miR-340靶向Nrf2-ARE信号逆转肝癌细胞顺铂耐药的分子机制研究
发布时间：2019-06-28 18:04
【摘要】：近年来,肿瘤细胞化疗药物耐药是引起治疗失败的重要原因,研究肿瘤细胞耐药性的产生机制和探索逆转耐药的途径是目前肿瘤治疗研究的热点领域。肿瘤对化疗药物的耐药性可分为原发性耐药和获得性耐药,临床上后者多见。产生多药耐药的机制非常复杂,一般认为基因遗传学水平的变异与肿瘤原发性耐药密切相关,而基因表观遗传学水平失调往往与获得性耐药有着特征性联系。在肿瘤获得性耐药发生过程中,相关耐药基因的表达调控主要通过DNA位点甲基化、组蛋白修饰和非编码RNA来进行。microRNA(miRNA)以转录后调控的方式广泛参与了生物进化与发育,细胞分化与恶变,细胞增殖与凋亡等各种生命活动过程。近年来的研究表明,miRNA除了可以作为生物学标记物对肿瘤诊断和预后作出评价,更重要的是肿瘤耐药性与miRNA异常表达有着特征性的联系。抑制和重新表达耐药相关miRNA可以提高抗肿瘤药物的疗效,目前肝癌耐药相关miRNAs的报道较少,已有研究发现：在肝癌细胞中miR-221和miR-222过度表达,分别下调上述miRNAs可恢复PTEN和TIMP3的表达,进而抑制AKT通路,提高肿瘤坏死因子相关诱导凋亡配体(TNF-related apoptosis-inducing ligand, TRAIL)的敏感性,增强化疗药物的作用,并降低肿瘤细胞的侵袭性。为进一步分析肝癌顺铂耐药相关miRNAs的作用与分子机制,本实验进行了如下研究：目的方法：1.利用高通量miRNA芯片联合实时荧光定量RT-PCR比较肝癌耐药细胞HepG2/CDDP和其亲本细胞HepG2 miRNA表达谱差异；采用靶基因预测软件筛选出能与Nrf2-3'-UTR区相互作用的miRNAs;通过以上两种方法结果的比对,最终确定miR-340为我们所要研究的目的miRNA。2.运用瞬时转染miR-340模拟物和抑制物的方法分别上调和下调顺铂耐药和亲本肝癌HepG2细胞系中miR-340的表达,并设阴性对照、空白对照和联合转染(miR-340模拟物+miR-340抑制物)组,并用通过实时荧光定量RT-PCR法对转染效率予以验证。通过CCK8法检测细胞增殖、药物半数致死剂量(IC50)、流式细胞术检测细胞凋亡等实验分析miR-340对肝癌细胞耐药表型的影响。3.运用双荧光素酶报告基因技术验证miR-340与Nrf2的靶向关系,并通过实时荧光定量RT-PCR和Westernblot等技术检测上述各组转染成功细胞株内Nrf2及下游基因表达水平的变化,从而揭示miR-340调控肝癌HepG2细胞顺铂耐药的分子机制。结果：1.高通量miRNA芯片比较发现：HepG2/CDDP和其亲本细胞HepG2相比有17条显著差异表达的miRNAs,其中9条显著上调(=2.0 fold),8条显著下调(0.5 fold),实时荧光定量PCR结果显示：相对于亲本细胞HepG2,miR-340在顺铂耐药细胞系中的表达水平明显下调,且其下调幅度(change fold=0.07,P0.01)在所有下调表达的miRNAs中最为显著。2.转染miR-340模拟物可显著上调顺铂耐药肝癌HepG2细胞中miR-340表达水平,并显著增加顺铂对HepG2/CDDP细胞诱导的凋亡率,进而降低耐药细胞对化疗药物的IC50；相反,转染miR-340抑制物可显著下调亲本肝癌细胞珠中miR-340表达水平,并显著降低顺铂对HepG2细胞诱导的凋亡率,进而提高亲本细胞对化疗药物的IC503.双荧光素酶实验证实Nrf2基因的3'-UTR携带有miR-340的直接结合位点,并且上调miR-340表达能显著抑制耐药肝癌细胞HepG2/CDDP中Nrf2及其下游基因的表达；相反在亲本细胞株中下调miR-340表达可促进亲本HepG2细胞中Nrf2及其下游基因的表达。结论：通过高通量miRNA芯片比较,我们首次发现了一组肝癌顺铂耐药相关miRNAs分子,而miR-340可能是这组分子中影响肝癌顺铂耐药性状的关键miRNAs分子；miR-340在肝癌顺铂耐药细胞中表达水平显著低于相应亲本细胞,上调miR-340表达可以通过拮抗Nrf2抗氧化应激通路进而部分逆转肝癌耐药细胞株对顺铂的耐药。
[Abstract]:In recent years, the drug resistance of tumor cell chemotherapy is an important cause of the failure of the treatment, and the mechanism of the mechanism of the drug resistance of the tumor cells and the way to explore the reverse resistance are the hot spot in the research of tumor therapy. The drug resistance of the tumor to the chemotherapy drugs can be divided into the primary drug resistance and the acquired resistance, and the latter is more common in the clinic. The mechanism of multi-drug resistance is very complicated. It is generally considered that the genetic level of the gene is closely related to the primary drug resistance of the tumor, and the apparent genetic level of the gene is often associated with the acquired resistance. In the course of the occurrence of tumor-acquired resistance, the expression regulation of the related drug-resistant gene is mainly carried out by DNA site methylation, histone modification and non-coding RNA. MicroRNA (miRNA) is widely involved in the processes of biological evolution and development, cell differentiation and malignant transformation, cell proliferation and apoptosis in a way of post-transcriptional regulation. In recent years, the research has shown that the miRNA can be used as a biological marker to evaluate the diagnosis and prognosis of the tumor, and more importantly, the resistance of the tumor and the abnormal expression of the miRNA have a characteristic connection. The inhibition and reexpression of the resistance-related miRNAs can improve the curative effect of the anti-tumor drug, and the reports of the related miRNAs related to the drug resistance of the liver cancer are less, and the research has found that the miR-221 and the miR-222 are overexpressed in the liver cancer