Dysferlin肌病miRNA表达模式的改变及miR-708在成肌细胞生长中的作用
发布时间：2018-09-18 15:37
【摘要】：【目的】系统检测mi RNA在Dysferlin肌病中的表达,初步探讨mi RNA与Dysferlin肌病的关系;结合靶基因预测及细胞学实验,探讨mi R-708 mimic对成肌细胞的影响。【方法】1.结合临床表现、HE染色和肌酶特殊染色、免疫组化等方法,筛选Dysferlin肌病病例为实验组,非特异性改变肌组织为对照组,取部分组织行mi RNA芯片分析。2.靶基因预测网站预测作用于DYSF基因的mi RNA,结合mi RNA芯片结果,选择与dysferlin肌病关系密切的mi RNA,Taqman探针实时定量PCR(RT-PCR)检测其在人肌肉组织及小鼠成肌细胞C2C12增殖和分化过程中的表达。3.合成mi R-708 mimic,转染成肌细胞C2C12,光学显微镜下观察成肌细胞生长分化情况,利用CCK-8检测mi R-708 mimic对C2C12细胞生长增殖的影响,用流式细胞术检测转染mi R-708 mimic后的增殖期C2C12细胞周期和凋亡情况,RT-PCR、Western blot检测肌细胞增殖分化相关基因的表达。【结果】1.免疫组化显示:dysferlin蛋白在正常肌细胞的肌膜上呈线性表达,而在Dysferlin肌病组织中,dysferlin蛋白表达明显减弱或缺失。光镜下Dysferlin肌病组织病理学改变:肌纤维呈轻-重度圆形萎缩伴多量肥大肌纤维形成,散在肌纤维见镶边空泡,个别肌纤维溶解坏死,内核纤维明显增多,间质纤维脂肪组织增生。2.mi RNA芯片结果显示:Dysferlin肌病组织,116个mi RNA表达上调,176个mi RNA表达下调,部分mi RNA表达水平异常与肌病时细胞内各种酶活性的调节、细胞代谢的调控及MAPK、Wnt信号通路等病理过程相关。结合芯片结果与靶基因预测结果,选择与DYSF基因关系密切的3个mi R(-122、-346、-708)。其中mi R-122、-346下调,mi R-708上调。3.筛选9例Dysferlin肌病标本进行mi RNA检测,与非特异性改变肌组织对比,mi R-122、-346、-708表达均升高(P值分别为0.04,0.04,0.03),其中mi R-708在Dysferlin肌病患者肌组织的表达模式与芯片结果一致。4.检测mi R-122、-346、-708在C2C12成肌细胞生长分化过程中的表达,显示增殖期mi R-122、-346、-708表达量较低,分化早期mi R-122、-346、-708均升高,但随着C2C12分化时间的延长,mi R-122表达水平基本不变,mi R-346、-708表达下调,其中mi R-708下调更明显,达到增殖期水平。5.C2C12细胞转染mi R-708 mimic后,光学显微镜下可见成肌细胞增殖受到明显抑制。CCK-8检测显示mimic组C2C12抑制率为(15.40±0.0052)%,(P=0.004),明显高于NC组(2.87±0.0034)%;流式细胞术结果显示:转染mi R-708 mimic后,C2C12细胞周期变化明显,S期明显缩短(20.87%),G1期明显延长(68.64%),NC组分别为45.66%、42.69%,而细胞凋亡指数mimic组与NC组无显著差异;RT-PCR及Western blot结果显示:与阴性对照(NC组)相比,转染mi R-708mimic后MYOD、MYOG、MHC、DYSF蛋白表达水平无明显变化。【结论】1.Dysferlin肌病可引起多种mi RNA表达模式的改变,这些mi RNA可能通过肌动蛋白骨架形成的调控、细胞内各种酶活性的调节、细胞代谢的调控及MAPK、Wnt信号通路的调节等环节参与Dysferlin肌病病理生理过程。2.Mi R-708可能是成肌细胞增殖的调控分子,但与成肌细胞分化和凋亡无明显相关,其在Dysferlin肌病中的作用机制有待于进一步探讨。
[Abstract]:[Objective] To detect the expression of MI RNA in Dysferlin myopathy and explore the relationship between MI RNA and Dysferlin myopathy, and to explore the effect of MI R-708 mimic on myoblasts by target gene prediction and cytological experiments. [Methods] 1. To screen Dysferlin myopathy by combining clinical manifestations, HE staining, enzyme specific staining and immunohistochemistry. The patients were the experimental group and the non-specific muscle tissues were the control group. Some tissues were taken for microarray analysis. 2. The target gene prediction website predicted the MI RNA of DYSF gene, and combined with the results of microarray, selected the MI RNA closely related to dysferlin myopathy. Taqman probe real-time quantitative PCR (RT-PCR) was used to detect the MI RNA in human muscle tissues and mice. Expression of C2C12 in myocytes during proliferation and differentiation Immunohistochemistry showed that dysferlin protein was linearly expressed on the sarcolemma of normal myocytes, while in Dysferlin myopathy, the expression of dysferlin protein was significantly decreased or deleted. Mild to severe round atrophy accompanied by a large number of hypertrophic muscle fibers, scattered muscle fibers with rimmed vacuoles, individual muscle fibers dissolved and necrosis, the number of nuclear fibers increased significantly, interstitial fibrous adipose tissue proliferation. 2. MiRNA microarray results showed that in Dysferlin myopathy, 116 mi RNA expression was up-regulated, 176 mi RNA expression was down-regulated, and some mi RNA expression levels were down-regulated. Abnormalities were associated with the regulation of intracellular enzymes, cellular metabolism, MAPK and Wnt signaling pathways in myopathy. Three miRs (-122, -346, -708) closely related to DYSF gene were selected by combining the results of microarray and target gene prediction. Among them, mi R-122, -346 were down-regulated, and MI R-708 was up-regulated. 3. Nine Dysferlin myopathy specimens were screened. MiR-122, -346, and-708 expressions were increased in non-specific muscle tissues (P values were 0.04, 0.04, 0.03 respectively). The expression pattern of MI R-708 in muscle tissues of Dysferlin myopathy patients was consistent with that of microarray. 4. The expression of MI R-122, -346, and-708 in C2C12 myoblasts during proliferation and differentiation was detected. 2, -346, -708 expression was low, and the expression of MI R-122, -346, -708 increased in the early stage of differentiation. However, with the prolongation of differentiation time of C2C12, the expression of MI R-122 remained unchanged, and the expression of MI R-346, -708 was down-regulated. MiR-708 was down-regulated more significantly, reaching the proliferative stage level. CCK-8 assay showed that the inhibition rate of C2C12 in mimic group was (15.40 +0.0052)%, (P = 0.004), which was significantly higher than that in NC group (2.87 +0.0034)%. Flow cytometry showed that the cell cycle of C2C12 was significantly changed, S phase was significantly shortened (20.87%), G1 phase was significantly prolonged (68.64%), NC group was 45.66%, and apoptosis index was 42.69%. The expression of MYOD, MYOG, MHC and DYSF was not significantly changed after transfection of MI R-708 Mi mic compared with the negative control group (NC group). [Conclusion] 1. Dysferlin myopathy can induce changes in expression patterns of multiple mi RNA, which may be regulated by actin cytoskeleton formation. Mi R-708 may be involved in the pathophysiological process of Dysferlin myopathy. 