本文選題：miR-490-3p + 心肌梗疾病��； 參考：《武漢大學(xué)》2017年碩士論文

【摘要】：microRNAs是一類小的、內(nèi)源性的非編碼RNAs,長度約17～25 bp,廣泛存在于動植物體內(nèi),可以調(diào)控基因的表達,在人類基因組中30%的基因都受到miRNAs的調(diào)控。研究表明miRNAs表達的改變是腫瘤發(fā)生的共同特征之一,miRNAs在腫瘤細胞中參與細胞周期、細胞遷移、細胞侵襲和細胞凋亡等過程的調(diào)控。miRNAs可以作為原癌基因或抑癌基因參與腫瘤的發(fā)生發(fā)展過程,抑制抑癌基因表達的miRNAs在腫瘤中上調(diào),導(dǎo)致抑癌基因的沉默;相反,抑制原癌基因表達的miRNAs在腫瘤中下調(diào),敲除或突變會導(dǎo)致原癌基因的過表達。此外,除了具有最常見的原癌基因或抑癌基因功能,miRNAs還與腫瘤轉(zhuǎn)移有關(guān)。miRNAs在臨床上還可以作為腫瘤早期診斷的分子標志物,特別是腫瘤不同階段病人生存率的診斷。miR-490-3p在多種腫瘤中均表現(xiàn)出下調(diào),可能是腫瘤生長的抑制因子,同時它可能與心臟的健康密切相關(guān),miR-490-3p的表達量在心臟疾病中表現(xiàn)出了顯著變化。但是,對于miR-490-3p在腫瘤以及心臟病變中的具體作用卻有待進一步闡明。本研究旨在探討miR-490-3p對腫瘤生長相關(guān)特性的調(diào)控以及在心臟正常發(fā)育和病變過程中可能的作用。通過RT-qPCR檢測心肌梗病人外周血血清中miR-490-3p與正常對照中的表達,發(fā)現(xiàn)miR-490-3p在心肌梗病人中表達量上調(diào),表明miR-490-3p作為診斷心肌梗疾病分子標志物的可能性。過表達miR-490-3p后,測定大鼠心肌細胞H9C2的細胞培養(yǎng)液葡萄糖和乳酸濃度,發(fā)現(xiàn)葡萄糖攝取能力和乳酸生成能力下降。同時為了研究miR-490-3p對腫瘤細胞生長、遷移、細胞周期、上皮間質(zhì)轉(zhuǎn)換及代謝的影響,在本研究中通過外源感染miR-490-3p,在子宮頸癌細胞HeLa和腎癌細胞786-0中過表達miR-490-3p,利用Western blotting和RT-qPCR檢測下游基因和蛋白的表達,miR-490-3p能抑制hnRNPA1(核不均一核糖核蛋白 A1,heterogeneous nuclear ribonucleoprotein A1)mRNA 和蛋白的表達,促進PKM1(丙酮酸激酶亞型1,pyruvate kinase,muscle 1)的表達,下調(diào)PKM2/PKM1表達的比例。隨后通過葡萄糖和乳酸試劑盒檢測細胞培養(yǎng)液葡萄糖和乳酸濃度,發(fā)現(xiàn)葡萄糖攝取能力和乳酸生成能力下降。此外,細胞計數(shù)和流式細胞儀分析結(jié)果表明miR-490-3p抑制細胞生長,使HeLa細胞阻滯在G2/M期,786-0細胞阻滯在G1/G0期。劃痕實驗顯示miR-490-3p能夠抑制細胞遷移和上皮間質(zhì)轉(zhuǎn)化。因此,miR-490-3p通過下調(diào)hnRNPA1的表達,上調(diào)PKM1的水平,下調(diào)PKM2/PKM1比例,改變細胞的代謝方式,從而抑制細胞的增殖和遷移,可以作為腫瘤的潛在治療靶點和分子標記物,在腫瘤治療和臨床上具有重要的實踐意義。
[Abstract]:MicroRNAs is a kind of small, endogenous non-coding RNs, which is about 1725 BP in length. It can regulate the expression of genes in animals and plants. 30% of the genes in human genome are regulated by miRNAs. It has been shown that the change of miRNAs expression is one of the common characteristics of tumorigenesis, in which miRNAs are involved in cell cycle and cell migration. The regulation of cell invasion and apoptosis. MiRNAs can be used as proto-oncogene or tumor suppressor gene to participate in the carcinogenesis and development of tumor. MiRNAs, which inhibits the expression of tumor suppressor gene, is up-regulated in tumor, leading to the silencing of tumor suppressor gene. MiRNAs, which inhibits proto-oncogene expression, is down-regulated in tumor, and knockout or mutation may lead to overexpression of proto-oncogene. In addition, in addition to having the most common proto-oncogene or suppressor gene function, miRNAs are also associated with tumor metastasis. MiRNAs can also be used as molecular markers for early diagnosis of tumors. In particular, the diagnosis of survival rate of patients at different stages of tumor. MiR-490-3p is down-regulated in many kinds of tumors, and may be an inhibitor of tumor growth. At the same time, the expression of miR-490-3p may be closely related to heart health. However, the role of miR-490-3p in cancer and heart disease needs to be further elucidated. The aim of this study was to investigate the role of miR-490-3p in the regulation of tumor growth related characteristics and in the normal development and pathological process of the heart. The expression of miR-490-3p in peripheral blood of patients with myocardial infarction was detected by RT-qPCR. It was found that the expression of miR-490-3p was up-regulated in patients with myocardial infarction, indicating the possibility of miR-490-3p as a molecular marker for diagnosing myocardial infarction. After overexpression of miR-490-3p, the concentration of glucose and lactic acid in the cell culture medium of rat cardiomyocytes H9C2 was measured, and the glucose uptake and lactic acid production ability were decreased. In order to study the effects of miR-490-3p on the growth, migration, cell cycle, epithelial interstitial transition and metabolism of tumor cells, In this study, miR-490-3p was overexpressed in cervical cancer cell line HeLa and renal cancer cell line 786-0 by exogenous infection of miR-490-3p. the expression of downstream genes and proteins was detected by Western blotting and RT-qPCR. MiR-490-3p could inhibit the expression of hnRNPA1 (nuclear heterogeneous nuclear ribonucleoprotein A1)mRNA and protein). Promote the expression of PKM1 (pyruvate kinase subtype 1 pyruvate kinasemuscle 1) and down-regulate the expression of PKM2/PKM1. Then glucose and lactic acid concentration were detected by glucose and lactic acid kit, and the glucose uptake and lactic acid production ability were decreased. In addition, the results of cell count and flow cytometry showed that miR-490-3p inhibited cell growth and blocked HeLa cells at G 2 / M phase in G1/G0 phase. Scratch test showed that miR-490-3p could inhibit cell migration and epithelial mesenchymal transformation. Therefore, miR-490-3p can inhibit cell proliferation and migration by down-regulating the expression of hnRNPA1, upregulating the level of PKM1, down-regulating the proportion of PKM2/PKM1 and changing the metabolic pattern of cells, which can be used as a potential therapeutic target and molecular marker for tumor. It has important practical significance in tumor treatment and clinical practice.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R73-3

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1 王亞波;miR-490-3p對腫瘤細胞增殖及心肌細胞代謝的影響[D];武漢大學(xué);2017年

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本文編號：1868832