MiR-27a促肝癌增殖及其作用機(jī)制的研究
發(fā)布時(shí)間：2019-06-07 10:22
【摘要】：研究背景：肝細(xì)胞肝癌（hepatocellular carcinoma, HCC）是臨床上較常見的惡性腫瘤之一，具有較高的發(fā)病率和死亡率。目前公認(rèn)的治療方法是以手術(shù)為主的綜合治療，但由于肝癌早期缺乏特異性體征，患者發(fā)現(xiàn)腫瘤時(shí)往往處于癌癥的中晚期，而肝癌術(shù)后又極易出現(xiàn)復(fù)發(fā)和轉(zhuǎn)移，所以多數(shù)患者的預(yù)后并不理想。因此，人們急需尋找新的治療靶點(diǎn)以提高肝癌的治療效果。微小RNA（MicroRNA）是一組非編碼小RNA，以轉(zhuǎn)錄后調(diào)節(jié)的方式影響著細(xì)胞的生長、增殖、分化過程，并通過抑制下游靶基因的表達(dá)發(fā)揮調(diào)節(jié)功能，可同時(shí)調(diào)控多種下游靶基因。作為microRNA家族中的重要成員之一，MiR-27a不僅參與脂質(zhì)、骨代謝以及血液系統(tǒng)疾病的調(diào)節(jié)，還被證明在不同類型腫瘤中都能起到促癌功能。過氧化物酶增殖物激活受體-γ（PPAR-γ），是脂代謝過程中重要的調(diào)節(jié)因子，同時(shí)也被證明能夠抑制包括肝癌在內(nèi)的多種腫瘤的發(fā)展。目前僅有少量文獻(xiàn)報(bào)道MiR-27a在肝癌中具有高表達(dá)現(xiàn)象，，但其與肝癌發(fā)生發(fā)展的關(guān)系尚不清楚。 研究目的：闡述miR-27a在肝癌中的表達(dá)及作用機(jī)制，同時(shí)探討miR-27a通過抑制靶基因PPAR-γ的表達(dá)影響肝癌增殖和發(fā)展的機(jī)制，為開發(fā)肝癌診治的新靶點(diǎn)提供了理論依據(jù)。 研究內(nèi)容： Realtime-PCR方法分析正常人與肝癌患者血清及肝癌組織中miR-27a表達(dá)差異；Realtime-PCR方法檢測人正常肝細(xì)胞系HL-7702與人肝癌細(xì)胞系HepG2、Bel-7402、Bel-7404中miR-27a表達(dá)水平；應(yīng)用MiR-27a人工合成類似物mimics及抑制劑inhibitors分別轉(zhuǎn)染HepG2細(xì)胞系，通過MTT方法檢測其對肝癌細(xì)胞生長增殖的影響；流式細(xì)胞術(shù)檢測轉(zhuǎn)染后肝癌細(xì)胞凋亡及細(xì)胞周期變化；熒光素酶報(bào)告基因檢測PPAR-γ是否為miR-27a的下游靶點(diǎn)；免疫組化技術(shù)檢測臨床病理切片中PPAR-γ表達(dá)；應(yīng)用PPAR-γ配體激動劑羅格列酮作用于HepG2細(xì)胞系，通過Western Blot方法檢測miR-27a與羅格列酮刺激后PPAR-γ蛋白表達(dá)水平。 研究結(jié)果： 1. miR-27a類似物對HepG2細(xì)胞特性影響 肝癌患者血清、組織樣本及肝癌細(xì)胞系中miR-27a高表達(dá)，轉(zhuǎn)染miR-27amimics可加速肝癌細(xì)胞生長，誘導(dǎo)G1/S期轉(zhuǎn)化，促進(jìn)細(xì)胞增殖；轉(zhuǎn)染miR-27ainhibitors可誘發(fā)肝癌細(xì)胞G1期阻滯，抑制細(xì)胞增殖，增加凋亡比例。 2.miR-27a對PPAR-γ的影響 PPAR-γ的3’-UTR區(qū)存在miR-27a的互補(bǔ)序列，是miR-27a的下游靶基因；轉(zhuǎn)染miR-27a mimics后肝癌細(xì)胞內(nèi)PPAR-γmRNA及蛋白表達(dá)明顯受抑制；羅格列酮刺激能夠相對逆轉(zhuǎn)miR-27a對肝癌細(xì)胞增殖特性及PPAR-γ蛋白表達(dá)水平的影響。 研究結(jié)論： MiR-27a在肝癌高表達(dá)，能夠通過靶基因PPAR-γ，誘使G1/S期轉(zhuǎn)化，促進(jìn)肝癌細(xì)胞增殖，抑制miR-27a的表達(dá)能夠?qū)е录?xì)胞出現(xiàn)G1期阻滯，抑制肝癌細(xì)胞增殖，同時(shí)促進(jìn)肝癌細(xì)胞凋亡。PPAR-γ配體激活劑羅格列酮能夠增強(qiáng)PPAR-γ的表達(dá)，抑制肝癌細(xì)胞增殖，相對逆轉(zhuǎn)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- 緯) 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- 緯, 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- 緯 was a downstream target of miR-27a, and the expression of PPAR- 緯 in clinicopathological sections was detected by immunohistochemistry. HepG2 cell line was treated with PPAR- 緯 ligand agonist rosiglitazone. The expression of PPAR- 緯 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- 緯 there was a complementary sequence of miR-27a in the 3'-UTR region of PPAR- 緯, which was the downstream target gene of miR-27a, and the expression of PPAR- 緯 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- 緯 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- 緯, 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-緯, could enhance the expression of PPAR- 緯, inhibit the proliferation of HCC cells and reverse the effect of miR-27a.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R735.7
本文編號：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- 緯) 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- 緯, 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- 緯 was a downstream target of miR-27a, and the expression of PPAR- 緯 in clinicopathological sections was detected by immunohistochemistry. HepG2 cell line was treated with PPAR- 緯 ligand agonist rosiglitazone. The expression of PPAR- 緯 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- 緯 there was a complementary sequence of miR-27a in the 3'-UTR region of PPAR- 緯, which was the downstream target gene of miR-27a, and the expression of PPAR- 緯 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- 緯 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- 緯, 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-緯, could enhance the expression of PPAR- 緯, inhibit the proliferation of HCC cells and reverse the effect of miR-27a.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R735.7
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
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;周勝華;;microRNA生物合成的調(diào)控進(jìn)展——TGFβ/Smad信號途徑的作用[J];現(xiàn)代生物醫(yī)學(xué)進(jìn)展;2011年14期
本文編號：2494723
本文鏈接：http://sikaile.net/yixuelunwen/zlx/2494723.html
最近更新
教材專著