cell, the expression of the PTEN and TIMP3 can be recovered by the miRNAs respectively, and the AKT path can be further inhibited, The sensitivity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is improved, and the effect of chemotherapy drugs is enhanced, and the invasiveness of tumor cells is reduced. In order to further analyze the role and molecular mechanism of the drug-resistant miRNAs in the liver cancer, the following research was carried out:1. using a high-throughput miRNA chip and a real-time fluorescence quantitative RT-PCR to compare the difference of the expression profiles of the HepG2/ CDDP and the HepG2 miRNA of a parent cell of the liver cancer; screening the miRNAs capable of interacting with the Nrf2-3 '-UTR region by using the target gene prediction software; and comparing the miRNAs obtained by the two methods, It is finally determined that miR-340 is the target miRNA for which we are to study. The expression of the miR-340 in the cisplatin-resistant and the parent liver cancer HepG2 cell line is up-regulated and down-regulated by the method of transient transfection of the miR-340 mimetic and the inhibitor, and the negative control, the blank control and the combined transfection (miR-340 mimetic + miR-340 inhibitor) group are arranged, The transfection efficiency was verified by real-time fluorescence quantitative RT-PCR. The effect of miR-340 on the resistance phenotype of hepatocellular carcinoma cells was analyzed by means of CCK8 method for detecting cell proliferation, half lethal dose of drug (IC50), flow cytometry and cell apoptosis. The target relationship between miR-340 and Nrf2 was verified by using the double-luciferase reporter gene technique, and the expression level of Nrf2 and downstream gene in the above-mentioned group was detected by real-time fluorescence quantitative RT-PCR and Western blot, so as to reveal the molecular mechanism of miR-340 to regulate the cisplatin resistance of HepG2 cells. Results:1. The comparison of high-throughput miRNA chips showed that there were 17 significant differences in the expression of miRNAs in HepG2/ CDDP and its parent cell HepG2, of which 9 were significantly up-regulated (= 2.0 fold) and 8 were significantly reduced (0.5 fold), and the real-time fluorescence quantitative PCR results showed that, with respect to the parent cell HepG2, The expression level of miR-340 in the cisplatin-resistant cell line was down-regulated and its down-regulation amplitude (change fold = 0.07, P0.01) was the most significant in all the down-regulated miRNAs. The transfection of the miR-340 mimetic can obviously increase the expression level of the miR-340 in the cisplatin-resistant liver cancer HepG2 cells, obviously increase the apoptosis rate induced by the cisplatin on the HepG2/ CDDP cells, and further reduce the IC50 of the drug-resistant cells to the chemotherapeutic drug; on the contrary, the transfection of the miR-340 inhibitor can significantly reduce the expression level of the miR-340 in the parent liver cancer cell bead, And obviously reducing the apoptosis rate induced by the cisplatin on the HepG2 cells, and further improving the IC503 of the parent cell to the chemotherapy drug. The double-luciferase experiment confirmed that the 3 '-UTR of the Nrf2 gene carries the direct binding site of the miR-340, and the up-regulation of the miR-340 expression can obviously inhibit the expression of Nrf2 and the downstream gene of the drug-resistant liver cancer cell HepG2/ CDDP; In contrast, the down-regulation of miR-340 expression in the parent cell line can