Study On The Mechanism Of Vitamin B6 Regulating Hair Follicle Development Of Rex Rabbits By MiRNA

Rex rabbits are famous for fur production,and the fur quality has important economic values.The density of hair follicle is one particularly important indicators for evaluating the quality of Rex rabbits skin.In recent years,how to improve the hair density of Rex rabbits has become the most important concern in rabbit production.The hair is produced by the hair follicle.Hair follicles is an appendage from the vertebrate skin epithelium,it can control the growth of hair and has unique physiological structure and periodic growth characteristics.In the process of hair follicles formation and differentiation,involving dermal papilla,hair matrix,inner root sheath and outer root sheath cells.Dermal papilla cells(DPC)is a population of mesenchymal cells aggregated in hair bulbs and the key dermal component of the hair follicle,it can directly regulate hair follicle development,growth and regeneration.microRNA(miRNA)is a kind of non-coding small molecular RNA about 20~24 nucleotides in size.Recent studies have reported that miRNA plays an important role in regulating hair follicle morphogenesis,proliferation,differentiation and apoptosis of hair follicle stem cells in mice,rats,goat and sheep.In order to provide a theoretical basis for improving the fur quality of Rex rabbits,we isolated and identified the DPC from Rex rabbits skin,and analyzed the miRNA expression profile of the DPC from different hair density Rex rabbits.Finally,the mechanism of vitamin B6 regulating hair follicle density of Rex rabbits by miRNA were studied.The main contents as follows:1.Isolation,culture and identification of dermal papilla cells from Rex rabbits skinPiece of the dorsal skin were excised from the 30-day-old Rex rabbits,and the skin was rinsed with sterile phosphate buffer solution(PBS)containing 100 U/mL penicillin and 0.1mg/mL streptomycin,disinfected with iodine and destained with 75% ethanol and cut into2cm*5cm strips before digesting with 0.25 mg/mL Dispase II overnight at 4 ? and with an additional 30 minutes incubation at 37 ? on the following day.The tissues were then minced with ophthalmological scissors and digested with 0.1 mg/ mL Collagenase D for 4~6 hours at37 ? when dissociation of dermal papillas(DPs)from liquid fats was observed bymicroscopy.Subsequently,DPs were purified by a series of washing with PBS till the supernatant was completely clear,and filtered with a 70 ?m filter to remove single cells.Following purification,DPs were trypsinized to obtain single cells which were then cultured in dulbecco's modified eagle media(DMEM)containing 10% fetal bovine serum(FBS)at37? in a humidified atmosphere of 95% air and 5% CO2.After morphological observation,growth curve drawing and immunochemical identification of specific expression vimentin(Vim)and ? smooth muscle actin(?-SMA),the cultured cells were dermal papilla cells of Rex rabbit.2.Analysis of the expression profile of small RNA in dermal papilla cells from different hair density Rex rabbitsThe dermal papilla cells small RNA library from the skin of 30-day-old Rex rabbits with high and low hair densities(LD and HD group)was constructed,and high-throughput sequencing was performed.We obtained 37930744,39442011,40965907,38502653,40622117 and 41149163 clean reads in total from the six samples,and the vast majority of reads have a length of 23 nucleotides,which was consistent with the size of mammalian miRNA.Comparing clean reads with known small RNA databases,more than 90 % of the reads can be matched,which were 91.91 %,91.05 %,92.46 %,92.77 %,90.13 %,and91.61 %,respectively.The results of the small RNA classification commentary showed that miRNA accounted for 80.80 %,82.50 %,81.60 %,85.80 %,76.50 %,and 81.80 %,respectively.Through differential expressed genes screen(DEGs),240 difference expressions of miRNAs were screened,including 122 miRNAs for up regulate and 118 miRNAs for down regulate.Among them,ocu-miR-205 was low expressed in dermal papilla cells of Rex rabbits with high hair density(HD)and high expressed in dermal papilla cells