The Mechanisms Of Period2 Gene On The Mammary Casein Synthesis

Milk protein is a key indicator of milk quality and safety of dairy products,and hence,its regulation is at the core of the competitiveness of the dairy industry.Circadian rhythms exist in biological organisms and play a certain regulation role in normal physiological and biochemical activities.Previous in vitro research from our laboratory showed that the casein synthesis of bovine mammary epithelial cells was affected by circadian clock gene Period2,but the specific regulatory mechanisms are still unclear.Therefore,this study investigates the molecular mechanisms of transcription and translation signaling pathways that Period2 down-regulation affects cell proliferation,mammary gland development and casein synthesis via gene silencing in vitro and gene knockout in vivo technique.Experiment 1:Signaling mechanisms of Period2 gene silence affecting cellular proliferation and casein synthesis of bovine mammary epithelial cellsThis section explores the signaling mechanisms that Period2 gene silencing promotes casein synthesis of bovine mammary epithelial cells(BMEC)in vitro.The fourth passage BMEC were transiently-transfected with siRNA specific for Period2 to down-regulate transcription.After incubating for 24 h,the alterations in proliferative activity of BMEC by cell counting Kit-8,cellular apoptosis and cell cycle distribution by flow cytometry,and abundance of molecules on casein synthesis signaling pathways by qRT-PCR and Western blot were detected.The results showed that the cell proliferation activity of the silencing group was greater than that of the control after 24 h and 30 h siRNA transfection(P<0.05).Period2 gene silencing inhibited the apoptotic rate of BMEC(P<0.05)accompanied by decreased mRNA abundance of pro-apoptotic genes P53,Bax,Casp8,and Casp3 and increased that of anti-apoptosis gene Bcl-2(P<0.05).Compared with the control,the proportion of cells at G0/G1 phase in the silencing group was decreased(P<0.01),and cells at S and G2/M phases showed an upward trend(P>0.05).Period2 gene silencing reduced the gene expressions of Weely Cdkn2a,Cdknla,Ccndl,and Cdk2(P<0.05),and increased that of E2fl,Cdk4,c-Myc,and RbN(P<0.05),which could explain the transition of G1 to S phase.In addition,the mRNA abundance of Jak2,Stat5a,Pik3c3,Pdk1,and Akt2 genes was promoted after Period2 gene silencing(P<0.05)while the expression of 4ebp1 gene was decreased(P<0.05).Protein abundance of total mTOR,p-mTOR,and the proportion of p-mTOR to total mTOR showed an increased trend(P>0.05),To sum up,Period2 gene silencing regulates casein synthesis probably by promoting cell proliferation,inhibiting apoptosis,and redistributing cell cycle distribution,which all provides a physiological basis for milk component synthesis,and by manipulating expression of key molecules on JAK2-STAT5 and PI3K-AKT-mTOR signaling pathway to regulate directly the transcription and translation process of casein proteins.Experiment 2:Study on diurnal rhythms of mouse mammary casein synthesis and lactation-related hormonesTo further study the effects and mechanisms of the circadian clock gene Period2 on mammary casein synthesis,this part of experiment uses mice to explore diurnal rhythm of mammary casein protein abundance and lactation-related hormones and their correlation with Period2 gene expression.Wild C57BL/6N mice were fed and bred on a 12 h light:12 h dark(12L:12D)cycle.Six zeitgeber time points(ZTO,ZT4,ZT8,ZT12,ZT16,ZT20)were selected at intervals of 4 h within 24 h to collect the serum and mammary tissues of puerperium mice(L14±2,n=4).The abundance of Period2 gene,casein proteins and their synthesis signaling pathway molecules of mammary gland tissue,and the level of lactation-related hormones in serum were detected.The results showed that there was a diurnal variation of cosine function in Period2 gene expression that went to the highest at ZTO and ZT4(P<0.05),and then gradually decreased to the minimum at ZT20.The mRNA abundance of Csn1s1,Csn2,and Csn3 and the protein abundances of as1-casein,?-casein and ?-casein showed significant daily rhythm changes(P<0.05).And correlation and regression analysis found that the mRNA abundance of Period2 showed a negative correlation with that of Csn1s1 gene throughout the whole day,the light period and the dark period(P>0.05),and was linearly positive correlated with the gene expression of Csn2(P<0.05)and Csn3(P>0.05).In contrast,Period2 gene expression was positively correlated with asl-casein abundance(P>0.05),but negatively correlated with abundance of ?-casein(P>0.05)and ?