promote the expression of Nrf2 and its downstream gene in the parent HepG2 cell. Conclusion: Compared with the high-flux miRNA chip, we first discovered a group of miRNAs related to the cisplatin resistance of the liver cancer, and the miR-340 may be the key miRNAs molecule which can influence the drug resistance of the cisplatin in the group of molecules, and the expression level of the miR-340 in the cisplatin-resistant cells of the liver cancer is significantly lower than that of the corresponding parent cell, The up-regulation of miR-340 expression can reverse the resistance to cisplatin by antagonizing the Nrf2 anti-oxidative stress pathway.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7
本文编号：2507503
[Abstract]:In recent years, the drug resistance of tumor cell chemotherapy is an important cause of the failure of the treatment, and the mechanism of the mechanism of the drug resistance of the tumor cells and the way to explore the reverse resistance are the hot spot in the research of tumor therapy. The drug resistance of the tumor to the chemotherapy drugs can be divided into the primary drug resistance and the acquired resistance, and the latter is more common in the clinic. The mechanism of multi-drug resistance is very complicated. It is generally considered that the genetic level of the gene is closely related to the primary drug resistance of the tumor, and the apparent genetic level of the gene is often associated with the acquired resistance. In the course of the occurrence of tumor-acquired resistance, the expression regulation of the related drug-resistant gene is mainly carried out by DNA site methylation, histone modification and non-coding RNA. MicroRNA (miRNA) is widely involved in the processes of biological evolution and development, cell differentiation and malignant transformation, cell proliferation and apoptosis in a way of post-transcriptional regulation. In recent years, the research has shown that the miRNA can be used as a biological marker to evaluate the diagnosis and prognosis of the tumor, and more importantly, the resistance of the tumor and the abnormal expression of the miRNA have a characteristic connection. The inhibition and reexpression of the resistance-related miRNAs can improve the curative effect of the anti-tumor drug, and the reports of the related miRNAs related to the drug resistance of the liver cancer are less, and the research has found that the miR-221 and the miR-222 are overexpressed in the liver cancer cell, the expression of the PTEN and TIMP3 can be recovered by the miRNAs respectively, and the AKT path can be further inhibited, The sensitivity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is improved, and the effect of chemotherapy drugs is enhanced, and the invasiveness of tumor cells is reduced. In order to further analyze the role and molecular mechanism of the drug-resistant miRNAs in the liver cancer, the following research was carried out:1. using a high-throughput miRNA chip and a real-time fluorescence quantitative RT-PCR to compare the difference of the expression profiles of the HepG2/ CDDP and the HepG2 miRNA of a parent cell of the liver cancer; screening the miRNAs capable of interacting with the Nrf2-3 '-UTR region by using the target gene prediction software; and comparing the miRNAs obtained by the two methods, It is finally determined that miR-340 is the target miRNA for which we are to study. The expression of the miR-340 in the cisplatin-resistant and the parent liver cancer HepG2 cell line is up-regulated and down-regulated by the method of transient