LYRM1基因沉默致心肌样细胞凋亡的线粒体机制研究

发布时间：2018-08-16 16:00

【摘要】：心脏发育的过程是极其复杂的,涉及不同时间和空间的一系列胚胎发育相关基因的顺序表达。心脏结构和功能对心脏发育相关基因的扰动相当敏感。在心脏发育相关基因网络中,任何一个基因突变都可能导致先天性心脏病的发生。因此,先天性心脏病遗传学病因的探索仍然是预防和治疗该病的关键。LYRM1(LYR motif containing 1)基因是郭锡熔教授课题组发现并克隆得到的一条人类新基因。令人惊讶的是,多组织表达谱分析发现LYRM1基因除了在人类脂肪组织中高丰度表达外,在心脏组织中同样呈现高丰度表达。另外,研究表明LYRM1是LYR超家族复合体1的成员,而LYR蛋白主要是线粒体蛋白质,影响线粒体内稳态。郭锡熔教授课题小组成员发现LYRM1在3T3-L1前体脂肪细胞的增值、凋亡及分化过程均扮演重要角色,且具有损伤3T3-L1成熟脂肪细胞线粒体功能作用。鉴于心脏和脂肪均来源于中胚层,来源于相同胚层的组织具有相似的结构与功能,因此提示LYRM1也可能参与心脏发生发育的生理过程。本研究小组探索了LYRM1基因过表达在P19细胞向心肌样细胞分化过程的作用,结果显示LYRM1基因过表达虽然不影响P19细胞向心肌样细胞的分化,但是能显著诱导P19细胞的增殖、明显抑制P19细胞凋亡。因此,我们推测LYRM1基因在心脏发育中可能占有重要作用。为进一步探讨心脏发育过程中LYRM1基因的功能角色,我们选择P19细胞作为研究对象,运用RNA干扰技术使LYRM1基因表达沉默,进而探索LYRM1基因表达沉默对P19细胞增殖、凋亡和分化的影响,以及对P19诱导分化的心肌样细胞线粒体功能的影响。深度分析LYRM1基因对心肌样细胞凋亡的影响及凋亡作用与线粒体功能之间的关系,进一步论证在胚胎心脏发育过程中LYRM1基因的重要作用及致畸的可能机制,这将为先天性心脏病的病理生理学的基础研究提供新线索。第一部分LYRM1基因表达沉默对P19细胞增殖凋亡及分化的影响目的：探索LYRM1基因表达沉默对P19细胞增殖、凋亡及分化的影响。方法：设计LYRM1干扰靶序列,构建LYRM1基因短发夹状RNA的慢病毒载体pgLV-U6-puro-LYRM1-shRNA,对照组为pgLV-U6-puro-NC-shRNA.将pgLV-U6-puro-LYRM1-shRNA或pgLV-U6-puro-NC-shRNA转染到HEK-293T细胞并获得病毒上清,感染P19细胞并用嘌呤霉素筛选得到稳定转染的P19细胞。定量PCR检测LYRM1基因沉默干扰效果；细胞计数盒(CCK-8)检测其对P19细胞增殖的影响;流式细胞仪检测细胞周期、分析Caspase-3的活性及检测细胞凋亡；倒置显微镜观察P19细胞向心肌样细胞分化过程中细胞形态的改变；定量PCR及Westem blot检测心肌细胞分化过程中的心肌标志物基因(GATA4和Nkx2-5)的表达水平。结果:1) LYRM1基因沉默明显抑制P19细胞增殖,表现为：促进细胞进入G1期,受阻于S期；2)LYRM1基因沉默显著促进P19细胞凋亡；3)沉默LYRM1基因的P19细胞分化过程中,细胞边缘模糊,不规整。心肌标志物GATA4和Nkx2-5的基因表达水平均在心肌细胞分化第6天和第10天显著下降,蛋白表达水平在分化第6天前均无明显变化,但第10天明显下降。结论:LYRM1基因表达沉默具有抑制P19细胞的增值、促进其凋亡以及抑制P19细胞向心肌样细胞分化的作用,提示LYRM1基因在胚胎心脏发育过程可能占有重要作用。第二部分LYRM1基因表达沉默对P19细胞诱导分化的心肌样细胞线粒体功能的影响目的：探讨LYRM1基因表达沉默对P19细胞诱导分化的心肌样细胞线粒体功能的影响。方法：稳定转染LYRM1基因沉默病毒的P19细胞株,经1%DMSO诱导分化成为心肌样细胞。以诱导分化第10天的心肌样细胞为研究对象,采用荧光定量PCR技术检测线粒体DNA拷贝数；ATP检测试剂盒检测细胞ATP含量；采用DCFDA (2,7-Dichlorofluorescein diacetate, DCFDA)荧光探针、荧光显微镜、流式细胞仪检测细胞活性氧(reactive oxygen species, ROS)的改变；应用JC-1(Mitochondrial membrane potential assay kit with JC-1)荧光探针、荧光显微镜、流式细胞仪检测线粒体膜电位的变化。结果：1) LYRM1基因沉默对心肌样细胞的线粒体DNA拷贝数无明显影响；2) LYRM1基因沉默的心肌样细胞中ATP含量显著降低；3) LYRM1基因沉默显著增加心肌样细胞中ROS的产生；4) LYRM1基因沉默明显降低心肌样细胞线粒体膜电位水平。结论：]LYRM1基因沉默具有损伤心肌样细胞线粒体功能作用,提示LYRM1基因在心肌样细胞线粒体功能维持过程中可能扮演重要角色。
[Abstract]:The process of heart development is extremely complex, involving the sequential expression of a series of embryonic development-related genes in different time and space. Cardiac structure and function are sensitive to the disturbance of heart development-related genes. Any gene mutation in the heart development-related gene network can lead to congenital heart disease. The LYRM1 (LYR motif containing 1) gene is a new human gene discovered and cloned by Professor Guo Xirong's research group. Surprisingly, multiple tissue expression profiling analysis revealed that LYRM1 gene is highly expressed in human adipose tissue in addition to human adipose tissue. In addition, LYRM1 is a member of LYR superfamily complex 1, and LYR proteins are mainly mitochondrial proteins that affect mitochondrial homeostasis. Professor Guo Xirong's team members found that LYRM1 plays an important role in the proliferation, apoptosis and differentiation of 3T3-L1 preadipocytes. LYRM1 may also be involved in the physiological process of cardiogenesis and development. Our team explored the over-expression of LYRM1 gene in P19 cells, which is myocardially fine. The results showed that LYRM1 gene overexpression did not affect the differentiation of P19 cells into cardiomyocyte-like cells, but could significantly induce the proliferation of P19 cells and inhibit the