Study On The Regulation Of Mtr-miR156a On Milk Protein Synthesis By Targeting PPP2R5D And PIK3R2 In BMECs

The biosynthesis of milk protein affects milk quality.The aim of this study is to analyze the regulatory effect of alfalfa miR156a(mtr-miR156a)on milk protein synthesis in BMECs,and to provide technical support for improving milk protein production and theoretical basis for miRNA transboundary regulation.Firstly,the expression patterns of mtr-miR156a in alfalfa,whole blood and whey of dairy cows were explored by qRT-PCR and DHI milk composition determination.Secondly,CCK-8,qRT-PCR,EdU and flow cytometry were used to detect the effect of mtr-miR156a overexpression on the viability,proliferation and apoptosis of BMECs.Then the target genes of mtr-miR 156a were predicted and screened by Lianchuan Biological cloud platform,TargetScan and RNAhybrid online websites,and the target genes were identified by dual-luciferase assay.Finally,qRT-PCR and ELISA were used to detect the effects of overexpression of mtr-miR156a on the expression of milk protein synthesis related genes in PI3K-AKT/mTOR signaling pathway and the regulation of casein composition in BMECs.Main findings are as follows:(1)qRT-PCR results showed that mtr-miR156a can be detected in the stems and leaves of dried and fresh samples of Zhongmu NO.1 and Xinyan alfalfa.It was found that the expression of mtr-miR 156a in whole blood and whey of dairy cows were also correlated with the level of milk protein.Oxidation test verified that mtr-miR 156a is a plant exogenous miRNA.Therefore,mtr-miR156a may enter cows with alfalfa feeding and play a cross-border regulatory role in milk protein synthesis in cows.(2)CCK-8,qRT-PCR,EdU and flow cytometry results showed that the viability of BMECs was weakened after overexpression of mtr-miR156a.The expression of PCNA,CDK4,Cyclin D1 and Cyclin D2 were extremely significantly decreased(P<0.01).The proliferation of BMECs was inhibited and the apoptosis was promoted.miRNAs play regulatory roles by targeting mRNAs,so the target genes of mtr-miR 156a were predicted and identified.(3)It was found that mtr-miR156a specifically targeted the 3’UTR of PPP2R5D and PIK3R2 genes in BMECs by dual-luciferase assay.Further qRT-PCR results showed that PPP2R5D and PIK3R2 genes were extremely significantly inhibited after overexpression of mtr-miR156a(P<0.01),indicating that the target genes of mtr-miR156a were PPP2R5D and PIK3R2 genes.(4)After overexpression of mtr-miR156a,the mRNA expression levels of casein-related genes CSN1S1,CSN1S2,CSN2 and CSN3 were extremely significantly upregulated(P<0.01),and theα-casein,β-casein and κ-casein per milliliter in the cell culture medium supernatant were also significantly increased(P<0.05).These results indicated that mtr-miR156a could promote casein synthesis in BMECs.In conclusion,mtr-miR156a can reduce cell viability,inhibit cell proliferation and promote cell apoptosis in BMECs.The target genes of mtr-miR156a in BMECs are PPP2R5D and PIK3R2.The mtr-miR156a can bind to target genes,significantly upregulate the expression levels of AKT1 and eIF4EBP1 genes in PI3K/AKT-mTOR signaling pathway(P<0.01),and significantly downregulate the expression levels of PDK1,S6K1,EIF4B,RPS6,TSC2,RHEB,mTOR and eIF4E(P<0.01).The synthesis of α-casein,β-casein and κ-casein in BMECs can be promoted when mtr-miR156a was overexpressed.The results of the study reveals functions of mtr-miR156a in BMECs at the cellular level,and the regulatory effects of mtr-miR156a on milk protein synthesis of dairy cows at the mRNA and protein levels,which would provide a reference to improve milk protein of dairy cows by using alfalfa components efficiently in production.