MiR-27a促肝癌增殖及其作用机制的研究
发布时间：2019-06-07 10:22
【摘要】：研究背景：肝细胞肝癌（hepatocellular carcinoma, HCC）是临床上较常见的恶性肿瘤之一，具有较高的发病率和死亡率。目前公认的治疗方法是以手术为主的综合治疗，但由于肝癌早期缺乏特异性体征，患者发现肿瘤时往往处于癌症的中晚期，而肝癌术后又极易出现复发和转移，所以多数患者的预后并不理想。因此，人们急需寻找新的治疗靶点以提高肝癌的治疗效果。微小RNA（MicroRNA）是一组非编码小RNA，以转录后调节的方式影响着细胞的生长、增殖、分化过程，并通过抑制下游靶基因的表达发挥调节功能，可同时调控多种下游靶基因。作为microRNA家族中的重要成员之一，MiR-27a不仅参与脂质、骨代谢以及血液系统疾病的调节，还被证明在不同类型肿瘤中都能起到促癌功能。过氧化物酶增殖物激活受体-γ（PPAR-γ），是脂代谢过程中重要的调节因子，同时也被证明能够抑制包括肝癌在内的多种肿瘤的发展。目前仅有少量文献报道MiR-27a在肝癌中具有高表达现象，，但其与肝癌发生发展的关系尚不清楚。 研究目的：阐述miR-27a在肝癌中的表达及作用机制，同时探讨miR-27a通过抑制靶基因PPAR-γ的表达影响肝癌增殖和发展的机制，为开发肝癌诊治的新靶点提供了理论依据。 研究内容： Realtime-PCR方法分析正常人与肝癌患者血清及肝癌组织中miR-27a表达差异；Realtime-PCR方法检测人正常肝细胞系HL-7702与人肝癌细胞系HepG2、Bel-7402、Bel-7404中miR-27a表达水平；应用MiR-27a人工合成类似物mimics及抑制剂inhibitors分别转染HepG2细胞系，通过MTT方法检测其对肝癌细胞生长增殖的影响；流式细胞术检测转染后肝癌细胞凋亡及细胞周期变化；荧光素酶报告基因检测PPAR-γ是否为miR-27a的下游靶点；免疫组化技术检测临床病理切片中PPAR-γ表达；应用PPAR-γ配体激动剂罗格列酮作用于HepG2细胞系，通过Western Blot方法检测miR-27a与罗格列酮刺激后PPAR-γ蛋白表达水平。 研究结果： 1. miR-27a类似物对HepG2细胞特性影响 肝癌患者血清、组织样本及肝癌细胞系中miR-27a高表达，转染miR-27amimics可加速肝癌细胞生长，诱导G1/S期转化，促进细胞增殖；转染miR-27ainhibitors可诱发肝癌细胞G1期阻滞，抑制细胞增殖，增加凋亡比例。 2.miR-27a对PPAR-γ的影响 PPAR-γ的3’-UTR区存在miR-27a的互补序列，是miR-27a的下游靶基因；转染miR-27a mimics后肝癌细胞内PPAR-γmRNA及蛋白表达明显受抑制；罗格列酮刺激能够相对逆转miR-27a对肝癌细胞增殖特性及PPAR-γ蛋白表达水平的影响。 研究结论： MiR-27a在肝癌高表达，能够通过靶基因PPAR-γ，诱使G1/S期转化，促进肝癌细胞增殖，抑制miR-27a的表达能够导致细胞出现G1期阻滞，抑制肝癌细胞增殖，同时促进肝癌细胞凋亡。PPAR-γ配体激活剂罗格列酮能够增强PPAR-γ的表达，抑制肝癌细胞增殖，相对逆转miR-27a的作用。
[Abstract]:Background: hepatocellular carcinoma (HCC) (hepatocellular carcinoma, HCC) is one of the most common malignant tumors in clinic, with high incidence and mortality. At present, it is recognized that the treatment method is surgery-based comprehensive treatment, but because of the lack of specific signs in the early stage of liver cancer, patients often find the tumor in the middle and late stages of cancer, and the recurrence and metastasis of liver cancer are easy to occur after operation. Therefore, the prognosis of most patients is not ideal. Therefore, it is urgent to find new therapeutic targets to improve the therapeutic effect of liver cancer. MicroRNA (MicroRNA) is a group of non-coding small RNAs, which affect the growth, proliferation and differentiation of cells by post-transcriptional regulation, and play a regulatory role by inhibiting the expression of downstream target genes, which can regulate a variety of downstream target genes at the same time. As an important member of microRNA family, MiR-27a is not only involved in the regulation of lipid, bone metabolism and blood system diseases, but also has been shown to play a role in promoting cancer in different types of tumors. Peroxisome proliferator-activated receptor-gamma (PPAR- 纬) is an important regulator of lipid metabolism and has been shown to inhibit the development of a variety of tumors, including liver cancer. At present, only a small number of literatures have reported that MiR-27a is highly expressed in HCC, but its relationship with the occurrence and development of HCC is not clear. Objective: to elucidate the expression and mechanism of miR-27a in hepatocellular carcinoma (HCC), and to explore the mechanism of miR-27a affecting the proliferation and development of HCC by inhibiting the expression of target gene PPAR- 纬, which provides a theoretical basis for the development of new targets for the diagnosis and treatment of HCC. Content: the difference of miR-27a expression in serum and liver cancer tissue between normal subjects and patients with liver