MiR-100参与激活突变SHP-2酪氨酸磷酸酶促进砷诱导的MEFs恶性转化

发布时间：2018-03-19 13:44

本文选题：SHP-2　切入点：miR-100　出处：《安徽医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：肿瘤是严重威胁人类健康的重大疾病,不仅发病率和死亡率逐年上升,近年来更呈现出高发和早发的趋势。而肿瘤的发生是多因素作用,经过多阶段的长期的复杂过程,受环境因素与基因因素的共同调控。因此,研究两者共同作用下肿瘤发生发展的分子机制对解决肿瘤的高发和早发问题至关重要。SHP-2是由PTPN11编码的非受体型酪氨酸磷酸酶,参与了包括P13K.MAPK. JAK/STAT等多种信号通路的活化。最初在努南综合征中发现其突变体的存在,后来在多种血液病及肿瘤中也发现了这一突变体的存在,并且这一突变体对肿瘤的发生发展有着至关重要的作用。SHP-2最常见的激活突变体是D61G/D61Y,这也是我们一直致力于研究的一个突变体。砷主要存在于水源和土壤中,是国际公认的的化学致癌物,与皮肤癌、肺癌等多种肿瘤的发生发展密切相关。我们实验室前期的研究过程中发现激活突变SHP-2酪氨酸磷酸酶促进砷诱导的MEFs恶性转化。在对其机制进行初步探究的过程中发现：As203急性处理MEF细胞48h后,miR-100在As203诱导前后的细胞或SHP2激活突变与否的细胞出现减少,且受三氧化二砷和SHP2激活突变影响变化一致。MiR-100是小的非编码RNA,已经被报道在不同肿瘤中通过靶向不同的靶蛋白而发挥着致癌基因或是抑癌基因的作用。包括乳腺癌、肺癌、宫颈癌、淋巴瘤在内的多种肿瘤的发生发展都与miR-100的异常表达有关。但是关于环境污染与基因突变共同作用下肿瘤的发生过程中miR-100的作用机制并不明确。我们设想miR-100可能参与了激活突变SHP-2酪氨酸磷酸酶促进砷诱导的MEFs恶性转化。为了验证这一假说,首先,我们采用RT-PCR技术检测了SHP-2+/+、SHP-2D61G/+以及其对应的As203慢性处理40代的SHP-2+/+-40、SHP-2D61G/+-40细胞中miR-100的表达水平,结果发现miR-100在四种细胞中的表达逐渐下降,提示miR-100参与了MEF细胞的恶性转化过程。接下来,为了明确miR-100在其中发挥的效应,本实验通过转染miR-100inhibitor或miR-100 mimics改变相应的MEF细胞中miR-100的表达水平,结果发现miR-100可以减少细胞的克隆形成能力和非锚定生长能力以及体外迁移和增殖能力。双荧光素酶报告基因实验发现IGF1Rβ和nTOR是miR-100靶基因,WB实验表明IGF1Rβ在MEFs细胞中的表达随着As2O3诱导SHP-2D61G/+激活突变表达逐渐升高。最后,采用携带有目的基因的慢病毒载体感染细胞建立稳转细胞系,细胞学实验得到与上述同样的结果。以上结果表明：miR-100参与激活突变SHP-2酪氨酸磷酸酶促进砷诱导的MEFs恶性转化过程,并在其中发挥抑癌基因的作用。
[Abstract]:Cancer is a serious disease that threatens human health seriously. Not only the incidence and mortality rate increase year by year, but also the tendency of high incidence and early onset in recent years. Therefore, it is very important to study the molecular mechanism of tumorigenesis and development under the interaction of environmental and genetic factors. SHP-2 is a non-recipient tyrosine phosphatase encoded by PTPN11. Involved in the activation of multiple signaling pathways, including P13K.MAPK. JAK/STAT. The mutant was initially found in Nunan syndrome and later found in various hematological diseases and tumors. The most common active mutants of SHP-2 are D61G / D61Y, which is one of the mutants that we have been working on. Arsenic is mainly found in water and soil. Is an internationally recognized chemical carcinogen, associated with skin cancer, SHP-2 tyrosine phosphatase is found to promote arsenic induced malignant transformation of MEFs. In the course of preliminary study on the mechanism, we found that the activation mutation of SHP-2 tyrosine phosphatase promotes arsenic induced malignant transformation of MEFs. It was found that after 48 hours of acute treatment of MEF cells with 1: As203, the cells of miR-100 before and after As203 induction or those with or without SHP2 activation mutation were decreased. MiR-100, a small noncoding RNAs, has been reported to play the role of oncogene or tumor suppressor gene by targeting different target proteins in different tumors, including breast cancer and lung cancer. Cervical cancer, The occurrence and development of various tumors, including lymphoma, are related to the abnormal expression of miR-100. However, the mechanism of miR-100 in tumorigenesis under the action of environmental pollution and gene mutation is not clear. We assume that miR-100 may play an important role in the development of tumor. Participate in the activation of mutated SHP-2 tyrosine phosphatase to promote arsenic induced malignant transformation of MEFs. Firstly, we detected the expression of miR-100 in the SHP-2 / -40 SHP-2D61G / -40 cells treated with SHP-2 / HP-2D61G/ and its corresponding As203 chronically by RT-PCR technique. The results showed that the expression of miR-100 in four kinds of cells decreased gradually. The results suggest that miR-100 is involved in the malignant transformation of MEF cells. In order to understand the effect of miR-100, the expression of miR-100 in MEF cells was altered by transfection of miR-100inhibitor or miR-100 mimics. The results showed that miR-100 could reduce the ability of cell clone formation, unanchored growth, migration and proliferation in vitro. Double luciferase reporter gene assay showed that IGF1R 尾 and nTOR were miR-100 target genes in MEFs cells. The expression of SHP-2D61G/ activation mutation increased with the induction of As2O3. Finally, Stable transformed cell lines were established by infection with lentivirus vector carrying the target gene, and the same results were obtained by cytological experiments. The above results indicated that: miR-100 was involved in the activation of mutated SHP-2 tyrosine phosphatase to promote arsenic induced malignant transformation of MEFs. And play the role of tumor suppressor gene.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R730.2

【相似文献】