miR-340靶向Nrf2-ARE信號(hào)逆轉(zhuǎn)肝癌細(xì)胞順鉑耐藥的分子機(jī)制研究
發(fā)布時(shí)間：2019-06-28 18:04
【摘要】：近年來,腫瘤細(xì)胞化療藥物耐藥是引起治療失敗的重要原因,研究腫瘤細(xì)胞耐藥性的產(chǎn)生機(jī)制和探索逆轉(zhuǎn)耐藥的途徑是目前腫瘤治療研究的熱點(diǎn)領(lǐng)域。腫瘤對(duì)化療藥物的耐藥性可分為原發(fā)性耐藥和獲得性耐藥,臨床上后者多見。產(chǎn)生多藥耐藥的機(jī)制非常復(fù)雜,一般認(rèn)為基因遺傳學(xué)水平的變異與腫瘤原發(fā)性耐藥密切相關(guān),而基因表觀遺傳學(xué)水平失調(diào)往往與獲得性耐藥有著特征性聯(lián)系。在腫瘤獲得性耐藥發(fā)生過程中,相關(guān)耐藥基因的表達(dá)調(diào)控主要通過DNA位點(diǎn)甲基化、組蛋白修飾和非編碼RNA來進(jìn)行。microRNA(miRNA)以轉(zhuǎn)錄后調(diào)控的方式廣泛參與了生物進(jìn)化與發(fā)育,細(xì)胞分化與惡變,細(xì)胞增殖與凋亡等各種生命活動(dòng)過程。近年來的研究表明,miRNA除了可以作為生物學(xué)標(biāo)記物對(duì)腫瘤診斷和預(yù)后作出評(píng)價(jià),更重要的是腫瘤耐藥性與miRNA異常表達(dá)有著特征性的聯(lián)系。抑制和重新表達(dá)耐藥相關(guān)miRNA可以提高抗腫瘤藥物的療效,目前肝癌耐藥相關(guān)miRNAs的報(bào)道較少,已有研究發(fā)現(xiàn)：在肝癌細(xì)胞中miR-221和miR-222過度表達(dá),分別下調(diào)上述miRNAs可恢復(fù)PTEN和TIMP3的表達(dá),進(jìn)而抑制AKT通路,提高腫瘤壞死因子相關(guān)誘導(dǎo)凋亡配體(TNF-related apoptosis-inducing ligand, TRAIL)的敏感性,增強(qiáng)化療藥物的作用,并降低腫瘤細(xì)胞的侵襲性。為進(jìn)一步分析肝癌順鉑耐藥相關(guān)miRNAs的作用與分子機(jī)制,本實(shí)驗(yàn)進(jìn)行了如下研究：目的方法：1.利用高通量miRNA芯片聯(lián)合實(shí)時(shí)熒光定量RT-PCR比較肝癌耐藥細(xì)胞HepG2/CDDP和其親本細(xì)胞HepG2 miRNA表達(dá)譜差異；采用靶基因預(yù)測軟件篩選出能與Nrf2-3'-UTR區(qū)相互作用的miRNAs;通過以上兩種方法結(jié)果的比對(duì),最終確定miR-340為我們所要研究的目的miRNA。2.運(yùn)用瞬時(shí)轉(zhuǎn)染miR-340模擬物和抑制物的方法分別上調(diào)和下調(diào)順鉑耐藥和親本肝癌HepG2細(xì)胞系中miR-340的表達(dá),并設(shè)陰性對(duì)照、空白對(duì)照和聯(lián)合轉(zhuǎn)染(miR-340模擬物+miR-340抑制物)組,并用通過實(shí)時(shí)熒光定量RT-PCR法對(duì)轉(zhuǎn)染效率予以驗(yàn)證。通過CCK8法檢測細(xì)胞增殖、藥物半數(shù)致死劑量(IC50)、流式細(xì)胞術(shù)檢測細(xì)胞凋亡等實(shí)驗(yàn)分析miR-340對(duì)肝癌細(xì)胞耐藥表型的影響。3.運(yùn)用雙熒光素酶報(bào)告基因技術(shù)驗(yàn)證miR-340與Nrf2的靶向關(guān)系,并通過實(shí)時(shí)熒光定量RT-PCR和Westernblot等技術(shù)檢測上述各組轉(zhuǎn)染成功細(xì)胞株內(nèi)Nrf2及下游基因表達(dá)水平的變化,從而揭示miR-340調(diào)控肝癌HepG2細(xì)胞順鉑耐藥的分子機(jī)制。結(jié)果：1.高通量miRNA芯片比較發(fā)現(xiàn)：HepG2/CDDP和其親本細(xì)胞HepG2相比有17條顯著差異表達(dá)的miRNAs,其中9條顯著上調(diào)(=2.0 fold),8條顯著下調(diào)(0.5 fold),實(shí)時(shí)熒光定量PCR結(jié)果顯示：相對(duì)于親本細(xì)胞HepG2,miR-340在順鉑耐藥細(xì)胞系中的表達(dá)水平明顯下調(diào),且其下調(diào)幅度(change fold=0.07,P0.01)在所有下調(diào)表達(dá)的miRNAs中最為顯著。2.轉(zhuǎn)染miR-340模擬物可顯著上調(diào)順鉑耐藥肝癌HepG2細(xì)胞中miR-340表達(dá)水平,并顯著增加順鉑對(duì)HepG2/CDDP細(xì)胞誘導(dǎo)的凋亡率,進(jìn)而降低耐藥細(xì)胞對(duì)化療藥物的IC50；相反,轉(zhuǎn)染miR-340抑制物可顯著下調(diào)親本肝癌細(xì)胞珠中miR-340表達(dá)水平,并顯著降低順鉑對(duì)HepG2細(xì)胞誘導(dǎo)的凋亡率,進(jìn)而提高親本細(xì)胞對(duì)化療藥物的IC503.雙熒光素酶實(shí)驗(yàn)證實(shí)Nrf2基因的3'-UTR攜帶有miR-340的直接結(jié)合位點(diǎn),并且上調(diào)miR-340表達(dá)能顯著抑制耐藥肝癌細(xì)胞HepG2/CDDP中Nrf2及其下游基因的表達(dá)；相反在親本細(xì)胞株中下調(diào)miR-340表達(dá)可促進(jìn)親本HepG2細(xì)胞中Nrf2及其下游基因的表達(dá)。結(jié)論：通過高通量miRNA芯片比較,我們首次發(fā)現(xiàn)了一組肝癌順鉑耐藥相關(guān)miRNAs分子,而miR-340可能是這組分子中影響肝癌順鉑耐藥性狀的關(guān)鍵miRNAs分子；miR-340在肝癌順鉑耐藥細(xì)胞中表達(dá)水平顯著低于相應(yīng)親本細(xì)胞,上調(diào)miR-340表達(dá)可以通過拮抗Nrf2抗氧化應(yīng)激通路進(jìn)而部分逆轉(zhuǎn)肝癌耐藥細(xì)胞株對(duì)順鉑的耐藥。
[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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.7
本文編號(hào)：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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.7
【參考文獻(xiàn)】
相關(guān)期刊論文 前3條
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2 ;Inhibitory effects of miRNA-200c on chemotherapy-resistance and cell proliferation of gastric cancer SGC7901/DDP cells[J];癌癥;2010年12期
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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