長白豬和藍塘豬肌肉發(fā)育差異miRNA和mRNA表達譜的整合分析
發(fā)布時間：2018-09-18 14:31
【摘要】：長白豬和藍塘豬產(chǎn)肉量、生長速度明顯不同,且從胚胎期肌肉發(fā)育到出生后肌肉肥大的不同階段兩者在肌纖維發(fā)育程度與表型均有很大的差異。本研究選擇長白豬和藍塘豬胚胎期35、49、63、77天與出生后2、28、90、180天等8個不同時期的背最長肌進行Solexa測序,通過比較兩個豬種在肌肉發(fā)育過程中miRNA的表達差異,結(jié)合靶基因預測和轉(zhuǎn)錄組數(shù)據(jù),對骨骼肌發(fā)育的機制進行研究,并篩選成肌候選miRNA進行功能驗證。結(jié)果表明:1.出生前后差異miRNAs(DE-miRNAs)表達模式不同通過比較8個不同時期長白豬與藍塘豬的miRNA表達譜,我們發(fā)現(xiàn)了257個miRNA,其中差異表達的miRNA(DE-miRNA)152個。主成分分析(PCA)將DE-miRNAs清晰的分為出生前后兩個部分,說明出生前后miRNA表達模式存在差異。因此,我們將8個不同時期分為出生前后2組,分別命名為G1、G2。挑選G1、G2組中高豐度表達量的miRNAs進行熱圖分析,結(jié)果發(fā)現(xiàn)超過2/3的miRNA在品種間存在表達差異,在G1組,藍塘豬的miRNA表達量明顯高于長白豬,尤其是胚胎期35、49天;在G2組,長白豬的miRNA表達量要高于藍塘豬。此外,與成肌相關(guān)的miR-1、miR-206和miR-133在G1、G2組中品種間差異明顯。2.不同品種同一時期miRNAs表達模式不同在G1、G2組中每個時期分別挑選10個高豐度表達的DE-miRNAs,將其分為G1top(up)、G1top(down)、G2top(up)、G2top(down)等4個亞組進行比較,發(fā)現(xiàn)胚胎期35、49、63天和出生后2天高豐度DE-miRNAs的表達量在品種間存在較大差異。在G1top(up)亞組,胚胎期49天miR-378、mi R-30a、miR-148a和miR-127等品種間表達差異較大,胚胎期49、63、77天mi R-206在長白豬中表達量明顯高于藍塘豬。在G1top(down)亞組,胚胎期35、49、63天mi R-20、miR-499、miR-451和miR-335等miRNA品種間表達差異較大。在G2top(up)亞組,出生后2天miR-148a在長白豬中的表達量明顯高于藍塘豬。在G2top(down)亞組,品種間miRNAs表達差異不明顯。3.DE-miRNAs與轉(zhuǎn)錄組整合分析為了研究DE-miRNA調(diào)控肌肉發(fā)育過程的分子機制,我們將全部DE-miRNA分為G1all(up)、G1all(down)、G2all(up)和G2all(down)等4個亞組進行韋恩圖繪制,發(fā)現(xiàn)G1all(down)和G2all(up)組中DE-miRNAs的數(shù)量占全部DE-miRNAs數(shù)量的88%。對G1all(down)亞組的miRNAs進行聚類分析,發(fā)現(xiàn)主要的生物過程是肌肉發(fā)育、肌肉分化;對G2all(up)亞組的miRNAs進行聚類分析,發(fā)現(xiàn)主要的生物過程是肌肉系統(tǒng)的過程、肌纖維肥大,該結(jié)果與胚胎期主要進行肌纖維發(fā)育而出生后主要進行肌纖維肥大的表型吻合。采用Targetscan預測G1all(down)、G2all(up)亞組中成肌相關(guān)的miRNAs靶基因,用STEM軟件分析靶基因在長白豬和藍塘豬出生前后的表達趨勢圖,共計獲得26個趨勢圖,其中有9個趨勢圖顯著富集,涵蓋138個靶基因。用STRING網(wǎng)站預測138個靶基因的互作關(guān)系,并使用Cytoscape軟件進行制圖,最終形成由22個基因和35個miRNAs組成的互作網(wǎng)絡圖。網(wǎng)絡圖中包括已知的miR-206、mi R-133b、miR135-5p、MEF2A、MEF2C等成肌相關(guān)miRNAs和基因,MYH7、TPM2、DES等肌纖維形成相關(guān)基因。篩選出的未知功能的miRNA或者基因可為今后研究成肌調(diào)控機制提供數(shù)據(jù)。4.成肌相關(guān)的DE-miRNA(miR-127、miR-30a)功能驗證為了驗證生物信息學預測的miRNA在肌肉發(fā)育過程中的作用,本研究挑選了miR-127、miR-30a進行功能驗證。結(jié)果表明,miR-127過表達后,處于S期的細胞顯著增加,進入G2/M期的細胞顯著減少,同時,細胞中與細胞周期相關(guān)因子cyclinA2、cyclinE2、CDK4的表達量顯著降低,cyclin-CDK復合物的抑制因子P21顯著增加,說明轉(zhuǎn)染miR-127后,C2C12細胞的分裂能力減弱。雙熒光報告實驗表明miR-127通過作用于SEPT7基因3’UTR區(qū),從而抑制細胞的增殖。miR-30a過表達后,處于G1期的細胞減少,而S期和G2/M期的細胞增加,與之相對應的細胞周期蛋白cyclinA2及蛋白激酶CDK4的表達量也相應的上升,而抑制細胞周期的腫瘤抑制基因Rb的表達量下降,說明轉(zhuǎn)染miR-30a mimic后,C2C12細胞的增殖能力增強。本研究結(jié)果表明,品種間miRNA的表達差異在出生前大于出生后,其中藍塘豬胚胎期35、49天上調(diào)的miRNA通過促進成肌細胞過早分化,最終導致產(chǎn)肉量減少;出生后長白豬上調(diào)的miRNA促進肌肥大,從而增加產(chǎn)肉量。論文篩選得到miR-378、miR-127、miR-30a等8個成肌相關(guān)候選mi RNA,并對miR-127、miR-30a進行了功能驗證,發(fā)現(xiàn)miR-127作用于SEPT7基因3’UTR區(qū)抑制C2C12細胞的增殖,miR-30a促進C2C12細胞的增殖。
[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.
【學位授予單位】：中山大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：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.
【學位授予單位】：中山大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S828
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