2. Mi R-708 may be a regulator of myoblast proliferation, but it is not related to myoblast differentiation and apoptosis. Its mechanism in Dysferlin myopathy remains to be further explored. Please.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R685
本文编号：2248354
[Abstract]:[Objective] To detect the expression of MI RNA in Dysferlin myopathy and explore the relationship between MI RNA and Dysferlin myopathy, and to explore the effect of MI R-708 mimic on myoblasts by target gene prediction and cytological experiments. [Methods] 1. To screen Dysferlin myopathy by combining clinical manifestations, HE staining, enzyme specific staining and immunohistochemistry. The patients were the experimental group and the non-specific muscle tissues were the control group. Some tissues were taken for microarray analysis. 2. The target gene prediction website predicted the MI RNA of DYSF gene, and combined with the results of microarray, selected the MI RNA closely related to dysferlin myopathy. Taqman probe real-time quantitative PCR (RT-PCR) was used to detect the MI RNA in human muscle tissues and mice. Expression of C2C12 in myocytes during proliferation and differentiation Immunohistochemistry showed that dysferlin protein was linearly expressed on the sarcolemma of normal myocytes, while in Dysferlin myopathy, the expression of dysferlin protein was significantly decreased or deleted. Mild to severe round atrophy accompanied by a large number of hypertrophic muscle fibers, scattered muscle fibers with rimmed vacuoles, individual muscle fibers dissolved and necrosis, the number of nuclear fibers increased significantly, interstitial fibrous adipose tissue proliferation. 2. MiRNA microarray results showed that in Dysferlin myopathy, 116 mi RNA expression was up-regulated, 176 mi RNA expression was down-regulated, and some mi RNA expression levels were down-regulated. Abnormalities were associated with the regulation of intracellular enzymes, cellular metabolism, MAPK and Wnt signaling pathways in myopathy. Three miRs (-122, -346, -708) closely related to DYSF gene were selected by combining the results of microarray and target gene prediction. Among them, mi R-122, -346 were down-regulated, and MI R-708 was up-regulated. 3. Nine Dysferlin myopathy specimens were screened. MiR-122, -346, and-708 expressions were increased in non-specific muscle tissues (P values were 0.04, 0.04, 0.03 respectively). The expression pattern of MI R-708 in muscle tissues of Dysferlin myopathy patients was consistent with that of microarray. 4. The expression of MI R-122, -346, and-708 in C2C12 myoblasts during proliferation and differentiation was detected. 2, -346, -708 expression was low, and the expression of MI R-122, -346, -708 increased in the early stage of differentiation. However, with the prolongation of differentiation time of C2C12, the expression of MI R-122 remained unchanged, and the expression of MI R-346, -708 was down-regulated. MiR-708 was down-regulated more significantly, reaching the proliferative stage level. CCK-8 assay showed that the inhibition rate of C2C12 in mimic group was (15.40 +0.0052)%, (P = 0.004), which was significantly higher than that in NC group (2.87 +0.0034)%. Flow cytometry showed that the cell cycle of C2C12 was significantly changed, S phase was significantly shortened (20.87%), G1 phase was significantly prolonged (68.64%), NC group was 45.66%, and apoptosis index was 42.69%. The expression of MYOD, MYOG, MHC and DYSF was not significantly changed after transfection of MI R-708 Mi mic compared with the negative control group (NC group). [Conclusion] 1. Dysferlin myopathy can induce changes in expression patterns of multiple mi RNA, which may be regulated by actin cytoskeleton formation. Mi R-708 may be involved in the pathophysiological process of Dysferlin myopathy. 2. Mi R-708 may be a regulator of myoblast proliferation, but it is not related to myoblast differentiation and apoptosis. Its mechanism in Dysferlin myopathy remains to be further explored. Please.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R685
【参考文献】
相关期刊论文 前1条
1 王敏;笪宇威;卢岩;徐敏;刘璐;贾建平;;Dysferlin蛋白缺陷的Miyoshi肌病的临床与病理学特点[J];临床神经病学杂志;2009年01期
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