异常黏液质证候大鼠阴茎组织差异性表达microRNA的筛选及生物信息学分析
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摘要
目的筛选异常黏液质证候及药物干预大鼠阴茎组织差异表达microRNA(miRNA),并验证miR-140-3p和miR-486两项关键指标,探讨其生物学意义。方法取80只性功能正常SD雄性大鼠,从中随机抽取10只大鼠设为正常对照组,余70只为造模组,用芫荽实+菠菜实+湿寒性环境的干预条件建立异常黏液质证候模型,通过生物学表征确认模型成功后,随机分为证候模型组、证候药物组。伊木萨克片干预3周后,提取阴茎组织总RNA进行miRNA Illumina高通量测序,筛选出异常黏液质证候及药物干预相关的候选差异表达miRNA,实时荧光定量PCR验证测序结果的可靠性,并进行生物信息学分析。结果筛选出证候相关miRNA候选标志物15个(上调13个,下调2个),伊木萨克片干预相关miRNA候选靶点标志物3个(均下调),验证结果显示miR-486在证候模型组较正常对照组表达升高(P<0.05),miR-486和miR-140-3p在证候药物干预组较证候模型组表达降低(P<0.05)。结论miR-486可作为异常黏液质证标志物,miR-140-3p、miR-486可作为伊木萨克片靶点标志物,但仍需大样本量验证。
Objective To select the differently expressed microRNAs(miRNA) on abnormal Phlegmatic syndrome and it's medicine intervention rats' s penile tissue, validate miR-486 and miR-140-3p, discuss the significance of biology of miR-486 and miR-140-3p. Methods Eighty healthy mature male SD rats were enrolled in this study, 10 of them were taken randomly as the control group, the other rats(70) were in the model group. Animal models of abnormal phlegmatic syndrome were established by treated with spinach and coriander diet in the cold humid environment. Biological characterization of rats were observed weekly until the models were successfully established. The rats were randomly divided into abnormal phlegmatic syndrome model group and abnormal phlegmatic syndrome model's medicine intervention group. Counterevidence was performed for 3 weeks, then penis tissue of the rats was collected to extract total RNA, and then subjected to Illumina Hiseq technology for screening differentially expressed miRNA, and then cluster analysis was performed, the candidate miRNA was selected and validated by q PCR. Results Total of 15 differentially expressed miRNAs related to Phlegmatic syndrome were identified, including 13 up-regulated miRNAs and 2 down-regulated miRNA; Three downregulation miRNAs related to target of medicine intervention were inversely regulated by Yimusake tablets treatment. The results of RT-q PCR showed that compare to control group, miR-486 was up-regulated in abnormal phlegmatic syndrome model group(P <0.05), miR-486 and miR-140-3p were down-regulated in abnormal phlegmatic syndrome model's medicine intervention group as compared to the abnormal phlegmatic syndrome model group(P <0.05). Conclusion miR-486 may be associated with the development of abnormal Phlegmatic syndrome. miR-140-3p, miR-486 may be as targets of Yimusake intervention.
Objective To select the differently expressed microRNAs(miRNA) on abnormal Phlegmatic syndrome and it's medicine intervention rats' s penile tissue, validate miR-486 and miR-140-3p, discuss the significance of biology of miR-486 and miR-140-3p. Methods Eighty healthy mature male SD rats were enrolled in this study, 10 of them were taken randomly as the control group, the other rats(70) were in the model group. Animal models of abnormal phlegmatic syndrome were established by treated with spinach and coriander diet in the cold humid environment. Biological characterization of rats were observed weekly until the models were successfully established. The rats were randomly divided into abnormal phlegmatic syndrome model group and abnormal phlegmatic syndrome model's medicine intervention group. Counterevidence was performed for 3 weeks, then penis tissue of the rats was collected to extract total RNA, and then subjected to Illumina Hiseq technology for screening differentially expressed miRNA, and then cluster analysis was performed, the candidate miRNA was selected and validated by q PCR. Results Total of 15 differentially expressed miRNAs related to Phlegmatic syndrome were identified, including 13 up-regulated miRNAs and 2 down-regulated miRNA; Three downregulation miRNAs related to target of medicine intervention were inversely regulated by Yimusake tablets treatment. The results of RT-q PCR showed that compare to control group, miR-486 was up-regulated in abnormal phlegmatic syndrome model group(P <0.05), miR-486 and miR-140-3p were down-regulated in abnormal phlegmatic syndrome model's medicine intervention group as compared to the abnormal phlegmatic syndrome model group(P <0.05). Conclusion miR-486 may be associated with the development of abnormal Phlegmatic syndrome. miR-140-3p, miR-486 may be as targets of Yimusake intervention.
