miR-150在肝癌增殖和转移中的作用及相关机制的研究

发布时间：2018-06-29 08:22

本文选题：肝细胞肝癌 + miR-150　；参考：《第四军医大学》2016年博士论文

【摘要】：【背景】肝细胞肝癌是全世界最常见的恶性肿瘤之一,是世界第三位、我国第二位的肿瘤相关性死亡原因。近年来,尽管外科手术技术不断提高、现代影像技术和非手术局部治疗技术不断进步,大大提高了肝癌的治疗效果,然而,肝癌患者的预后并没有因此得到很大的改善。主要原因是肝癌早期诊断困难、容易发生复发和转移等。因此,深入研究肝癌发生、发展的分子机制,以期寻找能够用于肝癌早期诊断的分子标志物以及寻找有效治疗肝癌的新靶点是一项亟待解决的问题。micro RNA是一类非编码小RNA,主要通过与靶基因m RNA 3’非编码区相互结合进而负向调控靶基因的表达,从而参与多种细胞学功能,包括细胞增殖、凋亡、分化、代谢和调节内分泌系统等。mi RNA表达异常和包括肿瘤在内的多种疾病相关。在肿瘤的研究中发现,mi RNA参与了肿瘤细胞增殖、凋亡、分化、耐药和侵袭转移等多个方面的调控,其中,mi R-150在肿瘤的发生发展过程中也发挥了重要作用。在结肠癌、胰腺癌、卵巢癌中mi R-150表达下调,发挥抑癌作用,而在胃癌、乳腺癌中mi R-150高表达,促进肿瘤的进展。因此,mi R-150在不同的肿瘤类型中发挥着不同的作用。有研究表明,通过基因芯片分析CD133+和CD133-的肝癌细胞中micro RNA的表达差异发现,mi R-150在CD133+的肝癌干细胞中表达下调。另有一篇文献报道,通过比较肝癌转移灶和原位肝癌中的micro RNA表达差异,发现mi R-150在转移灶中表达下调,推测mi R-150可能参与了肝癌的侵袭转移过程。然而,mi R-150是否参与调控了肝癌的发生、发展过程?其在肝癌中的具体作用及机制是什么?至今尚不明确,仍待进一步研究。【目的】1、检测肝癌组织和对应癌旁组织中mi R-150的表达,分析mi R-150表达水平与患者临床病理特征及患者预后之间的关系。2、研究mi R-150对肝癌细胞增殖和侵袭转移能力的影响。3、探讨mi R-150调控肝癌细胞恶性表型的分子机制。【方法】1、通过荧光实时定量PCR方法检测mi R-150在84对肝癌组织和对应癌旁组织中的表达情况,分析mi R-150的表达和肝癌患者临床病理特征以及患者预后之间的关系。2、构建mi R-150过表达慢病毒载体,感染mi R-150表达相对较低的肝癌细胞系,建立稳定表达mi R-150和对照NC的肝癌细胞。通过CCK-8增殖实验、平板克隆形成实验和裸鼠皮下成瘤实验检测mi R-150对肝癌细胞增殖能力的影响;通过Transwell侵袭、迁移实验和裸鼠肺转移模型实验检测mi R-150对肝癌细胞侵袭、转移能力的影响。3、通过生物信息学软件预测mi R-150的下游靶基因,经综合分析,筛选GAB1可能是mi R-150的直接靶基因之一。通过双荧光素酶报告基因实验、荧光实时定量PCR和western blot实验进行验证。检测肝癌组织中GAB1的表达,分析其与mi R-150表达水平的相关性。采用RNAi技术沉默肝癌细胞中GAB1的表达,观察其对肝癌细胞增殖、侵袭、迁移能力的影响,过表达GAB1后观察肝癌细胞的功能恢复情况。进一步通过western blot和免疫组织化学染色方法检测mi R-150对GAB1表达水平、ERK1/2磷酸化水平和EMT标志物蛋白表达水平的影响,深入研究其中的分子机制。【结果】1、mi R-150在肝癌组织中的表达水平显著低于对应癌旁组织,结合肝癌患者临床病理资料分析,mi R-150低表达与肿瘤大小、静脉侵犯和转移密切相关。进一步研究发现,mi R-150低表达组患者的总体生存时间显著低于mi R-150高表达组患者。低mi R-150表达能够作为肝癌患者预后较差的独立指标。2、mi R-150在肝癌细胞系中的表达水平显著低于永生化人肝细胞系HL-7702,选用mi R-150表达相对较低的SMMC-7721和MHCC97-H细胞用于后续实验。成功构建mi R-150过表达慢病毒载体后,感染细胞,构建过表达mi R-150的肝癌细胞系。CCK-8实验、平板克隆实验、裸鼠皮下成瘤实验结果显示,过表达mi R-150能够显著抑制肝癌细胞的增殖能力、克隆形成能力和裸鼠皮下成瘤能力。3、Transwell实验证实,过表达mi R-150能够显著抑制肝癌细胞的侵袭、迁移能力。裸鼠体内实验结果显示,过表达mi R-150能够显著抑制肝癌细胞的肺转移能力。4、通过生物信息学软件预测,GAB1可能是mi R-150的靶基因。双荧光素酶报告基因实验显示,野生型GAB1-WT-3’UTR报告基因载体与mi R-150 mimic共转染可使荧光素酶活性显著降低,而mi R-150 mimic与突变型GAB1-MUT-3’UTR共转染后荧光素酶活性无明显改变。进一步实验证实,过表达mi R-150能够显著降低GAB1m RNA和蛋白表达水平。在肝癌组织中GAB1 m RNA表达上调,通过分析发现肝癌组织中mi R-150表达和GAB1 m RNA表达呈负相关关系。表明GAB1是mi R-150的直接靶基因。5、GAB1-si RNA下调GAB1表达能够抑制肝癌细胞的增殖、侵袭和迁移能力,模拟了mi R-150过表达对肝癌细胞的抑制作用,而恢复GAB1的表达后,部分逆转了mi R-150的抑制作用。6、western blot实验表明,过表达mi R-150抑制肝癌细胞GAB1表达的同时还抑制了ERK1/2的磷酸化,并且上调上皮样细胞分子标记物E-cadherin蛋白的表达,下调间质样细胞分子标记物N-cadherin和Vimentin蛋白的表达,提示过表达mi R-150能够抑制肝癌细胞的EMT过程。最后在裸鼠移植瘤组织水平验证了过表达mi R-150能够抑制GAB1的表达和ERK1/2的磷酸化。【结论】1、mi R-150在肝癌组织中低表达,并且mi R-150低表达和肝癌的恶性表型以及患者预后密切相关。2、过表达mi R-150能够在体外和体内水平抑制肝癌细胞的增殖、侵袭和转移能力。3、GAB1是mi R-150的直接靶基因,mi R-150通过负性调节GAB1-ERK1/2轴抑制肝癌细胞的增殖、侵袭、转移能力。临床标本进一步证实mi R-150的表达水平与GAB1 m RNA表达水平呈负相关关系。因此,mi R-150-GAB1-ERK1/2轴有可能成为肝癌治疗的新靶点。
[Abstract]:[background] hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. It is the third world in the world and the cause of tumor related death in the second of our country. In recent years, although the surgical technique has been improved, modern imaging technology and non-surgical local treatment technology have been progressing, the treatment effect of liver cancer has been greatly improved. However, the patients with liver cancer have been greatly improved. The main reason is that the early diagnosis of liver cancer is difficult, and the recurrence and metastasis are easy to occur. Therefore, it is urgent to study the molecular mechanism of the development of liver cancer and to find the molecular markers that can be used in the early diagnosis of liver cancer and to find new targets for effective treatment of liver cancer. Problem.Micro RNA is a class of non coding small RNA, which is mainly mediated by the non coding region of the target gene m RNA 3 'and then negatively regulates the expression of target genes, thus