【摘要】 旨在通过探究bta-miRNA-31与其靶基因RASA1(Ras GTP酶活化蛋白1,Ras GTPase-activating protein 1)对奶牛母体胎盘组织胶原蛋白降解的影响，进而阐明bta-miRNA-31可通过靶向RASA1影响胎衣不下奶牛发生的调控作用与机制。依据产后12 h内胎衣能否正常排出作为胎衣不下组（retained fetal membranes,RFM）奶牛和健康组（NRFM）奶牛的判定标准，选择胎衣不下组奶牛与健康组奶牛各6头。采用茎环法实时荧光定量PCR检测胎衣不下组与健康组奶牛母体胎盘组织bta-miRNA-31表达水平，采用qRT-PCR和Western blot方法检测两组奶牛母体胎盘组织中RASA1、MMP-2、MMP-9、COL-Ⅲ、COL-Ⅳ的mRNA及蛋白表达水平。同时运用生物信息学分析预测bta-miRNA-31的靶基因，并通过双荧光素酶试验验证其与RASA1的靶向关系。分别过表达和沉默btamiRNA-31，采用茎环法实时荧光定量PCR检测细胞中奶牛子宫内膜上皮细胞中bta-miRNA-31的表达变化；采用qRT-PCR和Western blot检测细胞中RASA1、MMP-2、MMP-9、COL-Ⅲ、COL-Ⅳ的mRNA与蛋白表达水平的变化。采用免疫荧光技术检测COL-Ⅳ在各组细胞中的表达变化。结果显示：奶牛发生胎衣不下时，其母体胎盘组织中bta-miRNA-31 mRNA表达极显著升高（P<0.01）,RASA1、MMP-2、MMP-9的mRNA及蛋白表达极显著降低（P<0.01）,COL-Ⅲ、COL-Ⅳ的mRNA及蛋白表达极显著升高（P<0.01）。生物信息学分析显示，bta-miRNA-31有39个潜在靶基因，双荧光素酶试验证实RASA1与bta-miRNA-31存在靶向关系；进一步通过qRT-PCR和Western blot检测过表达/沉默bta-miRNA-31的细胞发现，过表达bta-miRNA-31可使靶基因RASA1及MMP-2、MMP-9的mRNA与蛋白表达极显著降低（P<0.01）,COL-Ⅲ、COL-Ⅳ的mRNA与蛋白表达极显著升高（P<0.01），沉默bta-miRNA-31则呈相反结果（P<0.01）；免疫荧光结果亦显示，与对照组相比，bta-miRNA-31 mimics组COL-Ⅳ表达极显著升高（P<0.01）,inhibitor组COL-Ⅳ表达极显著降低（P<0.01）。高水平的bta-miRNA-31可通过靶向调控RASA1影响降低MMPs的表达、胶原蛋白的降解减少从而引起细胞外基质降解障碍，是引起胎衣不下发生的重要因素。更多还原

【Abstract】 This study aimed to explore the effect of bta-miRNA-31 and its target gene RASA1(Ras GTPase-activating protein 1) on collagen degradation in the maternal placental tissue of dairy cows,thereby clarifying the regulatory role and mechanism by which bta-miRNA-31 contributes to retained fetal membranes(RFM).Dairy cows were classified as RFM or non-retained fetal membranes(NRFM) based on whether fetal membranes were expelled within 12 h postpartum(n=6 per group).The expression of bta-miRNA-31 in the maternal placental tissues was quantified by stem-loop quantitative real-time PCR.of cows in the RFM and NRFM groups.The mRNA and protein levels of RASA1,MMP-2,MMP-9,COL-Ⅲ,and COL-Ⅳ were determined by qRT-PCR and Western blot.Meanwhile,bioinformatics analysis was used to predict the target genes of bta-miRNA-31,and a dual-luciferase assay was conducted to verify its targeting relationship with RASA1.After overexpressing and silencing bta-miRNA-31 respectively,stem-loop quantitative real-time PCR was used to detect the expression changes of bta-miRNA-31 in bovine endometrial epithelial cells;qRTPCR and Western blot were used to detect the changes in the mRNA and protein levels of RASA1,MMP-2,MMP-9,COL-Ⅲ,and COL-Ⅳ in the cells.Immunofluorescence technology was used to detect the expression changes of COL-Ⅳ in cells of each group.In RFM cows,the mRNA expression of bta-miRNA-31 in maternal placental tissues was significantly increased(P<0.01),while the mRNA and protein levels of RASA1,MMP-2,and MMP-9 were markedly decreased(P<0.01),and the mRNA and protein lvels of COL-Ⅲ and COL-Ⅳ were highly increased(P<0.01).Bioinformatics analysis showed that bta-miRNA-31 had 39 potential target genes,and the dualluciferase assay confirmed that RASA1 had a targeting relationship with bta-miRNA-31.In vitro,overexpression of bta-miRNA-31 significantly reduced RASA1 as well as MMP-2 and MMP-9 at both mRNA and protein levels(P<0.01) while increasing COL-Ⅲ and COL-Ⅳ(P<0.01);the opposite changes were observed upon bta-miRNA-31 inhibition(P<0.01).Immunofluorescence results were concordant,showing markedly increased COL-Ⅳ in the bta-miRNA-31 mimic group and decreased COL-Ⅳ in the inhibitor group(both P<0.01) relative to controls.Elevated btamiRNA-31 contributes to RFM by targeting and downregulating RASA1,thereby suppressing MMP expression,reducing collagen degradation,and impairing extracellular matrix remodeling.更多还原