Role And Mechanisms Of RNA Binding Protein MSI2 In Mastitis And Milk Fat Synthesis In Dairy Cows

The dairy industry is the pillar industry of animal husbandry.Milk and dairy products are rich in easy-to-absorb high-quality proteins,fats,vitamins,and minerals,which play an important role in improving the dietary structure and improving the health of the people.Mastitis is one of the most serious diseases in dairy cows,which seriously affects the lactation ability of cows and product quality and safety,and impairs production efficiency.Milk fat,as one of the three major nutrients in milk composition,provides a variety of essential fatty acids and fat-soluble vitamins required by the human body.Unsaturated fatty acids are a guarantee for the healthy development of the brain and immune system of embryos and infants.Therefore,an in-depth investigation of the causative factors of mastitis in dairy cows and its pathogenesis,and an analysis of the relevant molecular mechanisms of milk fat synthesis and fatty acid regulation are of great significance for safeguarding the quality and safety of dairy products,enhancing the nutritional value of milk,and improving the economic benefits of the dairy industry.RNA-binding proteins are widely involved in every stage of the RNA life cycle and play a regulatory role at the post-transcriptional level by binding to target genes to mediate RNA maturation,splicing,translocation,translation,etc.Musashi-2(MSI2),a highly conserved RNA-binding protein among mammals,can bind to target genes to mediate the regulation of post-transcriptional translation of RNA,affecting a wide range of biological processes.It has been found that MSI2 is closely associated with cancer development through the regulation of cellular epithelial-mesenchymal transition(EMT)and the inflammatory microenvironment.EMT and inflammation are the key pathological factors that promote mastitis,but so far,there is no report on the regulation of mastitis and milk fat synthesis by the RNA-binding protein MSI2.In this study,we used LPS to construct a mastitis model of bovine mammary epithelial cells(BMECs),established transcriptome differential gene expression profiles,and screened and identified MSI2,a responsive gene for mastitis in dairy cows;The RIP,RT-q PCR,WB,and ELISA were used,BMECs and mouse mastitis models as the research object,we clarified that MSI2 affects mastitis in dairy cows through activating the TGFβ signaling pathway,and analyze the interactions between MSI2 and TGFβR1;further in BMECs,through GC-MS and RIP experiments,we elucidated the regulation of different types of fatty acid synthesis by MSI2,and revealed the interactions between MSI2 and FASN m RNA,to finally comprehensively analyze the roles of MSI2 and its molecular mechanisms in dairy mastitis and milk fat synthesis.The results of the study are as follows.The specific results are as follows:1.Successfully constructed a mastitis model of BMECs and depicted differential gene expression profiles.310 differentially expressed genes were identified,of which286 were up-regulated and 24 were down-regulated;the differentially expressed genes were mainly enriched in inflammation-related signaling pathways,cytokine-cytokine receptor interactions,and chemokines and other related signaling pathways.Through differential gene screening and mammary tissue validation in mastitis cows,a new mastitis regulator,RNA binding protein MSI2,was unearthed.2.MSI2 was highly expressed in the mammary tissue of BMECs mastitis model and mastitis cows,and it could up-regulate the expression of inflammatory factors such as IL-6,IL-1β,and TNFα in BMECs to promote inflammation,and down-regulate the expression of tight junction proteins ZO-1,Occludin and Claudin-3 to disrupt the integrity of the mammary blood-milk barrier.3.MSI2 specifically binds to the 3’-UTR of TGFβR1 m RNA,an important receptor for TGFβ,activating the TGFβ/Smad signaling pathway and promoting inflammation;silencing MSI2 inhibits the stability and translation of m TGFβR1,suppressing the TGFβ signaling pathway and alleviating mastitis in cows.4.Successfully constructed a mouse mastitis model,and verified that in vivo silencing of MSI2 can inhibit the TGFβ signaling pathway,down-regulate the expression of inflammatory factors,and up-regulate the expression of tight junction proteins to alleviate mastitis.5.MSI2 was highly expressed in mammary tissues of dry-period cows;Overexpression of MSI2 in BMECs reduces the expression of milk fat synthesis genes FASN and SREBP1,decreases milk fat synthesis,silencing MSI2 promotes the production of unsaturated fatty acids,and increases the content and proportion of unsaturated fatty acids in milk fat.6.MSI2 specifically binds to MBE within the 3’-UTR of FASN m RNA and inhibits the expression of FASN;silencing MSI2 promotes the expression and extra-nuclear translocation of FASN,and plays a role in promoting milk fat synthesis.In summary,this study took the regulatory role of RNA-binding protein MSI2 in the mammary gland of dairy cows as the entry point and clarified the binding relationship of MSI2 with TGFβR1 and FASN.The molecular mechanisms by which MSI2 regulates mastitis in dairy cows by activating the TGFβ signaling pathway through binding to TGFβR1 and MSI2 regulates fatty acid synthesis through binding to FASN were systematically elucidated.The role and molecular mechanism of MSI2 in mastitis and milk fat synthesis in dairy cows were revealed.The results of this study provide a new idea for the study of the molecular regulation mechanism of mastitis and milk fat synthesis in dairy cows and provide a theoretical basis for ensuring the safety of dairy products,improving milk composition,and enhancing milk quality.