本文主要研究内容
作者彭术军（2019）在《circSNX29通过Wnt5a/Ca2+信号通路调控牛肌细胞增殖、分化的机制研究》一文中研究指出：肌生成是一个复杂而精确的过程,并受到一些非编码RNA和信号通路的高度调控。环状RNAs（circRNAs）是一类新型内源性非编码RNA,在绝大多数真核生物转录后水平发挥调节功能。虽然在多个物种和组织中已鉴定出一些cirRNAs,但其调控的分子机制仍不太明确。在本研究中,利用牛骨骼肌circRNAs测序数据,筛选并鉴定了一个来自宿主SNX29基因的候选circRNA,并将其命名为circSNX29,并进一步探究其在肌肉发育过程中的调控作用。超表达circSNX29促进成肌细胞分化,抑制细胞增殖,而干扰circSNX29抑制其分化,促进增殖。随后,利用RNAhybrid进行生物信息学预测发现,circSNX29可能吸附到miR-744,具有9个潜在的结合位点。利用双荧光素酶报告分析实验,结果显示,circSNX29能够直接与miR-744竞争性结合,并有效地扭转miR-744对Wnt5a和CaMKIIδ的抑制作用。重要的是,通过KEGG通路富集分析、Western blotting、钙离子荧光探针和CamKII活性检测等实验,结果发现,超表达Wnt5a和circSNX29通过增加CamKII激酶的活性和PKC的磷酸化水平激活非经典Wnt/Ca2+通路,进而调控牛成肌细胞的增殖分化。这些结果有助于进一步理解circRNAs和miRNAs在肌生成中的作用。主要研究结果如下:1.miR-744通过靶向抑制Wnt5a基因调控牛成肌细胞增殖和分化从前期秦川牛骨骼肌miRNAs测序数据中,我们随机筛选了10个miRNAs,RT-qPCR结果显示,miR-744在胎牛时期显著高于其他miRNAs,利用EdU、MTT、CCK-8、RT-qPCR、Immunofluorescence Staining及Western Blotting等技术研究miR-744对牛肌细胞的影响,研究发现,超表达miR-744可以增加成肌细胞增殖系数和marker基因CyclinD1、PCNA和CDK2的表达水平,暗示促进牛成肌细胞增殖。相反,干扰miR-744可以抑制牛成肌细胞增殖。而超表达miR-744显著降低牛成肌细胞分化因子MyoG、MyoD及MyHC的表达量,暗示可以抑制牛成肌细胞分化、肌管的产生。此外,双荧光素酶报告分析试验显示,miR-744通过调节Wnt5a基因的转录后水平促进牛成肌细胞增殖,抑制其分化。2.circSNX29作为竞争性内源性miRNA-744的海绵,减弱其对Wnt5a的抑制作用为了鉴定牛骨骼肌中特定的circRNAs,从circRNA测序数据中随机选择9个circRNAs,RT-qPCR结果显示,circRNA243在胚胎期的表达量远高于成年期。由于circRNA243是由SNX29基因转录而来,将其命名为circSNX29。通过在线软件RNAhybrid预测发现,circSNX29与miR-744可能有9个结合位点,而后,双荧光素酶报告系统试验进一步表明circSNX29与miR-744确实结合,且超表达circSNX29能显著降低miR-744的表达量。通过EdU、MTT、CCK-8、RT-qPCR、Immunofluorescence Staining及Western Blot等技术研究circSNX29对牛肌细胞增殖和分化的影响。结果显示,circSNX29能显著抑制牛肌细胞增殖,促进分化。而这些结果与超表达miR-744对细胞增殖及分化的影响恰好相反,同时,超表达circSNX29能增加miR-744靶基因Wnt5a的表达水平。因此,circSNX29通过吸附miR-744来减轻其对Wnt5a基因表达的调控作用,进而影响牛成肌细胞的增殖与分化。本研究揭示一个新的circSNX29可作为内源性miR-744的海绵,进而激活Wnt5a/Ca2+通路促进牛成肌细胞分化,这可能被用作调节肌细胞生成的潜在靶点。
Abstract
ji sheng cheng shi yi ge fu za er jing que de guo cheng ,bing shou dao yi xie fei bian ma RNAhe xin hao tong lu de gao du diao kong 。huan zhuang RNAs（circRNAs）shi yi lei xin xing nei yuan xing fei bian ma RNA,zai jue da duo shu zhen he sheng wu zhuai lu hou shui ping fa hui diao jie gong neng 。sui ran zai duo ge wu chong he zu zhi zhong yi jian ding chu yi xie cirRNAs,dan ji diao kong de fen zi ji zhi reng bu tai ming que 。zai ben yan jiu zhong ,li yong niu gu ge ji circRNAsce xu shu ju ,shai shua bing jian ding le yi ge lai zi su zhu SNX29ji yin de hou shua circRNA,bing jiang ji ming ming wei circSNX29,bing jin yi bu tan jiu ji zai ji rou fa yo guo cheng zhong de diao kong zuo yong 。chao biao da circSNX29cu jin cheng ji xi bao fen hua ,yi zhi xi bao zeng shi ,er gan rao circSNX29yi zhi ji fen hua ,cu jin zeng shi 。sui hou ,li yong RNAhybridjin hang sheng wu xin xi xue yu ce fa xian ,circSNX29ke neng xi fu dao miR-744,ju you 9ge qian zai de jie ge wei dian 。li yong shuang ying guang su mei bao gao fen xi shi yan ,jie guo xian shi ,circSNX29neng gou zhi jie yu miR-744jing zheng xing jie ge ,bing you xiao de niu zhuai miR-744dui Wnt5ahe CaMKIIδde yi zhi zuo yong 。chong yao de shi ,tong guo KEGGtong lu fu ji fen xi 、Western blotting、gai li zi ying guang tan zhen he CamKIIhuo xing jian ce deng shi yan ,jie guo fa xian ,chao biao da Wnt5ahe circSNX29tong guo zeng jia CamKIIji mei de huo xing he PKCde lin suan hua shui ping ji huo fei jing dian Wnt/Ca2+tong lu ,jin er diao kong niu cheng ji xi bao de zeng shi fen hua 。