Screening And Identification Of MiRNA In The Maternal Luteal Abnormalities In Cloned Cattle

Mi RNA is a kind of small non-coding endogenous post-transcriptional regulatory RNA that can inhibit translation of target genes or directly cause degradation thereto by acting on 3?-UTR of the target genes to realize the purpose of regulation of the target genes.According to the statistics,mi RNA can regulate near 1/3 of protein coding m RNA of human genome.For this reason,discovery,identification and functional identification of mi RNAs are currently research hotspots in the field of molecular biology.Somatic cell cloning technology refers to a technique of transferring an animal's somatic cell nuclei into enucleated oocyte to form a reconstructed embryo which eventually develops into a complete individual.The early pregnancy rate of cloned cows is relatively high,but with the occasional occurrence of abortion and abnormal pregnancy?abdominal circumference is too large?,cloning efficiency is relatively low.By employing high-throughput sequencing technology,this experiment aims at analyzing differentially expressed mi RNAs in the maternal corpus luteum of abnormal pregnant somatic cell cloned cows and the normal pregnant cows,screening and identifying mi RNAs with significant difference in expression,to obtain candidate target genes related to pregnancy maintenance.In addition,this experiment also has preliminarily established a regulatory network for interaction between target genes and mi RNAs associated with pregnancy maintenance,to understand the regulatory signaling pathways of abnormal pregnancy.In this study,two abnormal cloned cows that had been pregnant for 180 days were selected as the Experimental Group,and three normal cows that had been pregnant for 180 days were selected as the Control Group to collect their corpus luteum respectively.By using the Illumina sequencing platform,2069 mi RNAs were identified and 1759 mi RNAs were differentially expressed,of which 11 were significantly upregulated and 22 were significantly downregulated.SOAP target gene analysis software was utilized to predict 5,746 target genes,and the result was that mi RNA target genes were enriched in the KEGG pathway and GO signaling pathway.Analysis found that hsa-mi R-874-5p,hsa-mi R-152-5p,bta-mi R-495,PC-5p-35049₁3 and PC-3p-41396₁0 interacted with Bax,Caspase-9,TP53,AIF,Caspase-3,Cyto c,Apaf-1,and ING2 target genes.By regulating the apoptotic pathway in the corpus luteum,abnormal level of hormone secretion was induced,thereby affecting the maintenance of the cows' pregnancy.Nine significantly differentially expressed mi RNAs with high expression quantity were selected to identify the expression level of the mi RNAs in the somatic cell cloning group and the normal pregnant group by fluorescent quantitative PCR.The results showed that 6 mi RNAs were identical with the sequencing results and 3 were inconsistent with the sequencing results.11 related genes in the apoptotic pathway were selected to detect the changes of its expression quantity by fluorescent quantitative PCR,by which the action mechanism of the apoptotic pathway in the corpus luteum when controlling cow pregnancy was preliminarily determined.This study found that there are a large number of differentially expressed mi RNAs in the Experimental Group and the Control Group.These abnormally expressed mi RNAs may be related to the abnormal pregnancy of the Experimental Group.Further identification of the expression levels of related target genes revealed that some mi RNAs may have a regulatory effect on the apoptotic pathway in the corpus luteum,which has laid a foundation for subsequent functional identification of mi RNAs and exploration of regulatory mechanism involved in the pregnancy maintenance in corpus luteum.