participating in a variety of cytological functions, including cell proliferation, apoptosis, differentiation, metabolism and regulation of the.Mi RNA expression, such as the endocrine system, and a variety of diseases, including tumors. In the study of tumor, MI RNA has been involved in the regulation of tumor cell proliferation, apoptosis, differentiation, resistance and invasion and metastasis. Among them, MI R-150 also plays an important role in the development of tumor. The expression of MI R-150 in colon, pancreatic and ovarian cancer is downregulated, and MI R in gastric cancer and breast cancer. -150 is highly expressed and promotes the progression of tumors. Therefore, MI R-150 plays a different role in different tumor types. Studies have shown that the expression of micro RNA in CD133+ and CD133- cells by gene chip analysis showed that MI R-150 was downregulated in the liver cancer stem cells of CD133+. The difference in the expression of micro RNA in cancer metastasis and in situ liver cancer showed that MI R-150 was down regulated in the metastasis, and that MI R-150 may be involved in the invasion and metastasis of liver cancer. However, whether mi R-150 participates in the regulation of the occurrence and development of liver cancer, what is its specific role and mechanism in the liver cancer? It is still not clear and still needs to be advanced. [Objective] [Objective] [Objective] 1 to detect the expression of MI R-150 in liver cancer tissues and adjacent tissues, to analyze the relationship between the expression of MI R-150 and the clinicopathological features of the patients and the prognosis of the patients.2, and to study the effect of MI R-150 on the proliferation and invasion and metastasis of hepatoma cells.3, and to explore the molecular mechanism of the MI R-150 regulation of the malignant phenotype of hepatoma cells. [method] 1, the expression of MI R-150 in 84 liver cancer tissues and adjacent tissues was detected by real time fluorescence quantitative PCR method, and the relationship between the expression of MI R-150 and the clinicopathological features of the patients with liver cancer and the prognosis of the patients was analyzed.2. The expression of MI R-150 by the overexpression of the lentivirus carrier and the infection of the low expression of the MI R-150 to the lower liver cancer cells The hepatoma cells that express mi R-150 and control NC were established. The effects of MI R-150 on the proliferation of hepatoma cells were detected by CCK-8 proliferation test, and the effect of MI R-150 on the proliferation of hepatoma cells. The invasion, migration test and lung metastasis model test of nude mice were used to detect the invasion and metastasis of MI R-150 to the liver cancer cells. Influence.3 and predict the downstream target gene of MI R-150 through bioinformatics software. Through comprehensive analysis, screening GAB1 may be one of the direct target genes of MI R-150. Through double luciferase reporter gene experiment, fluorescence real-time quantitative PCR and Western blot experiment were verified. The expression of GAB1 in liver cancer group was detected, and the expression level of MI R-150 was analyzed. RNAi technique was used to silence the expression of GAB1 in hepatoma cells, to observe the effect of its effect on the proliferation, invasion and migration of hepatoma cells. After overexpressing GAB1, the functional recovery of hepatoma cells was observed. The expression level of MI R-150 to GAB1, ERK1/2 phosphorylation level and E were further detected by western blot and immunohistochemical staining. The molecular mechanism of the expression level of MT marker protein was studied. [results] 1, the expression level of MI R-150 in the liver cancer tissues was significantly lower than that of the para