Role Of MiR-143 On Dairy Goat Mammary Gland Epithelial Cells By Targeting MMP9 And LOC108635657

Laoshan dairy goat is one of the excellent breeds of dairy goats.The process of breast growth and development is a complex process controlled by various factors;The key regulatory factors are genes,miRNAs and lncRNAs.The three of them are considered to participate in the regulation of various biological processes through the construction of ceRNA network.Based on the previous research of miR-143,this study transfected miR-143 mimics into three libraries: M1,M2 and M3 in the experimental group by taking mammary epithelial cells of milk goats as the research object.Control group without any treatment,and divided into C1,C2 and C3 three library,6 library transcriptome sequencing screening differentially expressed genes,subject to the regulation of bioinformatics analysis,forecast and the role of lncRNAs,finally obtain MMP9 gene,miR-143 and LOC108635657 as the research object,the nanny goat mammary gland epithelial cells in vitro culture,through the cell transfection,Quantitative real time polymerase chain reaction(qRT-PCR),Dual-Luciferase Reporter Assay and Western blot and other experiments,such as To explore the role of miR-143 on dairy goat mammary gland epithelial cells by targeting MMP9 and LOC108635657,and to elucidate the regulatory role of ceRNA network on mammary epithelial cells,providing a theoretical basis for the in-depth study on the function of mirna-143 in the growth and development of mammary epithelial cells of goats.The main results are as follows:(1)82,969,530,67,319,936,81,526,696,81,673,114,80,493,910 and 81,915,328 clean reads were obtained in the six libraries of M1,M2 and M3,C1,C2 and C3.The proportion of clean Reads in 6 samples before filtration are above 96%.Among them,70% of the exons can be compared to the goat genome,and the comparison rate has reached the qualified level.Analysis of differentially expressed mRNAs revealed that there were 1804,1973,1879,and1005 differentially expressed mRNAs in the M1-C1,M2-C2,M3-C3,and M-C groups,respectively,and a total of 693 differentially expressed mRNAs in the four groups;GO analysis found that differentially expressed mRNA was enriched in 146 entries in the Biological Process,20 entries in the Cellular Component,and 23 entries in the Molecular Function.These entries were mainly to maintain the extracellular environment and regulate Immune response and enzyme activity,etc;KEGG analysis found that differentially expressed mRNA was enriched in 7 pathways,which are mainly assimilation,alienation and synthesis of steroids.(2)MMP9 gene wild-type and mutant dual luciferase reporter gene vectors were constructed and transfected into 293 T cells with miR-143 mimics and mimics NC,respectively.The results showed that miR-143 mimics and MMP9 wild-type groups had luciferin The enzyme expression activity was significantly lower than the other three groups(p<0.01),which indicated that miR-143 could directly target the MMP9 gene.qRT-PCR results showed that the expression level of MMP9 gene was significantly reduced after miR-143 overexpression(p<0.01),which indicated that miR-143 could regulate the expression of MMP9 gene.Western blot results showed that the expression level of MMP9 gene was significantly reduced after miR-143 overexpression(p<0.01),which further verified the results of qRT-PCR.In summary,miR-143 regulates its expression through targeted binding to MMP9 gene.(3)By constructing LOC108635657 wild-type and mutant double luciferase reporter gene vectors and transfecting them into 293 T cells with miR-143 mimics and mimics NC,respectively,the results showed that miR-143 mimics and luciferase in the wild-type group of LOC108635657 The expression activity was extremely significantly lower than the other three groups(p<0.01),which indicated that miR-143 could directly target LOC108635657.The qRT-PCR results showed that after miR-143 was over-expressed,the expression level of LOC108635657 was significantly reduced(p<0.01),indicating that miR-143 can regulate the expression of LOC108635657.In summary,miR-143 regulates its expression through targeted binding to LOC108635657.(4)Detection of LOC108635657 silencing by qRT-PCR and Western blot.The qRT-PCR results showed that when LOC108635657 was silenced,the expression level of miRNA-143 was significantly increased(p<0.01),and the expression level of MMP9 gene was significantly reduced(p<0.05),which indicates that LOC108635657 can regulate the expression of MMP9 gene.Western blot results showed that the expression of MMP9 genewas significantly reduced after silencing of LOC108635657(p<0.01),which further verified the results of qRT-PCR.In summary,LOC108635657 can regulate the expression of MMP9 gene.Through the above experiments,it can be found that miRNA-143 can target the MMP9 gene and LOC108635657 respectively,and interact with the LOC108635657 and MMP9 genes to play a role together;LOC108635657 regulates the MMP9 gene through targeted binding to miR-143.