长白猪和蓝塘猪肌肉发育差异miRNA和mRNA表达谱的整合分析
发布时间：2018-09-18 14:31
【摘要】：长白猪和蓝塘猪产肉量、生长速度明显不同,且从胚胎期肌肉发育到出生后肌肉肥大的不同阶段两者在肌纤维发育程度与表型均有很大的差异。本研究选择长白猪和蓝塘猪胚胎期35、49、63、77天与出生后2、28、90、180天等8个不同时期的背最长肌进行Solexa测序,通过比较两个猪种在肌肉发育过程中miRNA的表达差异,结合靶基因预测和转录组数据,对骨骼肌发育的机制进行研究,并筛选成肌候选miRNA进行功能验证。结果表明:1.出生前后差异miRNAs(DE-miRNAs)表达模式不同通过比较8个不同时期长白猪与蓝塘猪的miRNA表达谱,我们发现了257个miRNA,其中差异表达的miRNA(DE-miRNA)152个。主成分分析(PCA)将DE-miRNAs清晰的分为出生前后两个部分,说明出生前后miRNA表达模式存在差异。因此,我们将8个不同时期分为出生前后2组,分别命名为G1、G2。挑选G1、G2组中高丰度表达量的miRNAs进行热图分析,结果发现超过2/3的miRNA在品种间存在表达差异,在G1组,蓝塘猪的miRNA表达量明显高于长白猪,尤其是胚胎期35、49天;在G2组,长白猪的miRNA表达量要高于蓝塘猪。此外,与成肌相关的miR-1、miR-206和miR-133在G1、G2组中品种间差异明显。2.不同品种同一时期miRNAs表达模式不同在G1、G2组中每个时期分别挑选10个高丰度表达的DE-miRNAs,将其分为G1top(up)、G1top(down)、G2top(up)、G2top(down)等4个亚组进行比较,发现胚胎期35、49、63天和出生后2天高丰度DE-miRNAs的表达量在品种间存在较大差异。在G1top(up)亚组,胚胎期49天miR-378、mi R-30a、miR-148a和miR-127等品种间表达差异较大,胚胎期49、63、77天mi R-206在长白猪中表达量明显高于蓝塘猪。在G1top(down)亚组,胚胎期35、49、63天mi R-20、miR-499、miR-451和miR-335等miRNA品种间表达差异较大。在G2top(up)亚组,出生后2天miR-148a在长白猪中的表达量明显高于蓝塘猪。在G2top(down)亚组,品种间miRNAs表达差异不明显。3.DE-miRNAs与转录组整合分析为了研究DE-miRNA调控肌肉发育过程的分子机制,我们将全部DE-miRNA分为G1all(up)、G1all(down)、G2all(up)和G2all(down)等4个亚组进行韦恩图绘制,发现G1all(down)和G2all(up)组中DE-miRNAs的数量占全部DE-miRNAs数量的88%。对G1all(down)亚组的miRNAs进行聚类分析,发现主要的生物过程是肌肉发育、肌肉分化;对G2all(up)亚组的miRNAs进行聚类分析,发现主要的生物过程是肌肉系统的过程、肌纤维肥大,该结果与胚胎期主要进行肌纤维发育而出生后主要进行肌纤维肥大的表型吻合。采用Targetscan预测G1all(down)、G2all(up)亚组中成肌相关的miRNAs靶基因,用STEM软件分析靶基因在长白猪和蓝塘猪出生前后的表达趋势图,共计获得26个趋势图,其中有9个趋势图显著富集,涵盖138个靶基因。用STRING网站预测138个靶基因的互作关系,并使用Cytoscape软件进行制图,最终形成由22个基因和35个miRNAs组成的互作网络图。网络图中包括已知的miR-206、mi R-133b、miR135-5p、MEF2A、MEF2C等成肌相关miRNAs和基因,MYH7、TPM2、DES等肌纤维形成相关基因。筛选出的未知功能的miRNA或者基因可为今后研究成肌调控机制提供数据。4.成肌相关的DE-miRNA(miR-127、miR-30a)功能验证为了验证生物信息学预测的miRNA在肌肉发育过程中的作用,本研究挑选了miR-127、miR-30a进行功能验证。结果表明,miR-127过表达后,处于S期的细胞显著增加,进入G2/M期的细胞显著减少,同时,细胞中与细胞周期相关因子cyclinA2、cyclinE2、CDK4的表达量显著降低,cyclin-CDK复合物的抑制因子P21显著增加,说明转染miR-127后,C2C12细胞的分裂能力减弱。双荧光报告实验表明miR-127通过作用于SEPT7基因3’UTR区,从而抑制细胞的增殖。miR-30a过表达后,处于G1期的细胞减少,而S期和G2/M期的细胞增加,与之相对应的细胞周期蛋白cyclinA2及蛋白激酶CDK4的表达量也相应的上升,而抑制细胞周期的肿瘤抑制基因Rb的表达量下降,说明转染miR-30a mimic后,C2C12细胞的增殖能力增强。本研究结果表明,品种间miRNA的表达差异在出生前大于出生后,其中蓝塘猪胚胎期35、49天上调的miRNA通过促进成肌细胞过早分化,最终导致产肉量减少;出生后长白猪上调的miRNA促进肌肥大,从而增加产肉量。论文筛选得到miR-378、miR-127、miR-30a等8个成肌相关候选mi RNA,并对miR-127、miR-30a进行了功能验证,发现miR-127作用于SEPT7基因3’UTR区抑制C2C12细胞的增殖,miR-30a促进C2C12细胞的增殖。
[Abstract]:The meat output and growth rate of Landrace * * and blue pond pigs are significantly different. The developmental degree and phenotype of the muscle fiber are different from the muscle development of embryo to the stage of postnatal muscle hypertrophy. * the longest length of 35,49,63,77 period and 8 days after birth 2,28,90180 in the Landrace and blue pond pigs were selected. Muscle Solexa sequencing * by comparing the miRNA expression differences between two pig breeds during muscle development, combined with target gene prediction and transcriptome data, the mechanism of skeletal muscle development was studied, and the myogenic candidate miRNA was screened for functional verification. The results showed that: 1. the difference of miRNAs (DE-miRNAs) expression pattern before and after birth was different by 8. MiRNA expression profiles of Landrace and * * blue pig in different periods were found. We found 257 miRNA, of which 152 were differentially expressed miRNA (DE-miRNA). Principal component analysis (PCA) clearly divided DE-miRNAs into two parts before and after birth, indicating that there were differences in miRNA expression pattern before and after birth. Therefore, we divided 8 different periods into 2 groups before and after birth. They were named G1, G2. selected G1, and miRNAs with high abundance expression in G2 group were analyzed by thermography. It was found that there were differences in miRNA expression among varieties. MiRNA in G1 group was significantly higher than that in Landrace * * *, especially in embryonic period. In the G2 group, the expression level of the expression of HLA in the Landrace was higher than that in the blue pond. Relevant microRNAs-1, microRNA206 and microRNA133 were significantly different among G1 and G2 groups. 