Role And Mechanism Of AKT1 Gene Methylation In The Decreased Lactation Ability Of Dairy Cows Induced By LPS

The purpose of this study was to investigate the mechanism of AKT1 gene methylation in dairy cows induced by LPS.The research mainly includes four parts.(1)Construction of an LPS-induced epithelial inflammatory cell model in dairy cows.By detecting the expression of inflammatory factors,the activity of cell proliferation and the synthesis and secretion of milk components,the successful construction of inflammatory cell model was evaluated.The results showed that the inhibitory rate of 1 μg/m L LPS was 3.52%,and that of 10 μg/m L LPS was 9.71%.The optimal concentration of 10 μg/m L was selected according to the criterion of cell inhibition rate(IR)≤10%.After LPS treatment for 12 h,the expressions of inflammatory cytokines TNF-α,IL-1β and IL-6 were significantly increased(P<0.01),and the synthesis ability of lactose,milk fat and milk protein and cell proliferation activity were significantly decreased(P<0.01),indicating that the mastitis cell model was successfully constructed.Then,q RT-PCR and Western blot were used to detect the effect of mastitis on the expression of lactation related genes in mammary epithelial cells,and bisulfite sequencing(BSP)was used to detect the methylation changes of AKT1 promoter,a key lactation gene.The results showed that the expression of lactation related genes such as AKT1,mTOR,S6K1,STAT5,GLUT1,SREBP1,PPAR-γ and CSN2 in BMECs was significantly decreased(P<0.01).The protein expressions of p-AKt1,p-mtor,P-S6K1 and P-STAT5 were significantly decreased(P<0.01).At the same time,the methylation level of AKT1 promoter increased after LPS treatment(P<0.05),and the methylation rate increased from 11.25% to 18.75%.(2)Verification of the relationship between targeted regulation of transcription factor S P1 and AKT1 gene.In this study,we first determined that SP1 was strongly bound to the AKT1 promoter at the predicted binding site S2 by Ch IP-PCR analysis.Secondly,aiming at the AKT1 promoter region,this study constructed a luciferase vector including the SP1 binding site.The results of double luciferase reporter gene test showed that SP1 could significantly enhance the activity of AKT1 promoter(P<0.01),and the binding site of SP1 was 5 ’-CGCCCGCCCTG-3’(-1792 ～-1782bp).In this study,the predicted potential binding sites were mutated by site-specific mutation to construct the mutant AKT1 promoter luciferase vector.The results of double luciferase detection showed that compared with wild-type AKT1 promoter luciferase vector,the luciferase activity of the mutant AKT1 promoter luciferase vector was significantly decreased after transfection into BMECs(P<0.01).The results indicated that the 5 ’-CGCCcgcc Cgg-3’ sequence in the-1792 ～-1782 bp region of AKT1 gene was the effective binding site of SP1.(3)The effect of SP1 on mammary epithelial cells of dairy cows.In this study,SP1 protein expression level in mammary gland tissues of Holstein cows during adolescence,lactation and dry milk period was detected.Western blot results showed that SP1 expression level in mammary gland tissues of Holstein cows during different periods was significantly different(P<0.01),and the highest expression level was found in lactation period.Quantitative fluorescent PCR and Western blot were used to detect the effects of LPS-induced breast epithelial cells on the expression of SP1 at different times.The results showed that the expression of SP1 firstly increased and then decreased with the increase of time.The above results indicated that SP1 protein had basic exp ression in BMECs.And its expression was affected by LPS-induced mastitis.In order to further understand the effect of transcription factor SP1 on BMECs,this study successfully constructed an overexpression vector of SP1,and transfected the constructed expression vector into normal BMECs and LPs-induced BMECs.The results showed that SP1 overexpression could significantly increase the proliferative activity of normal and inflammatory cells and the synthesis of lactose,milk fat and milk protein(P<0.01),and also significantly increase the expressions of lactation related genes and lactation related proteins(P<0.05 or P<0.01).These results suggest that transcription factor SP1 positively regulates the lactation process of mammary epithelial cells,and can restore the proliferative activity and lactation ability of inflammatory mammary epithelial cells induced by LPS to a certain extent.(4)The effect of DCas9-mediated directed methylation and demethylation modification on the regulatory relationship between transcription factor SP1 and AKT1 gene promoter.In this study,d Cas9 directed AKT1 promoter methylation vector guided by small guide RNA(sgRNA),which was successfully screened in the previous work of our laboratory,was transfected into normal BMEC s.The SP1 overexpression vector and AKT1 promoter luciferase vector were then co-transfected into the cells.The results showed that the activity of AKT1 promoter was significantly reduced,indicating that DNA methylation could inhibit the binding of SP1 to AKT1 promoter.In this study,a SGRNA4-guided demethylation vector of d Cas9 directed AKT1 promoter was successfully selected and transfected into LPs-induced BMECs.The methylation level of AKT1 promoter was significantly reduced(P<0.01)after BSP modification and Sanger sequencing.These results indicated that Sgr NA4-guided d Cas9 directed AKT1 promoter demethylation vector had efficient demethylation effect on LPS induced AKT1 promoter hypermethylation.Secondly,the SGRNA4-guided DCas9-directed AKT1 promoter demethylation vector and SP1 overexpression vector were co-transfected into LPs-induced BMECs,and the results showed that the proliferative activity of mammary epithelial cells was significantly increased(P<0.01).The synthesis of triglyceride,l actose and β-casein was significantly increased(P<0.01);The expressions of lactation related genes AKT1,mTOR,S6K1,STAT5,GLUT1,SREBP1,PPAR-γ and CSN2 were significantly increased(P<0.05 or P<0.01).These results suggest that the synergistic effect between DNA demethylation and SP1 overexpression has a certain restorative effect on reduced lactation function caused by LPs-induced mastitis.In conclusion,LPS can also inhibit the binding of transcription factor SP1 to AKT1 promoter by increasing the methylation level of lactation related gene AKT1,thus inhibiting the expression of AKT1 gene and the activities of AKT1/mTOR and JAK2/STAT5 signaling pathways,leading to the decrease of lactation function of dairy cows.