Study On The Mechanism Of Bta-miR-34b Regulating Milk Fat Synthesis In Dairy Cow Mammary Epithelial Cells

Milk is known as the "full-nutrition food" which meets all human nutritional needs.Milk fat is not only the main energy component of milk,but also determines the physical characteristics,manufacturing characteristics and sensory qualities of dairy products.Therefore,how to increase the milk fat rate has already been a scientific problem which need to be solved urgently in the domestic and foreign dairy industries.Moreover,the study of gene networks that regulating the milk fat synthesis via the perspective of molecular biology has also become a research hotspot in this field.More and more studies have shown that miRNA can take a further control in a variety of biological processes including milk fat synthesis by firstly combining its target genes and then participating in downstream signaling pathways.In this study,miR-34b mimic/inhibitor were transfected into BMECs by liposome transfection method.Firstly,oil red O staining and TAG content analysis were used to determine the intracellular triglyceride content,RT-qPCR and Western-blot were used to detect the mRNA and protein expression levels of milk fat synthesis-related genes.Secondly,KEGG analysis was used to analyze the differential genes between two groups of BMECs which were transfected with miR-34b mimic and miR mimic NC.Then we screened the downstream pathways and determined the signaling pathway that may play a significant role in the process which miR-34b decreased milk fat synthesis in BEMCs,and used pathway agonists to conduct rescue experiments against miR-34b mimic in BMECs.Moreover,we used Target Scan to predict the target genes of miR-34band used dual luciferase reporter vector verified the prediction results.Finally,we transfected the siRNA of the target gene into BMECs and verified the activity level of the downstream pathway.The experimental results are as follows:(1)The results of oil red O staining and TAG content determination showed that miR-34b overexpression can inhibit triglyceride synthesis in BMECs,qPCR and Western blot results showed that miR-34b overexpression can inhibit the expression of milk fat synthesis-related genes at the mRNA and protein levels.Moreover,the inhibition of miR-34b led to the completely opposite results.(2)Transcriptome sequencing results indicated that the overexpression of miR-34b in BMECs caused 1999 genes up-regulated and 2009 genes down-regulated.And the KEGG analysis showed that the inhibitory effect of miR-34b on milk fat synthesis in BMECs may occur via the Akt/mTOR signaling pathway.(3)The results of RT-qPCR and Western blot showed that the overexpression of miR-34b can inhibit the activity level of the Akt/mTOR signaling pathway by inhibiting the phosphorylation level of the key genes Akt,mTOR,4E-BP1 and P70S6 K.(4)Treated BMECs with mTOR specific agonist MHY1485 and miR-34b mimic,the results of oil red O staining and TAG content determination showed that the inhibitory effect of miR-34b mimic on milk fat synthesis can be rescued by MHY1485.(5)Target Scan was used to predict the target mRNA of miR-34b,it was found that RAI14 has a miR-34b binding site in its 3’-UTR,and the dual luciferase report assay confirmed that RAI14 is the target gene of miR-34b.(6)The knockdown of RAI14 in BMECs can decrease the activity level of Akt/mTOR signaling pathway,which showed that RAI14 is an important mediator between miR-34b and its downstream Akt/mTOR signaling pathway.In summary,miR-34b inhibits milk fat synthesis by targeting the RAI14-Akt/mTOR signaling pathway in BMECs,and miR-34b may become a biomarker and potential therapeutic target for regulating milk fat synthesis in the future.What’s more,this study provides an important reference for improving the production of beneficial milk components in dairy cows.