cancer was analyzed by Realtime-PCR. The expression of miR-27a in human normal liver cell line HL-7702 and human hepatocellular carcinoma cell line HepG2,Bel-7402,Bel-7404 was detected by Realtime-PCR. HepG2 cell line was transfected with MiR-27a synthetic analog mimics and inhibitor inhibitors respectively, and the effect of MTT on the growth and proliferation of HCC cells was detected by flow cytometry, and the apoptosis and cell cycle of HCC cells were detected by flow cytometry. Luciferase reporter gene was used to detect whether PPAR- 纬 was a downstream target of miR-27a, and the expression of PPAR- 纬 in clinicopathological sections was detected by immunohistochemistry. HepG2 cell line was treated with PPAR- 纬 ligand agonist rosiglitazone. The expression of PPAR- 纬 protein stimulated by miR-27a and rosiglitazone was detected by Western Blot. Results: 1. MiR-27a analogues affect the high expression of miR-27a in serum, tissue samples and HCC cell lines of patients with liver cancer. MiR-27amimics can accelerate the growth of HCC cells, induce the transformation of G1 / S phase and promote the proliferation of HCC cells. MiR-27ainhibitors can induce G 1 phase arrest, inhibit cell proliferation and increase the proportion of apoptosis in HCC cells. The effect of 2.miR-27a on PPAR- 纬 there was a complementary sequence of miR-27a in the 3'-UTR region of PPAR- 纬, which was the downstream target gene of miR-27a, and the expression of PPAR- 纬 mRNA and protein in HCC cells was significantly inhibited after miR-27a mimics transfer. Rosiglitazone stimulation could relatively reverse the effect of miR-27a on the proliferation and expression of PPAR- 纬 protein in HCC cells. Conclusion: the high expression of MiR-27a in HCC can induce the transformation of G1 / S phase through the target gene PPAR- 纬, promote the proliferation of HCC cells, and inhibit the expression of miR-27a, which can lead to G 1 phase arrest and inhibit the proliferation of HCC cells. At the same time, it promoted the apoptosis of HCC cells. Rosiglitazone, a ligand activating agent of PPARR-纬, could enhance the expression of PPAR- 纬, inhibit the proliferation of HCC cells and reverse the effect of miR-27a.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7
本文编号：2494723
[Abstract]:Background: hepatocellular carcinoma (HCC) (hepatocellular carcinoma, HCC) is one of the most common malignant tumors in clinic, with high incidence and mortality. At present, it is recognized that the treatment method is surgery-based comprehensive treatment, but because of the lack of specific signs in the early stage of liver cancer, patients often find the tumor in the middle and late stages of cancer, and the recurrence and metastasis of liver cancer are easy to occur after operation. Therefore, the prognosis of most patients is not ideal. Therefore, it is urgent to find new therapeutic targets to improve the therapeutic effect of liver cancer. MicroRNA (MicroRNA) is a group of non-coding small RNAs, which affect the growth, proliferation and differentiation of cells by post-transcriptional regulation, and play a regulatory role by inhibiting the expression of downstream target genes, which can regulate a variety of downstream target genes at the same time. As an important member of microRNA family, MiR-27a is not only involved in the regulation of lipid, bone metabolism and blood system diseases, but also has been shown to play a role in promoting cancer