Role Of MiR-29c In LPS Induced Inflammatory Response Of Bovine Mammary Epithelial Cells

Mastitis is one of the most common diseases in dairy cows,which can cause the decline of milk quality and undermine the lactation performance of dairy cows.Its high incidence has seriously restricted the economic benefits of the milk industry.In addition to environmental factors,the incidence of mastitis is closely related to the genetic level of dairy cows.Therefore,improving mastitis resistance has become one of the objectives of dairy cows breeding.miRNA are endogenous non-coding small RNA molecules that are highly conservative in evolution and can regulate gene expression at the post-transcriptional level.The previous high-throughput sequencing results showed that miR-29c was differentially expressed in the mammary gland of healthy dairy cows and dairy cows with mastitis induced by E.coli,but whether miR-29c participated in the regulation of mastitis is still unclear.Therefore,in this study,bovine mammary epithelial cells(bMECs)were used as the material to analyze the potential role of miR-29c in bMECs using transcriptome sequencing(RNA-seq)technology.Secondly,the lipopolysaccharide(LPS)-induced inflammation model of bMECs was constructed to detect the expression of miR-29c after LPS induction by quantitative real-time PCR(qRT-PCR).Besides,the function of miR-29c in LPS-induced inflammatory response of bMECs was explored by miR-29c overexpression/inhibition,double luciferase reporter gene assay,target gene overexpression/inhibition,qRT-PCR,enzyme-linked immunosorbent assay(ELISA)and CCK-8,etc.The main results are as follows:(1)RNA-seq was performed after miR-29c inhibitor was transfected into bMECs,and analysis showed that inhibition of miR-29c caused significant up-regulation of 42 genes and significant down-regulation of 27 genes,functional enrichment analysis indicated that miR-29c was a potential regulator of inflammatory response and oxidative stress of bMECs.(2)After LPS induced bMECs,mRNA expression and protein secretion of pro-inflammatory cytokines were detected by qRT-PCR and ELISA,the cell viability was measured by CCK-8,and the effect of LPS on the expression of miR-29c was detected.The results showed that LPS significantly up-regulated the mRNA expression and protein secretion of pro-inflammatory cytokines IL-1β,IL-6 and TNF-α in bMECs and inhibited cell viability,indicating that LPS activated the immune response of bMECs.Moreover,miR-29c was extremely significantly down-regulated in LPS-induced bMECs.(3)Mimic and inhibitor of miR-29c were respectively transfected into bMECs for overexpression and inhibition,and LPS was used for inflammation induction.The mRNA expressions of pro-inflammatory cytokines and apoptosis-related genes were detected by qRT-PCR,the secretion of pro-inflammatory cytokine proteins was detected by ELISA,and cell viability was detected by CCK-8.The results showed that overexpression of miR-29c could up-regulate the mRNA and protein expressions of pro-inflammatory cytokines,significantly up-regulated Bax and significantly inhibited Bcl-2 mRNA expression,and tended to inhibit cell viability.Inhibition of miR-29c,on the other hand,produced the opposite result to overexpression.(4)Eleven candidate target genes of miR-29c were predicted by TargetScan database,and the corresponding psi-CHECKTM-2-3’ UTR wild-type vector was successfully constructed,double luciferase reporter gene assay showed that LIF was the target gene of miR-29c.Further detected by qRT-PCR and ELISA revealed that miR-29c mainly regulates the expression of LIF protein in bMECs inflammatory response at the translation level.(5)The overexpression vector of LIF was constructed and specific siRNA was synthesized for the overexpression and inhibition of LIF in bMECs,respectively,and LPS was used for inflammation induction.The mRNA expressions of pro-inflammatory cytokines and apoptosis-related genes as well as cell viability were detected by qRT-PCR and CCK-8,respectively.The results showed that overexpression of LIF significantly inhibited the expression of IL-1β,IL-6,TNF-α and Bax,significantly up-regulated the expression of Bcl-2,and significantly enhanced cell viability.Inhibition of LIF significantly up-regulated the expressions of TNF-α and Bax and significantly inhibited the expression of Bcl-2,and significantly inhibited cell viability,but extremely significantly inhibited the expression of IL-6.In summary,this study showed that miR-29c promoted LPS-induced inflammatory response of bMECs by targeting LIF.The expression of miR-29c in LPS-induced bMECs was significantly down-regulated,reducing the inhibition of LIF expression,thereby helping to alleviate the excessive inflammatory response.The results provided theoretical information for analyzing the molecular regulatory mechanism of dairy mastitis and reference for breeding of dairy mastitis resistance.