-casein(P<0.05)in a linear form.In addition,the gene expression of Jak2,Mtor and 4ebp1 and concentrations of lactation-related hormones including PRL,PRH,GH and GHRH showed significant diurnal rhythms(P<0.05).Correlation and regression analysis showed that the mRNA abundance of Period2 was linearly negative correlated with the PRL level(P<0.05),and was positively correlated with levels of PRH,GH and GHRH(P>0.05).The concentration variation of PRL and PRH was negatively correlated with that of GH and GHRH(P>0.05),likely related to competitive binding of hormone receptors.In conclusion,the Period2 gene expression is positively correlated with the mRNA abundance of three casein genes,but negatively correlated with casein protein abundance,which may be related to the rhythm changes in casein synthetic signaling pathway molecules and concentration of lactation-related hormones and hypothalamic trophic hormone-releasing hormones in serum,jointly regulated by circadian clock gene.Experiment 3:Effects of Period2 gene knockout on mammary gland development and casein synthesis in postpartum miceBased on the results of in vitro study in BEMC cells and in vivo exploration on diurnal rhythms of mouse mammary casein protein abundance and lactation-related hormones,this section further verifies whether the gene knockout in vivo has consistent effects on mammary gland development and casein synthesis in postpartum mice.Period2 gene knockout homozygous mice(Period2-/-)and wild C57BL/6N mice(Period2+/+)were fed and bred on a 12L:12D cycle.Mammary gland tissues of 8 lactating female mice(L14±2)were collected from each group.Paraffin sections were used to determine the condition of mammary gland development,qRT-PCR and ELISA assays were used to detect gene abundance of apoptosis-related genes and three caseins.The results showed that the mammary Period2 expressions in both mRNA and protein levels were decreased after subjecting to Period2 gene knockout(P<0.05).Consistent with in vitro study on BMEC,mammary acinar of Period2 gene knockout mice had more regular shapes than those of the control group,and the area of acinar section was increased in the knockout group(P<0.05).Compared with the control group,Period2 gene knockout decreased the expressions of pro-apoptotic genes Bax and Casp8 while increased that of anti-apoptosis gene Bcl-2(P<0.05).At the same time,the abundance of three casein genes and proteins in Period2 gene knockout group was increased compared to the control(P<0.05).To sum up,Period2 gene knockout could promote the mammary gland development,inhibit tissular apoptosis,and increase mammary casein synthesis in postpartum mice.Experiment 4:Analysis of signaling pathways of Period2 gene knockout affecting mammary casein synthesis in postpartum miceThis part of experiment further explores the lactogenic hormone-mediated signaling pathways involved in Period2 gene knockout in vivo affecting mammary casein synthesis in postpartum mouse.Mammary gland tissues of postpartum mice(n=8)in Period2 knockout group(Period2-/-)and wild group(Period2+/+)were collected based on the design of experiment 3 above.qRT-PCR and Western blot assays were used to detect the expression of amino acid transporters,casein synthetic signaling pathway molecules,and lactation-related hormones and their receptors.The results showed that the mRNA abundance of Jak2,Stat5a,Stat5b,S6k1,and Rps6 in the Period2 knockout group was greater than those in the control(P<0.05).There was no significant difference in the abundance of Mtor gene and total mTOR protein between the two groups(P>0.05),but phosphorylation level of mTOR was significantly increased(P<0.05).In addition,Period2 gene knockout increased the mRNA abundance of Prl,Ghr,Igf-1,and Igf-1r compared with the control(P<0.05),but the Slc7al and Slc38a2 expression showed no difference between two groups(P>0.05).The results above show that Period2 gene knockout promotes mammary casein synthesis by promoting the expression of molecules on JAK2-STAT5 and mTOR pathway mediated by prolactin and growth hormone(including downstream target genes IGF-1 and its receptors).In conclusion,1)in vitro studies on BMEC cells have confirmed that Period2 gene silencing could promote cell proliferation,inhibit cellular apoptosis,affect cell cycle progression to provide a physiological basis for casein synthesis.At the same time,Period2 gene silencing directly regulated the transcription and translation process of casein proteins probably by manipulating expression of key molecules on JAK2-STAT5 and PI3K-AKT-mTOR signaling pathways.2)The mouse circadian clock gene Period2 showed a significant diurnal rhythm,which was inversely correlated with casein gene expression and casein protein abundance.Those might be related to the diurnal rhythms in casein synthesis signaling pathway molecules and lactating-related hormones,suggesting the potential interaction between casein synthesis and Period2.3)In vivo studies showed that Period2 gene knockout promoted mammary acinar growth and development,and increased the abundance of three casein proteins in postpartum mice.4)Those influences on casein synthesis might be achieved via its regulation on the growth and development of mammary gland indirectly and lactogenic hormones-mediated JAK2-STAT5 and mTOR pathway directly.