Effect And Gene Function Research Of ARID3A On Milk Fat Synthesis Regulation In Mouse Mammary Epithelial Cells

Milk is the main nutritional source for human infants and newborn mammals,and dairy products are also one of the important nutritional sources for different populations.The level of milk fat content in milk is one of the important indicators to measure milk quality.Therefore,exploring the mechanism of milk fat synthesis and establishing new biotechnology to improve the milk fat content has become a research hotspot in recent years.The milk secretion ability of the mammary gland mainly depends on the milk synthesis and proliferation ability of the mammary gland epithelial cells(MECs).Therefore,it would be beneficial for the establishment of new technical approaches for improving milk fat content through nutritional regulation,to uncover the molecular mechanism of milk fat synthesis in MECs.AT-rich interaction domain 3A(ARID3A)is a member of the A + T interaction domain(ARID)rich protein family.ARID3A can participate in the regulation of various biological processes,such as cell differentiation,development,cell cycle control,epigenetic regulation and chromatin remodeling,but it is still unknown whether ARID3A participates in the process of milk fat synthesis.Therefore,exploring the molecular mechanism of ARID3A in regulating milk fat synthesis in MECs would help to deeply clarify the regulatory mechanism of milk fat synthesis.Palmitic acid(PA)can stimulate the synthesis of milk fat in mouse MECs(HC11),but the detailed molecular mechanism is yet known little.The purpose of this study is to uncover the role and corresponding molecular mechanism of ARID3A in the process of milk fat synthesis in HC11 cells stimulated by PA.In this study,it was found that the expression level of ARID3A protein in mammary gland tissue of lactation mice was significantly higher than that in puberty and involution.siRNA transfection and CRISPR-d Cas9 technology were used to inhibit and activate the ARID3A gene respectively.CCK8 kit,triglyceride(TG)assay kit,total cholesterol(TC)assay kit,BODIPY fluorescence staining,and nonesterified Free fatty acids(NEFA)assay kit were used to detect cell proliferation ability,TG,TC,lipid droplet formation ability,and secreted NEFA content respectively.it was found that TG,TC,lipid droplets and se NEFA in HC11 cells were significantly reduced after ARID3A gene inhibition.Proliferation of HC11 cells was also significantly reduced as determined by CCK8 assay and cell counting.Opposite results were observed in ARID3A gene activation experiments.These data indicate that ARID3A is a key positive regulator in milk fat synthesis in HC11 cells and cell proliferation.In addition,after the inhibition of ARID3A gene,the expression of f SREBP1(full length form of SREBP1),nSREBP1(nuclear form of SREBP1)protein and SREBP1 mRNA in HC11 cells were significantly decreased by Western blotting and qRT-PCR assays.The activation of ARID3A gene had the opposite results.Taken together,these above results demonstrate that ARID3A can positively regulate SREBP1 mRNA expression and subsequent protein expression and maturation in HC11 cells.In a later study,it was observed that PA promoted ARID3A protein expression as well as SREBP1 protein expression and maturation in a dose-dependent manner,and has the best effect at 120 μM.To further explore the molecular mechanism underlying this process,HC11 cells were treated with PA together with the addition of LY294002(a specific PI3K inhibitor)treatment,and the results showed that the stimulatory effects of PA on ARID3A protein expression as well as Akt phosphorylation were almost completely abolished by LY294002 treatment.Significant increase in nuclear expression of ARID3A was found by immunofluorescence observation in HC11 cells after PA treatment.ARID3A gene was inhibited,and HC11 cells were also treated with PA.The results showed that ARID3A gene inhibition almost completely blocked the expression of ARID3A and expression and maturation of SREBP1 protein promoted by PA.Moreover,ARID3A gene inhibition also blocked the stimulation of PA on SREBP1 mRNA expression.The above data indicate that PA can stimulate SREBP1 protein expression and maturation through the PI3K/ARID3A signal pathway.ARID3A is a key positive regulator of PA-stimulated SREBP1 mRNA expression and subsequent protein expression and maturation.To sum up,this study reveals the role and molecular mechanism of ARID3A in the process of PA-stimulated milk fat synthesis.We demonstrate that ARID3A is a key mediator for PA to promote SREBP1 mRNA expression and subsequent protein expression and maturation,and also a key positive regulator of milk fat synthesis in HC11 cells and its proliferation.This study provides a new theoretical basis for the development of techniques for using PA to promote milk fat synthesis.