cancerous tissue. The low expression of MI R-150 was closely related to the size of the tumor and the invasion and metastasis of the tumor. The further study found that MI R-150 was found. The overall survival time of the low expression group was significantly lower than that of the MI R-150 high expression group. The low MI R-150 expression could be used as an independent indicator of the poor prognosis of the liver cancer patients.2. The expression level of MI R-150 in the hepatocellular carcinoma cell lines was significantly lower than that of the immortalized human hepatocyte line HL-7702, and MI R-150 was selected to express relatively low SMMC-7721 and MHCC97-H cells. After the successful construction of the MI R-150 overexpressed lentivirus vector, the infected cells were infected and the hepatoma cell line expressing mi R-150 was constructed by.CCK-8 experiment. The experiment of flat clones and subcutaneous tumor formation in nude mice showed that overexpression of MI R-150 could significantly inhibit the proliferation of hepatoma cells, the cloning ability and the subcutaneous tumor formation ability of nude mice. 3, Transwell experiments confirmed that overexpression of MI R-150 could significantly inhibit the invasion and migration of hepatoma cells. The experimental results in nude mice showed that overexpression of MI R-150 could significantly inhibit the lung metastasis of hepatoma cells.4, and GAB1 might be the target gene for MI R-150 through bioinformatics software. The double luciferase reporter gene experiment showed that the expression of GAB1 could be the target gene. The results showed that the co transfection of the wild type GAB1-WT-3 'UTR reporter gene carrier and MI R-150 mimic could significantly reduce the luciferase activity, while the MI R-150 mimic and the mutant GAB1-MUT-3' UTR co transfected luciferase activity did not change significantly. Further experiments showed that the overexpression of MI R-150 could significantly reduce the level of the protein expression and protein expression. The expression of GAB1 m RNA was up-regulated in the tissue. It was found that the expression of MI R-150 in the liver cancer tissues was negatively correlated with the expression of GAB1 m RNA, indicating that GAB1 is the direct target gene.5 of MI R-150, which inhibits the proliferation, invasion and migration of hepatoma cells. When the expression of GAB1 was restored, the inhibitory effect of MI R-150 on.6 was partly reversed. The Western blot experiment showed that overexpression of MI R-150 inhibited the GAB1 expression of liver cancer cells and inhibited the phosphorylation of ERK1/2, and up regulated the expression of E-cadherin protein of the molecular markers of the epithelioid cells, and down regulated the molecular marker N-cadherin of interstitial like cells. The expression of tin protein suggests that over expression of MI R-150 can inhibit the EMT process of hepatoma cells. Finally, the expression of MI R-150 can inhibit the expression of GAB1 and the phosphorylation of ERK1/2 in nude mice. [Conclusion] 1, MI R-150 is low expression in liver cancer tissues, and MI R-150 is low expression and malignant phenotype of liver cancer and patients with liver cancer. The prognosis is closely related to.2, and overexpression of MI R-150 can inhibit the proliferation, invasion and metastasis of hepatoma cells in vitro and in vivo,.3, GAB1 is the direct target gene of MI R-150, MI R-150 can inhibit the proliferation, invasion and metastasis of hepatoma cells by negative regulation GAB1-ERK1/2 axis. The expression level of M RNA is negatively correlated. Therefore, MI R-150-GAB1-ERK1/2 axis may become a new target for treatment of HCC.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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