引文
1阿地力江·伊明,范强,潘建春,等.维医异常黏液质阳痿病证动物模型的建立.中国男科学杂志2011;25(9):3-9
2 阿地力江·伊明,范强,哈木拉提·吾甫尔,等.维医异常黏液质证候与阳痿病证大鼠性行为学改变的研究.中国男科学杂志2011;25(9):20-23,28
3 张盼盼,刘凤霞,阿地力江·伊明,等.异常黏液质型阳痿病证大鼠性腺轴改变的形态学研究.中国男科学杂志2014;28(7):8-13
4 阿地力江·伊明,潘建春,哈木拉提·吾甫尔,等.维医异常黏液质证候与阳痿病证大鼠模型生殖激素的改变及其生物学意义.新疆医科大学学报2012;35(11):1438-1444
5 朱彦刘莹,买买提祖农·买苏尔,刘凤霞,等.伊木萨克片对异常黏液质型阳痿病证模型大鼠阴茎组织形态学的影响.中国男科学杂志2015;29(6):9-14
6 毛吾兰·买买提依明,张盼盼,刘凤霞,等.维医异常黏液质型阳痿病证大鼠模型阴茎组织中ET-1的改变及其生物学意义.新疆医科大学学报2016;39(5):535-537
7 刘凤霞,腊博雅,刘琪,等.Rho A/Rho激酶在异常黏液质证候及阳痿病证大鼠阴茎中的表达及伊木萨克片干预的研究.中国男科学杂志2016;30(9):5-9
8 Small EM,O'Rourke JR,Moresi V,et al.Regulation of P I3-kinase/Aktsignaling by muscle-enriched microRNA-486.Proc Natl Acad Sci USA 2010;107(9):4218-4223
9 Hitachi K,Nakatani M,Tsuchida K.Myostatin signaling regulates Akt activity via the regulation of miR-486expression.Int J Biochem Cell Biol 2014;47:93-103
10 曾静波,李玉秀,孙琦,等.罗格列酮和二甲双胍治疗对胰岛素抵抗KKAy糖尿病小鼠肝及横纹肌组织PTEN蛋白表达的影响.中华老年多器官疾病杂志2010;9(6):529-532
11 康英秀,高鑫,等.PTEN与胰岛素抵抗.国际内分泌代谢杂志2008;28(2):96-98
12 周霞,徐可树.转化生长因子-β3对肝脏和胰腺纤维化的影响.世界华人消化杂志2007;15(28):30-35
13 陈超,武德梅.转化生长因子-β3与肾脏纤维化的关系.医学综述2014;20(11):24-25
14 买买提祖农·买苏尔,朱彦刘莹,刘凤霞,等.异常黏液质证候大鼠模型阴茎组织形态学改变的研究.新疆医科大学学报2015;38(6):687-695
2 阿地力江·伊明,范强,哈木拉提·吾甫尔,等.维医异常黏液质证候与阳痿病证大鼠性行为学改变的研究.中国男科学杂志2011;25(9):20-23,28
3 张盼盼,刘凤霞,阿地力江·伊明,等.异常黏液质型阳痿病证大鼠性腺轴改变的形态学研究.中国男科学杂志2014;28(7):8-13
4 阿地力江·伊明,潘建春,哈木拉提·吾甫尔,等.维医异常黏液质证候与阳痿病证大鼠模型生殖激素的改变及其生物学意义.新疆医科大学学报2012;35(11):1438-1444
5 朱彦刘莹,买买提祖农·买苏尔,刘凤霞,等.伊木萨克片对异常黏液质型阳痿病证模型大鼠阴茎组织形态学的影响.中国男科学杂志2015;29(6):9-14
6 毛吾兰·买买提依明,张盼盼,刘凤霞,等.维医异常黏液质型阳痿病证大鼠模型阴茎组织中ET-1的改变及其生物学意义.新疆医科大学学报2016;39(5):535-537
7 刘凤霞,腊博雅,刘琪,等.Rho A/Rho激酶在异常黏液质证候及阳痿病证大鼠阴茎中的表达及伊木萨克片干预的研究.中国男科学杂志2016;30(9):5-9
8 Small EM,O'Rourke JR,Moresi V,et al.Regulation of P I3-kinase/Aktsignaling by muscle-enriched microRNA-486.Proc Natl Acad Sci USA 2010;107(9):4218-4223
9 Hitachi K,Nakatani M,Tsuchida K.Myostatin signaling regulates Akt activity via the regulation of miR-486expression.Int J Biochem Cell Biol 2014;47:93-103
10 曾静波,李玉秀,孙琦,等.罗格列酮和二甲双胍治疗对胰岛素抵抗KKAy糖尿病小鼠肝及横纹肌组织PTEN蛋白表达的影响.中华老年多器官疾病杂志2010;9(6):529-532
11 康英秀,高鑫,等.PTEN与胰岛素抵抗.国际内分泌代谢杂志2008;28(2):96-98
12 周霞,徐可树.转化生长因子-β3对肝脏和胰腺纤维化的影响.世界华人消化杂志2007;15(28):30-35
13 陈超,武德梅.转化生长因子-β3与肾脏纤维化的关系.医学综述2014;20(11):24-25
14 买买提祖农·买苏尔,朱彦刘莹,刘凤霞,等.异常黏液质证候大鼠模型阴茎组织形态学改变的研究.新疆医科大学学报2015;38(6):687-695