transfection of the miR-340 mimetic and the inhibitor, and the negative control, the blank control and the combined transfection (miR-340 mimetic + miR-340 inhibitor) group are arranged, The transfection efficiency was verified by real-time fluorescence quantitative RT-PCR. The effect of miR-340 on the resistance phenotype of hepatocellular carcinoma cells was analyzed by means of CCK8 method for detecting cell proliferation, half lethal dose of drug (IC50), flow cytometry and cell apoptosis. The target relationship between miR-340 and Nrf2 was verified by using the double-luciferase reporter gene technique, and the expression level of Nrf2 and downstream gene in the above-mentioned group was detected by real-time fluorescence quantitative RT-PCR and Western blot, so as to reveal the molecular mechanism of miR-340 to regulate the cisplatin resistance of HepG2 cells. Results:1. The comparison of high-throughput miRNA chips showed that there were 17 significant differences in the expression of miRNAs in HepG2/ CDDP and its parent cell HepG2, of which 9 were significantly up-regulated (= 2.0 fold) and 8 were significantly reduced (0.5 fold), and the real-time fluorescence quantitative PCR results showed that, with respect to the parent cell HepG2, The expression level of miR-340 in the cisplatin-resistant cell line was down-regulated and its down-regulation amplitude (change fold = 0.07, P0.01) was the most significant in all the down-regulated miRNAs. The transfection of the miR-340 mimetic can obviously increase the expression level of the miR-340 in the cisplatin-resistant liver cancer HepG2 cells, obviously increase the apoptosis rate induced by the cisplatin on the HepG2/ CDDP cells, and further reduce the IC50 of the drug-resistant cells to the chemotherapeutic drug; on the contrary, the transfection of the miR-340 inhibitor can significantly reduce the expression level of the miR-340 in the parent liver cancer cell bead, And obviously reducing the apoptosis rate induced by the cisplatin on the HepG2 cells, and further improving the IC503 of the parent cell to the chemotherapy drug. The double-luciferase experiment confirmed that the 3 '-UTR of the Nrf2 gene carries the direct binding site of the miR-340, and the up-regulation of the miR-340 expression can obviously inhibit the expression of Nrf2 and the downstream gene of the drug-resistant liver cancer cell HepG2/ CDDP; In contrast, the down-regulation of miR-340 expression in the parent cell line can promote the expression of Nrf2 and its downstream gene in the parent HepG2 cell. Conclusion: Compared with the high-flux miRNA chip, we first discovered a group of miRNAs related to the cisplatin resistance of the liver cancer, and the miR-340 may be the key miRNAs molecule which can influence the drug resistance of the cisplatin in the group of molecules, and the expression level of the miR-340 in the cisplatin-resistant cells of the liver cancer is significantly lower than that of the corresponding parent cell, The up-regulation of miR-340 expression can reverse the resistance to cisplatin by antagonizing the Nrf2 anti-oxidative stress pathway.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7
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相关期刊论文 前3条
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3 Caecilia HC Sukowati;Natalia Rosso;Lory S Crocè;Claudio Tiribelli;;Hepatic cancer stem cells and drug resistance: Relevance in targeted therapies for hepatocellular carcinoma[J];World Journal of Hepatology;2010年03期
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