Research On The Mechanism Of MiR-574-5p On Milk Synthesis By CircRNA-miRNA-mRNA Regulatory Network In Goat Mammary Epithelial Cells

The lactation of dairy goats is closely related to the development of the mammary glands.Mammalian mammary glands undergo a series of biological procedures such as cell growth,proliferation,differentiation,and apoptosis.Accumulated studies suggest that miRNAs and circ RNAs,as non-coding RNAs,play a key role in mammary development and lactation performance by regulating post-transcriptional levels of target genes.Previous laboratory studies find that miR-574-5p expresses differentially and is up-regulated significantly in mammary glands of dairy goats compared colostrum with peak lactation,but its specific regulatory mechanism has not been reported.Therefore,this experiment overexpressed miR-574-5p in goat mammary epithelial cells(GMECs)to construct RNA libraries by RNA sequencing(RNA-Seq)technology.Downstream target m RNAs and upstream circ RNAs of miR-574-5p were predicted and screened by bioinformatics.The molecular mechanism and the relationship of circ RNA,miRNA and m RNA were detected by methods of double luciferase gene reporters,RT-q PCR,Western Blot,CCK-8,Ed U,flow cytometry,ELISA,interference,overexpression and co-expression.The circ RNA-miRNA-m RNA regulatory network facilitates further probes on the function of miR-574-5p in GMECs.The main results are as follows:1.RNA sequencing of GMECs after miR-574-5p overexpressionRNA libraries of GMECs after miR-574-5p overexpression were constructed by RNA sequencing,and high-quality transcriptome information was obtained.A total of 332 differentially expressed genes(DEGs)were screened,with 74 up-regulated and 258down-regulated genes.GO annotation and KEGG signal pathway analysis revealed that these DEGs were involved in multiple biological functions.The m RNA expression levels of10 randomly selected genes(MAP3K9,DGKI,DGKH,RPS6KA2,SEL1 L,ADCY3,EVI5 L,SVEP1,STON2,SCARA3)in miR-574-5p overexpresssion and negative control groups were detected by RT-q PCR and the results were consistent with the sequencing trends of RNA-Seq.2.Regulatory effects of miR-574-5p on goat mammary epithelial cellsmiR-574-5p expressed in heart,spleen,liver,lung,mammary gland,ovary and uterus tissues of dairy goats.In mammary gland,the expression of miR-574-5p during peak lactation was higher than colostrum.miR-574-5p inhibited cell proliferation and promoted cell apoptosis in GMECs.miR-574-5p restrained the secretion of β-casein and triglycerides,and down-regulated m RNA levels of milk synthesis-related genes(CSN2(β-casein),CSN1S2(as2-casein),CSN3(kappa-casein),JAK2,STAT5,mTOR,S6K1,4EBP1,SREBP1,PPARG,FASN and FABP3)in GMECs.3.Regulatory effects of miR-574-5p via MAP3K9 on goat mammary epithelial cellsMAP3K9 expressed in heart,spleen,liver,lung,mammary gland,ovary,and uterus tissues of dairy goats.Compared with colostrum,the expression of MAP3K9 was lower in mammary gland during peak lactation.miR-574-5p targeted the 3’UTR of MAP3K9,which was also a DEG,and inhibited its expression.miR-574-5p restrained the JNK-c-Jun-cyclin D1 pathway resulting in weakening cell proliferation,demotivated the ERK-RSK1-Bad and JNK/ERK-Bcl-2-Bax pathways accompanied by enhanced cell apoptosis via MAP3K9.miR-574-5p also inactivated the PI3K-AKT-mTOR and JNK-mTOR-S6K1/4EBP1 pathways via MAP3K9,further inhibited the secretion of β-casein and triglycerides in GMECs.4.Regulatory effects of miR-574-5p via AKT3 on goat mammary epithelial cellsAKT3 expressed in heart,spleen,liver,lung,mammary gland,ovary,and uterus tissues of dairy goats.Compared with colostrum,the expression of AKT3 was lower in mammary gland during peak lactation.miR-574-5p targeted the 3’UTR of AKT3 and inhibited its m RNA and protein expression.miR-574-5p inactivated the mTOR-S6K1/4EBP1 pathway via AKT3,further inhibited the secretion of β-casein and triglycerides in GMECs.5.Regulatory effects of circ-016910 via sponging miR-574-5p on goat mammary epithelial cellsBased on the constructed circ RNAs libraries in our laboratory,the regulation of circ RNAs and miR-574-5p was predicted through bioinformatics,and circ-016910 was screened.circ-016910 targeted miR-574-5p’s specific binding sites to suppress its expression,and promoted the m RNA and protein levels of miR-574-5p’s target genes,MAP3K9 and AKT3,which showed its “miRNA sponge” function.circ-016910 activated the JNK-c-Jun-cyclin D1 pathway resulting in enhanced cell proliferation,motivated the ERK-RSK1-Bad and JNK/ERK-Bcl-2-Bax pathways accompanied by attenuated cell apoptosis via miR-574-5p.In addition,circ-016910 activated the PI3K-AKT-mTOR and JNK-mTOR-S6K1 pathways through miR-574-5p,then induced the secretion of β-casein and triglycerides in GMECs.In summary,this experiment focused on miR-574-5p,which differentially expressed significantly in mammary gland compared colostrum with peak lactation,and then constructed a regulatory network of circ RNA-miRNA-m RNA on GMECs.The molecularregulatory mechanism of mammary gland development and milk synthesis was exposed,providing a new theoretical basis for improving lactation performance in dairy goat.