in different types of tumors. Peroxisome proliferator-activated receptor-gamma (PPAR- 纬) is an important regulator of lipid metabolism and has been shown to inhibit the development of a variety of tumors, including liver cancer. At present, only a small number of literatures have reported that MiR-27a is highly expressed in HCC, but its relationship with the occurrence and development of HCC is not clear. Objective: to elucidate the expression and mechanism of miR-27a in hepatocellular carcinoma (HCC), and to explore the mechanism of miR-27a affecting the proliferation and development of HCC by inhibiting the expression of target gene PPAR- 纬, which provides a theoretical basis for the development of new targets for the diagnosis and treatment of HCC. Content: the difference of miR-27a expression in serum and liver cancer tissue between normal subjects and patients with liver cancer was analyzed by Realtime-PCR. The expression of miR-27a in human normal liver cell line HL-7702 and human hepatocellular carcinoma cell line HepG2,Bel-7402,Bel-7404 was detected by Realtime-PCR. HepG2 cell line was transfected with MiR-27a synthetic analog mimics and inhibitor inhibitors respectively, and the effect of MTT on the growth and proliferation of HCC cells was detected by flow cytometry, and the apoptosis and cell cycle of HCC cells were detected by flow cytometry. Luciferase reporter gene was used to detect whether PPAR- 纬 was a downstream target of miR-27a, and the expression of PPAR- 纬 in clinicopathological sections was detected by immunohistochemistry. HepG2 cell line was treated with PPAR- 纬 ligand agonist rosiglitazone. The expression of PPAR- 纬 protein stimulated by miR-27a and rosiglitazone was detected by Western Blot. Results: 1. MiR-27a analogues affect the high expression of miR-27a in serum, tissue samples and HCC cell lines of patients with liver cancer. MiR-27amimics can accelerate the growth of HCC cells, induce the transformation of G1 / S phase and promote the proliferation of HCC cells. MiR-27ainhibitors can induce G 1 phase arrest, inhibit cell proliferation and increase the proportion of apoptosis in HCC cells. The effect of 2.miR-27a on PPAR- 纬 there was a complementary sequence of miR-27a in the 3'-UTR region of PPAR- 纬, which was the downstream target gene of miR-27a, and the expression of PPAR- 纬 mRNA and protein in HCC cells was significantly inhibited after miR-27a mimics transfer. Rosiglitazone stimulation could relatively reverse the effect of miR-27a on the proliferation and expression of PPAR- 纬 protein in HCC cells. Conclusion: the high expression of MiR-27a in HCC can induce the transformation of G1 / S phase through the target gene PPAR- 纬, promote the proliferation of HCC cells, and inhibit the expression of miR-27a, which can lead to G 1 phase arrest and inhibit the proliferation of HCC cells. At the same time, it promoted the apoptosis of HCC cells. Rosiglitazone, a ligand activating agent of PPARR-纬, could enhance the expression of PPAR- 纬, inhibit the proliferation of HCC cells and reverse the effect of miR-27a.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7
【参考文献】
相关期刊论文 前2条
1 David Li;Josephine Kang;Benjamin J.Golas;Vincent W.Yeung;David C.Madoff;;Minimally invasive local therapies for liver cancer[J];Cancer Biology & Medicine;2014年04期
2 廖清池;周胜华;;microRNA生物合成的调控进展——TGFβ/Smad信号途径的作用[J];现代生物医学进展;2011年14期
本文编号：2494723
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