2. The expression patterns of microRNAs in G1 and G2 groups at the same time were different. Ten highly expressed DE-microRNAs were selected and divided into G1top (up), G1top (down), G2top (up), G2top (down), G2top (down) and four subgroups. The embryonic period was 35, 49, 63 days and G2top (down). There was a great difference in the expression of high abundance of DE-miRNAs between 2 days after birth. In G1top (up) subgroup, there was a great difference in the expression of miR-378, MI R-30a, miR-148a and miR-127 during the embryonic period of 49 days. The expression of 49,63,77 * mi * in the Landrace was significantly higher than that in the blue pond. -499, miR-451 and miR-335 showed significant differences in miRNA expression. In G2top (up) subgroup, miR-148a expression in Landrace pigs was significantly higher than that in blue * * pigs on the 2 day after birth. In G2top (down) subgroup, miRNAs expression was not significantly different among varieties..3.DE-miRNAs and transcriptome integration analysis was used to study the molecular mechanism of the regulation of muscle development by down. We divided all DE-microNAs into four subgroups, G1all (up), G1all (down), G2all (up) and G2all (down), and drew the Wayne map. We found that the number of DE-microNAs in G1all (down) and G2all (up) groups accounted for 88% of the total number of DE-microNAs. Cluster analysis of microRNAs in G2all (up) subgroup revealed that the main biological process was muscle system hypertrophy, which was consistent with the phenotype of muscle fiber hypertrophy in embryonic stage and in postnatal stage. Using STEM software to analyze the expression pattern of target genes in Landrace and * * blue pig before and after birth, a total of 26 trend maps were obtained. 9 trend maps were significantly enriched, covering 138 target genes. The interaction of 138 target genes was predicted by STRING website and charting by Cytoscape software, resulting in the formation of 22 genes and 35 miRNAs. The network diagram includes known microRNAs and genes related to myofibrillar formation such as microRNAs-206, microR-133b, microRNA135-5p, MEF2A, and MEF2C, and genes related to myofibrillar formation such as MYH7, TPM2, DES. (a) Functional validation To verify the role of microRNAs predicted by bioinformatics in muscle development, this study selected microRNAs - 127 and microRNAs - 30A for functional validation. The expression of E2 and CDK4 decreased significantly, and the inhibitor P21 of cyclin-CDK complex increased significantly, suggesting that the mitotic ability of C2C12 cells decreased after transfection of miR-127. Dual fluorescence assay showed that microwave-127 inhibited cell proliferation by acting on the 3'UTR region of SEPT7 gene. The expression of cyclin A2 and protein kinase CDK4 increased with the increase of cell stage, while the expression of tumor suppressor gene Rb decreased, which indicated that the proliferation of C2C12 cells was enhanced after transfection of microRNAs-30a mimic. Before the birth, the miRNA of 35,49 during the embryonic period of * * * blue pig promotes the premature differentiation of myoblasts, resulting in a reduction in meat production. The up regulation of miRNA in post born Landrace promotes muscle hypertrophy and thus increases meat production. The paper screened 8 RNA related RNA MI, RNA, miR-378, miR-127, miR-30a and miR-127. Functional tests showed that the 3'UTR region of SEPT7 gene inhibited the proliferation of C2C12 cells and the proliferation of C2C12 cells was stimulated by microwave irradiation.
