袭能力明显提高。结论:MiR-709在肝癌组织中可上调表达,过表达的miR-709与肝癌的pTNM分期正相关。进一步研究发现miR-709靶向作用GPC5基因可促使肝癌细胞的增殖、转移及侵袭。
[Abstract]:Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. Its incidence and fatality rate are fifth and third of the world's malignant tumors. The onset of liver cancer is hidden, the early diagnosis is difficult, the prognosis is poor and the recurrence is very easy to transfer. This also seriously affects the therapeutic effect of liver cancer. The molecular mechanism of birth development and metastasis is complicated and not completely clear that.MiRNA is a non coding single strand small molecule RNA found in eukaryotic organisms in recent years. MiRNA is highly conserved and plays a major role in the post transcriptional regulatory role in the body. It has been proved that miRNA plays an important role in the development of liver cancer,.MiR-709 is more important. The role of the rich.MiR-709 in liver cancer has not been reported in the organs such as brain, lymph node, heart, heart, lung, liver and kidney. Therefore, the function and mechanism of miR-709 in the development of liver cancer should be studied in depth. Objective: To explore the role of miR-709 in the development of HCC. Methods: using qRT-PCR To detect the miR-709 level of liver cells, hepatoma cells, liver cancer tissues and normal tissues adjacent to cancer, analyze the relationship between miR-709 and the clinicopathological features of liver cancer. MiR-709 mimic and miR-709 inhibitor were transfected into the hepatoma cells to detect the expression of miR-709, and CCK-8 proliferation test, Transwell migration test and Western blot test were used respectively. The changes in the proliferation, cycle, migration and invasion of the hepatocellular carcinoma cell lines. Then by using bioinformatics software to further detect the target gene of miR-709, and then through the luciferase reporter gene detection system, qRT-PCR and Western Blot to verify the target of miR-709. (1) (1) MiR-709 in the hepatoma cells Up-regulated expression: the expression of miR-709 in hepatoma cells (HepG2, SMMC7721, Hep3B and Bel7402) and the expression level of liver cell HL-7702 should be detected by using qRT-PCR to detect the expression of miR-709 in hepatoma cells. (2) MiR-709 is up regulated in the liver cancer tissue: 6 pairs of hepatocellular carcinoma tissues and miR-709 in the normal tissues adjacent to the cancer. The expression of miR-709 in liver cancer tissues was up-regulated by preliminary screening. Then, the expression of miR-709 in the 50 hepatoma tissues and the normal tissues adjacent to the cancer was detected. The expression of miR-709 in the liver cancer tissues was obviously up-regulated (P0.001). Among the 50 cases of liver cancer, 40 cases (80%) of the liver cancer patients had significantly higher levels of miR-709 in the liver cancer tissues. We found that high expression of miR-709 and pTNM staging of HCC, invasion and metastasis are positively correlated. (2) (1) overexpression of miR-709 can promote the proliferation of hepatoma cells: miR-709 mimic and inhibitor is transfected into hepatoma cells successfully, and the proliferation of liver cancer cells is detected by CCK-8 test and the decrease of miR-709 can inhibit the liver The proliferation of cancer cells, transfection of miR-709 mimic can promote the proliferation of hepatoma cells. The detection of Ki-67 protein and its mRNA level showed that transfection of miR-709 mimic could induce cell proliferation, and transfection of miR-709 inhibitor to inhibit proliferation. (2) overexpression of miR-709 can promote the metastasis and invasion of hepatoma cells: transfection m with miR-709 inhibitor hepatoma cells, transfection M IR-709 mimic overexpression of miR-709 can promote the metastasis and invasion of hepatoma cells. (3) GPC5 is the target gene of miR-709 action. Through the prediction of PicTar and TargetScan bioinformatics software, miR-709 can be paired with GPC5 3'-UTR. The wild type and mutant GPC5 3 are cloned into the luciferase reporter plasmid, and the wild type is constructed respectively. The luciferase reporter plasmids of the mutant type (Mut) and the miR-709 mimic and Scramble were transfected into the hepatoma cells to detect the activity of fluoro phosphomase to clarify the interaction between miR-709 and its target gene 3'UTR. In the HepG2 cells of the liver cancer cells, transfection of the luciferase of the wild type 3'UTR reporter gene plasmid of the miR-709 mimic against the gene GPC5. The activity has obvious inhibitory effect (P0.001), but the mutant 3'UTR reporter gene plasmid has no obvious effect on the activity of the fluoro enzyme. Gene GPC5 is the direct target gene of miR-709, and miR-709 can directly bind to the 3'UTR target of GPC5 and play a role in inhibiting its expression. The transfection of miR-709 mimic can obviously inhibit HepG2. The expression of GPC5 protein and mRNA in cells. The effect of pcDNA plasmids, pcDNA-GPC5 plasmids, pcDNA-GPC5+miR-709 transfected HepG2 cells on the proliferation and invasion ability of hepatoma cells, the proliferation and invasion ability of the hepatoma cells transfected with pcDNA-GPC5 plasmids were obviously weakened, and the proliferation and invasion of the hepatoma cells transfected with pcDNA-GPC5+miR-709 plasmids Conclusion: the expression of MiR-709 can be up-regulated in the liver cancer tissues, and the overexpressed miR-709 is positively related to the pTNM staging of liver cancer. Further studies have found that the targeting of miR-709 targeting GPC5 gene can promote the proliferation, metastasis and invasion of hepatoma cells.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7

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