miR-490-3p对肿瘤细胞增殖及心肌细胞代谢的影响

发布时间：2018-05-10 09:51

本文选题：miR-490-3p + 心肌梗疾病　；参考：《武汉大学》2017年硕士论文

【摘要】：microRNAs是一类小的、内源性的非编码RNAs,长度约17～25 bp,广泛存在于动植物体内,可以调控基因的表达,在人类基因组中30%的基因都受到miRNAs的调控。研究表明miRNAs表达的改变是肿瘤发生的共同特征之一,miRNAs在肿瘤细胞中参与细胞周期、细胞迁移、细胞侵袭和细胞凋亡等过程的调控。miRNAs可以作为原癌基因或抑癌基因参与肿瘤的发生发展过程,抑制抑癌基因表达的miRNAs在肿瘤中上调,导致抑癌基因的沉默;相反,抑制原癌基因表达的miRNAs在肿瘤中下调,敲除或突变会导致原癌基因的过表达。此外,除了具有最常见的原癌基因或抑癌基因功能,miRNAs还与肿瘤转移有关。miRNAs在临床上还可以作为肿瘤早期诊断的分子标志物,特别是肿瘤不同阶段病人生存率的诊断。miR-490-3p在多种肿瘤中均表现出下调,可能是肿瘤生长的抑制因子,同时它可能与心脏的健康密切相关,miR-490-3p的表达量在心脏疾病中表现出了显著变化。但是,对于miR-490-3p在肿瘤以及心脏病变中的具体作用却有待进一步阐明。本研究旨在探讨miR-490-3p对肿瘤生长相关特性的调控以及在心脏正常发育和病变过程中可能的作用。通过RT-qPCR检测心肌梗病人外周血血清中miR-490-3p与正常对照中的表达,发现miR-490-3p在心肌梗病人中表达量上调,表明miR-490-3p作为诊断心肌梗疾病分子标志物的可能性。过表达miR-490-3p后,测定大鼠心肌细胞H9C2的细胞培养液葡萄糖和乳酸浓度,发现葡萄糖摄取能力和乳酸生成能力下降。同时为了研究miR-490-3p对肿瘤细胞生长、迁移、细胞周期、上皮间质转换及代谢的影响,在本研究中通过外源感染miR-490-3p,在子宫颈癌细胞HeLa和肾癌细胞786-0中过表达miR-490-3p,利用Western blotting和RT-qPCR检测下游基因和蛋白的表达,miR-490-3p能抑制hnRNPA1(核不均一核糖核蛋白 A1,heterogeneous nuclear ribonucleoprotein A1)mRNA 和蛋白的表达,促进PKM1(丙酮酸激酶亚型1,pyruvate kinase,muscle 1)的表达,下调PKM2/PKM1表达的比例。随后通过葡萄糖和乳酸试剂盒检测细胞培养液葡萄糖和乳酸浓度,发现葡萄糖摄取能力和乳酸生成能力下降。此外,细胞计数和流式细胞仪分析结果表明miR-490-3p抑制细胞生长,使HeLa细胞阻滞在G2/M期,786-0细胞阻滞在G1/G0期。划痕实验显示miR-490-3p能够抑制细胞迁移和上皮间质转化。因此,miR-490-3p通过下调hnRNPA1的表达,上调PKM1的水平,下调PKM2/PKM1比例,改变细胞的代谢方式,从而抑制细胞的增殖和迁移,可以作为肿瘤的潜在治疗靶点和分子标记物,在肿瘤治疗和临床上具有重要的实践意义。
[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.
【学位授予单位】：武汉大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R73-3

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