| Lactation is an important economic trait for dairy goats.The content of milk affects its nutritional value,process behavior,the quality of dairy products and the economic benefits of the industry.Lactoprotein is an important indicator for evaluating milk quality.In goat milk,casein is the main component of lactoprotein accounting for more than 80% of the total milk protein.There are four types of casein,including αs1-,αs2-,β-and κ-casein.Theαs1-casein accounts for 5.6% of the total casein in goat milk.As we all know,the αs1-casein is the main allergen protein in milk,which easily induces allergic reactions such as infant eczema,abdominal pain and diarrhea in infants and young children.92% of allergic infants suffer from a variety of allergic symptoms,which could cause anaphylaxis shock in severe cases and endanger their lives.It has been found that miRNAs could participate in regulating the translation of mRNA by targeting the 3′UTR region of mRNAs,thereby regulating cell proliferation and apoptosis.In recent years,many studies have found that miRNAs play an important regulatory role in mammary gland growth,development and lactation.In the previous study,miR-2284b was found to be differentially expressed during pre-lactation and peak lactation in dairy goats,but its regulatory mechanism on the growth and development of mammary epithelial cells and lactation is not clear.Using bioinformatics analysis,miR-2284b was found to have a targetable binding site to the 3′UTR region of the CSN1S1 gene,so it was hypothesized that miR-2284b could regulate the synthesis of αs1-casein.In the present study,mammary epithelial cells(GMECs)were treated as the studying material.On the basis of constructing the CHECK2 vector and pcDNA3.1 overexpression vector of CSN1S1 gene,synthesizing mimics of miR-2284b and si RNA-CSN1S1,the molecular mechanism of miR-2284b regulating the synthesis of αs1-casein was researched using dual luciferase activity detection system,EdU,CCK8 and ELISA to provide experimental evidence for building a precise and rapid method to improve the milk quality of dairy goats.The main experimental results were as follows:1.MiR-2284b regulates apoptosis of and synthesis of αs1-casein GMECsCCK8 and flow cytometry analysis showed that miR-2284b significantly inhibited the proliferation(P<0.05)and promoted apoptosis of GMECs.ELISA assay results indicated that miR-2284b highly significantly inhibited the secretion of αs1-casein by GMECs(P<0.01).These results indicated that miR-2284b could inhibit the synthesis of αs1-casein by GMECs and affect the quality of milk.2.Discovered two novel CDS region of CSN1S1Contrasted with the known NCBI sequences,sequence alignment analysis showed CSN1S1-1 mutated from T to C at site 92,from C to G at sites 108 and 274.Amino acid sequence analysis showed the change of leucine to proline at position 31 and glutamine to glutamate at position 92;CSN1S1-2,in addition to the changes at the above sites,also had the deletion of C at site 242 and the deletion of TGAGG sequence at 249～253 sites,resulting in the change of serine to cysteine at position 83,glutamate to glutamine at position 84,and two serine deletions at positions 81 and 82,in addition to the above amino acid changes.3.Effects of CSN1S1-1/-2 on proliferation and αs1-casein synthesis of GMECsBoth CCK8 and EdU results showed that overexpression of CSN1S1-1/-2 significantly promoted the proliferation of GMECs(P<0.05);flow cytometry analysis indicated that overexpression of CSN1S1-1/-2 inhibited the apoptosis of GMECs;CSN1S1-1 had no effect on the synthesis of αs1-casein in GMECs,while CSN1S1-2 extremely significantly inhibited the synthesis of αs1-casein in GMECs(P<0.01)by ELISA assay.4.MiR-2284b regulates αs1-casein synthesis in GMECs by inhibiting CSN1S1 geneDual luciferase reporter assay results showed that miR-2284b could target binding to the 3′UTR region of CSN1S1 gene;RT-q PCR results and ELISA kit assay indicated that miR-2284b could extremely significantly reduce the expression of CSN1S1 at mRNA and protein level(P<0.01).Overexpression of CSN1S1-1/-2 could extremely significantly eliminate the inhibitory effect of miR-22284b on the proliferation of GMECs(P<0.01)through CCK8 assay.Meanwhile,overexpression of CSN1S1-2 could significantly eliminate the inhibitory effect of miR-2284b on the proliferation of GMECs by the EdU assay(P<0.05),but had no significant effect of overexpression of CSN1S1-1 on miR-2284b affecting the proliferation of GMECs(P>0.05).The results of flow cytometry showed that CSN1S1-1/-2 could eliminate the inhibitory effect of miR-2284b on the proliferation of GMECs.ELISA assay indicated that overexpression of CSN1S1-1 could extremely significantly eliminate the effect of miR-2284b on the synthesis of αs1-casein by GMECs(P<0.01)and overexpression of CSN1S1-2 could significantly eliminate the effect of miR-2284b on the synthesis ofαs1-casein by GMECs(P<0.05).Thus,it could be speculated that miR-2284b regulated the synthesis of αs1-casein in GMECs by regulating the expression of CSN1S1-1/-2.In conclusion,this experiment identified two novel sequences encoding αs1-casein genes,and verified the effect of miR-2284b on proliferation and αs1-casein synthesis in dairy goat mammary epithelial cells.It was revealed that miR-2284b might regulate the proliferation of GMECs and αs1-casein synthesis by regulating the expression of CSN1S1-1/-2. |
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