Regulation Of Melatonin And Lnc552 On The Proliferation And Differentiation Of Hair Follicle Stem Cells In Cashmere Goats

Cashmere is a kind of amyelinic fiber produced from secondary hair follicles,it is an upscale textile material with high economic value.Cashmere growth always undergoes periodic rhythm,which can be divided into three stages: anagen,catagen and telogen.Many factors like feeding condition,nutrition level,light duration,ambient temperature,humidity and feeding altitude may directly influence cashmere yields.Hair follicle stem cells(HFSCs),a kind of adult stem cells,are located in the bulge region of hair follicles which have extraordinary self-renewal ability and multidirectional differentiation potential.They are the protagonist of hair anagen-telogen transition.HFSCs replenish and renew the structural cells of hair follicles through continuous proliferation and differentiation.Understanding the regulation mechanism of HFSCs proliferation and differentiation has important guiding significance for a better understanding of the growth process of cashmere and accomplishing the increasing-cashmere goal of goat breeding.Existing studies had exhibited that melatonin subcutaneous implantation could promote the growth of cashmere,and the addition of melatonin in vitro culture medium could promote the growth of hair shafts,even causing the changes of long noncoding RNA(lncRNA)expression.However,the undergo mechanisms were clearly unknown.Therefore,this study intends to explore the effects of melatonin and lncRNA on the proliferation and differentiation of cultured goat HFSCs,which would provide a theoretical basis for elucidating the growth regulation mechanism of cashmere growth.The main results in this study were as follows:1.Using goat hair follicles as materials,the goat HFSCs and dermal papilla cells(DPCs)were successfully isolated and purified,and the related cell-specific marker were used to identify the cells.The cellular immunofluorescence results showed that the purified goat HFSCs were obviously positive for CD34,KRT15,and ITG?1 markers.And after cultured with adipogenic differentiation medium,it indicated that the isolated goat HFSCs had differentiation potential and could induce differentiation to form lipid droplets by oil red O staining.The isolated goat HFSCs still had good proliferation and differentiation ability after7 weeks of culture in vitro.At the same time,the isolated goat DPCs of goats were significant positive for SOX2,?-SMA and VCAN markers,which accorded with the identification characteristics of DPCs.2.Different concentrations of melatonin were added to the culture medium of goat HFSCs and treated with different lengths of time.It was found that 500 ng/L of melatonin could significantly increase the nucleus level of CTNNB1,the key protein in Wnt pathway,after 72 h continuous treatment.The Wnt pathway was activated and the expression of downstream genes TCF4,LEF1,C-MYC,C-JUN,and CYCLIND1 were affected,thereby promoting the proliferation of goat HFSCs.Melatonin receptors were tested by RT-qPCR in HFSCs,the result revealed that the expression of melatonin orphan nuclear receptor(ROR?)was significantly increased at m RNA level after 72 h melatonin treatment(P<0.01),but the expression of ROR? was not affected at protein level,which indicating that melatonin promoted the proliferation of HFSCs without depending melatonin receptor.The studies on stem cell differentiation and pluripotency related factors BMP4 and NOGGIN showed that the expressions of BMP4 and NOGGIN were related to melatonin treatment duration.Melatonin promoted their expression and maintained a higher expression level over time.At the same time,melatonin treatment could promote the protein expression levels of pluripotency marker proteins NANOG,OCT4 and CD34,indicating that melatonin regulated the pluripotency of goat HFSCs by promoting the expression of NOGGIN.3.The lncRNA-lnc552 which was related to the development of cashmere growth was screened and identified.The overexpressing adenovirus of lnc552 was successfully constructed and transfected to HFSCs.The results showed that lnc552 could significantly inhibit the aggregation of goat HFSCs,which significantly reduced the proportion of cells in the G2 phase,increased the cells in G1 phase,affected the cell cycle and inhibited cell proliferation.Lnc552 could promote the phosphorylation of SMAD1,a key downstream protein of BMP2,and affect the expression of the downstream differentiation-related gene RUNX2.As to the pluripotency of HFSCs,lnc552 effected the pluripotency of goat HFSCs by inhibiting the expression of pluripotency marker proteins NANOG,OCT4 and KRT15 in HFSCs,and promoting the differentiation of goat HFSCs into adipocytes by increasing the expression adipogenesis proteins of PPAR? and ADIPOQ.After co-treating lnc552 with melatonin,it was found that melatonin could weaken the effect of lnc552 on goat HFSCs.In conclusion,this study revealed the mechanism of melatonin and lnc552 in regulating the proliferation and differentiation of goat HFSCs through the Wnt signaling pathway,cell differentiation and pluripotency-related factors,which would provide theoretical basis for clarifying the molecular mechanism of cashmere growth and the molecular breeding of cashmere goat.