【学位授予单位】：中山大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S828
[Abstract]:The meat output and growth rate of Landrace * * and blue pond pigs are significantly different. The developmental degree and phenotype of the muscle fiber are different from the muscle development of embryo to the stage of postnatal muscle hypertrophy. * the longest length of 35,49,63,77 period and 8 days after birth 2,28,90180 in the Landrace and blue pond pigs were selected. Muscle Solexa sequencing * by comparing the miRNA expression differences between two pig breeds during muscle development, combined with target gene prediction and transcriptome data, the mechanism of skeletal muscle development was studied, and the myogenic candidate miRNA was screened for functional verification. The results showed that: 1. the difference of miRNAs (DE-miRNAs) expression pattern before and after birth was different by 8. MiRNA expression profiles of Landrace and * * blue pig in different periods were found. We found 257 miRNA, of which 152 were differentially expressed miRNA (DE-miRNA). Principal component analysis (PCA) clearly divided DE-miRNAs into two parts before and after birth, indicating that there were differences in miRNA expression pattern before and after birth. Therefore, we divided 8 different periods into 2 groups before and after birth. They were named G1, G2. selected G1, and miRNAs with high abundance expression in G2 group were analyzed by thermography. It was found that there were differences in miRNA expression among varieties. MiRNA in G1 group was significantly higher than that in Landrace * * *, especially in embryonic period. In the G2 group, the expression level of the expression of HLA in the Landrace was higher than that in the blue pond. Relevant microRNAs-1, microRNA206 and microRNA133 were significantly different among G1 and G2 groups. 2. The expression patterns of microRNAs in G1 and G2 groups at the same time were different. Ten highly expressed DE-microRNAs were selected and divided into G1top (up), G1top (down), G2top (up), G2top (down), G2top (down) and four subgroups. The embryonic period was 35, 49, 63 days and G2top (down). There was a great difference in the expression of high abundance of DE-miRNAs between 2 days after birth. In G1top (up) subgroup, there was a great difference in the expression of miR-378, MI R-30a, miR-148a and miR-127 during the embryonic period of 49 days. The expression of 49,63,77 * mi * in the Landrace was significantly higher than that in the blue pond. -499, miR-451 and miR-335 showed significant differences in miRNA expression. In G2top (up) subgroup, miR-148a expression in Landrace pigs was significantly higher than that in blue * * pigs on the 2 day after birth. In G2top (down) subgroup, miRNAs expression was not significantly different among varieties..3.DE-miRNAs and transcriptome integration analysis was used to study the molecular mechanism of the regulation of muscle development by down. We divided all DE-microNAs into four subgroups, G1all (up), G1all (down), G2all (up) and G2all (down), and drew the Wayne map. We found that the number of DE-microNAs in G1all (down) and G2all (up) groups accounted for 88% of the total number of DE-microNAs. Cluster analysis of microRNAs in G2all (up) subgroup revealed that the main biological process was muscle system hypertrophy, which was consistent with the phenotype of muscle fiber hypertrophy in embryonic stage and in postnatal stage. Using STEM software to analyze the expression pattern of target genes in Landrace and * * blue pig before and after birth, a total of 26 trend maps were obtained. 9 trend maps were significantly enriched, covering 138 target genes. The interaction of 138 target genes was predicted by STRING website and charting by Cytoscape software, resulting in the formation of 22 genes and 35 miRNAs. The network diagram includes known microRNAs and genes related to myofibrillar formation such as microRNAs-206, microR-133b, microRNA135-5p, MEF2A, and MEF2C, and genes related to myofibrillar formation such as MYH7, TPM2, DES. (a) Functional validation To verify the role of microRNAs predicted by bioinformatics in muscle development, this study selected microRNAs - 127 and microRNAs - 30A for functional validation. The expression of E2 and CDK4 decreased significantly, and the inhibitor P21 of cyclin-CDK complex increased significantly, suggesting that the mitotic ability of C2C12 cells decreased after transfection of miR-127. Dual fluorescence assay showed that microwave-127 inhibited cell proliferation by acting on the 3'UTR region of SEPT7 gene. The expression of cyclin A2 and protein kinase CDK4 increased with the increase of cell stage, while the expression of tumor suppressor gene Rb decreased, which indicated that the proliferation of C2C12 cells was enhanced after transfection of microRNAs-30a mimic. Before the birth, the miRNA of 35,49 during the embryonic period of * * * blue pig promotes the premature differentiation of myoblasts, resulting in a reduction in meat production. The up regulation of miRNA in post born Landrace promotes muscle hypertrophy and thus increases meat production. The paper screened 8 RNA related RNA MI, RNA, miR-378, miR-127, miR-30a and miR-127. Functional tests showed that the 3'UTR region of SEPT7 gene inhibited the proliferation of C2C12 cells and the proliferation of C2C12 cells was stimulated by microwave irradiation.
【学位授予单位】：中山大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S828
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