apoptosis of P19 cells. Because of the function of P19 cells, we used RNA interference to silence LYRM1 gene expression, and then explored the effects of LYRM1 gene silencing on the proliferation, apoptosis and differentiation of P19 cells, and on the mitochondrial function of P19-induced differentiated cardiomyocytes. The relationship between apoptosis and mitochondrial function, the important role of LYRM1 gene in embryonic heart development and the possible mechanism of teratogenesis will provide new clues for the basic study of pathophysiology of congenital heart disease. Objective: To explore the effects of LYRM1 gene silencing on proliferation, apoptosis and differentiation of P19 cells. Methods: LYRM1 interference target sequence was designed to construct a lentiviral vector pgLV-U6-puro-LYRM1-shRNA with short hairpin RNA of LYRM1 gene. The control group was pgLV-U6-puro-NC-shRNA. HEK-293T cells were infected with virus supernatant and purinomycin was used to screen stable transfected P19 cells.Quantitative PCR was used to detect the effect of LYRM1 gene silencing interference.Cell counting kit (CCK-8) was used to detect its effect on the proliferation of P19 cells.Flow cytometry was used to detect the cell cycle,Caspase-3 activity and apoptosis. Results: 1) LYRM1 gene silencing significantly inhibited the proliferation of P19 cells, which could promote the cells to enter G1 phase and be blocked by S phase. LYRM1 gene silencing significantly promoted P19 cell apoptosis; 3) LYRM1 gene silencing in P19 cell differentiation process, cell margin blurred, irregular. Myocardial markers GATA4 and Nkx2-5 gene expression levels were significantly decreased on the 6th and 10th day of cardiomyocyte differentiation, protein expression levels were not significantly changed before the 6th day of differentiation, but the 1st day. Conclusion: LYRM1 gene silencing can inhibit the proliferation of P19 cells, promote their apoptosis and inhibit the differentiation of P19 cells into cardiomyocyte-like cells, suggesting that LYRM1 gene may play an important role in the development of embryonic heart. AIM: To investigate the effect of LYRM1 gene silencing on mitochondrial function of cardiomyocyte-like cells induced by P19 cells. METHODS: P19 cells stably transfected with LYRM1 gene silencing virus were induced to differentiate into cardiomyocyte-like cells by 1% DMSO. Mitochondrial DNA copy number was detected by quantitative PCR, ATP assay kit, DCFDA (2,7-Dichlorofluorescein diacetate) fluorescence probe, fluorescence microscope and flow cytometry were used to detect the changes of reactive oxygen species (ROS) and JC-1 (Mitochondrial membrane potential assay k). It with JC-1) fluorescence probe, fluorescence microscope and flow cytometry were used to detect the changes of mitochondrial membrane potential. Results: 1) LYRM1 gene silencing had no significant effect on the mitochondrial DNA copy number of cardiomyocyte-like cells; 2) ATP content in LYRM1 gene silenced cardiomyocyte-like cells decreased significantly; 3) LYRM1 gene silencing significantly increased ROS content in cardiomyocyte-like cells. Conclusion: LYRM1 gene silencing can impair the mitochondrial function of cardiomyocytes, suggesting that LYRM1 gene may play an important role in the maintenance of mitochondrial function of cardiomyocytes.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R54

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