zhe xie jie guo you zhu yu jin yi bu li jie circRNAshe miRNAszai ji sheng cheng zhong de zuo yong 。zhu yao yan jiu jie guo ru xia :1.miR-744tong guo ba xiang yi zhi Wnt5aji yin diao kong niu cheng ji xi bao zeng shi he fen hua cong qian ji qin chuan niu gu ge ji miRNAsce xu shu ju zhong ,wo men sui ji shai shua le 10ge miRNAs,RT-qPCRjie guo xian shi ,miR-744zai tai niu shi ji xian zhe gao yu ji ta miRNAs,li yong EdU、MTT、CCK-8、RT-qPCR、Immunofluorescence Stainingji Western Blottingdeng ji shu yan jiu miR-744dui niu ji xi bao de ying xiang ,yan jiu fa xian ,chao biao da miR-744ke yi zeng jia cheng ji xi bao zeng shi ji shu he markerji yin CyclinD1、PCNAhe CDK2de biao da shui ping ,an shi cu jin niu cheng ji xi bao zeng shi 。xiang fan ,gan rao miR-744ke yi yi zhi niu cheng ji xi bao zeng shi 。er chao biao da miR-744xian zhe jiang di niu cheng ji xi bao fen hua yin zi MyoG、MyoDji MyHCde biao da liang ,an shi ke yi yi zhi niu cheng ji xi bao fen hua 、ji guan de chan sheng 。ci wai ,shuang ying guang su mei bao gao fen xi shi yan xian shi ,miR-744tong guo diao jie Wnt5aji yin de zhuai lu hou shui ping cu jin niu cheng ji xi bao zeng shi ,yi zhi ji fen hua 。2.circSNX29zuo wei jing zheng xing nei yuan xing miRNA-744de hai mian ,jian ruo ji dui Wnt5ade yi zhi zuo yong wei le jian ding niu gu ge ji zhong te ding de circRNAs,cong circRNAce xu shu ju zhong sui ji shua ze 9ge circRNAs,RT-qPCRjie guo xian shi ,circRNA243zai pei tai ji de biao da liang yuan gao yu cheng nian ji 。you yu circRNA243shi you SNX29ji yin zhuai lu er lai ,jiang ji ming ming wei circSNX29。tong guo zai xian ruan jian RNAhybridyu ce fa xian ,circSNX29yu miR-744ke neng you 9ge jie ge wei dian ,er hou ,shuang ying guang su mei bao gao ji tong shi yan jin yi bu biao ming circSNX29yu miR-744que shi jie ge ,ju chao biao da circSNX29neng xian zhe jiang di miR-744de biao da liang 。tong guo EdU、MTT、CCK-8、RT-qPCR、Immunofluorescence Stainingji Western Blotdeng ji shu yan jiu circSNX29dui niu ji xi bao zeng shi he fen hua de ying xiang 。jie guo xian shi ,circSNX29neng xian zhe yi zhi niu ji xi bao zeng shi ,cu jin fen hua 。er zhe xie jie guo yu chao biao da miR-744dui xi bao zeng shi ji fen hua de ying xiang qia hao xiang fan ,tong shi ,chao biao da circSNX29neng zeng jia miR-744ba ji yin Wnt5ade biao da shui ping 。yin ci ,circSNX29tong guo xi fu miR-744lai jian qing ji dui Wnt5aji yin biao da de diao kong zuo yong ,jin er ying xiang niu cheng ji xi bao de zeng shi yu fen hua 。ben yan jiu jie shi yi ge xin de circSNX29ke zuo wei nei yuan xing miR-744de hai mian ,jin er ji huo Wnt5a/Ca2+tong lu cu jin niu cheng ji xi bao fen hua ,zhe ke neng bei yong zuo diao jie ji xi bao sheng cheng de qian zai ba dian 。
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论文作者分别是来自西北农林科技大学的彭术军，发表于刊物西北农林科技大学2019-07-11论文，是一篇关于秦川牛论文,肌生成论文,西北农林科技大学2019-07-11论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自西北农林科技大学2019-07-11论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
标签：秦川牛论文; 肌生成论文; 西北农林科技大学2019-07-11论文;