of Rex rabbits with low hair density(LD).Further,ocu-miR-205 was highly expressed in the skin of Rex rabbit and has tissue specificity.The annotation of the targeted gene of differentially expressed miRNAs was performed using Gene Ontology(GO)enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis.A total of 205,661 target genes were enriched into GO terms,specific GO terms of the target genes were mainly involved in biological process(BP),cellular component(CC)and molecular function(MF).Following GO analysis,the target genes were also uploaded into the KEGG database to identify the pathway that wereactively regulated by miRNAs in dermal papilla cells.The result shown that a total of 325 pathways were predicted.Top enrichment pathways was Wnt signaling pathway,Notch signaling pathway,et al..3.Effects of ocu-miR-205 on dermal papilla cells of Rex rabbitsHBAD-GFP,HBAD-ocu-miR-205-GFP,HBAD-ocu-miR-205-5p-sponge-GFP were constructed and transfect dermal papilla cells as multiplicity of infection(MOI)200.Cell proliferation,cell cycle,cell apoptosis,ocu-miR-205-5p and hair follicle development-related gene and protein changes were detected 48 hours after transfection.The result shown that constructed adenovirus could successfully transfect dermal papilla cells,and the expression of ocu-miR-205-5p increased significantly after overexpression,while the expression of ocu-miR-205-5p decreased significantly after interference(P<0.05).ocu-miR-205 could increase cell proliferation,change cell cycle process and increase cell apoptosis ratio(P<0.05).ocu-miR-205 could inhibit the gene expression of Inppl1,Frk and Phlda3 in PI3K/Akt signaling pathway(P<0.05).ocu-miR-205 could promote DKK1 and inhibit Wnt10 b,Fzd4,Lrp5,Lrp6,CTNNB1,Axin,APC,GSK-3?,TCF3,Lef1 gene expression in Wnt signaling pathway(P<0.05).Notch1,Jagged1,Hes1 and Hes5 expression in Notch signaling pathway were suppressed by ocu-miR-205(P<0.05),and BMP2,BMP4 and TGF-?1 expression in BMP signaling pathway were promoted(P<0.05).The gene expression of hair follicle development related factors IGF1,EGF,FGF5,FGF7,SHH and TNF were affected by ocu-miR-205(P<0.05).ocu-miR-205 could inhibit the protein phosphorylation level of CTNNB1,GSK-3? and the protein expression level of noggin(NOG),promote Akt phosphorylation level(P<0.05).4.Effects of ocu-miR-205 on skin tissue of Rex rabbitsOne hundred 3-month-old Rex rabbits were randomly divided into 4 groups.After local shaving of the back skin,HBAD-GFP,HBAD-ocu-miR-205-GFP and HBAD-ocuocu-miR-205-5p-sponge-GFP 50 ?L per rabbit were injected intradermally.After 24 hours of transfection,one in each group was selected to observe the transfection,8 Rex rabbits were randomly selected from each group 7 days,14 days and 21 days after transfection.After slaughter,the injected skin was collected and placed in freezing tube and 4%polyformaldehyde stationary solution.The frozen samples were used to detect the changes ofocu-miR-205-5p and hair follicle development-related gene and proteins.The fixed samples were used to make paraffin sections and the hair density was counted after HE staining.The result shown that constructed adenovirus could successfully transfect the skin and hair follicles of Rex rabbits,and the expression of ocu-miR-205-5p increased significantly after overexpression,while the expression of ocu-miR-205-5p decreased significantly after interference(P<0.05).ocu-miR-205 could significantly affect gene expression in PI3K/Akt,Wnt,Notch and BMP signaling pathways,and gene expression of hair follicle development-related factors(P<0.05).ocu-miR-205 could significantly affect the protein phosphorylation level of CTNNB1,GSK-3?,Akt and the expression protein level of NOG(P<0.05).There was no significant change in primary follicle density(P>0.05),but the secondary follicle density and total follicle density(P<0.05)were changed after ocu-miR-205-5p interfered expression,and secondary/primary ratio(S/P)in ocu-miR-205-5p interfered expression group increased significant at 14 days after injection(P<0.05).5.Effects of vitamin B6 on dermal papilla cells of Rex rabbitsVitamin B6 at 0,10,20,40,80,160 ?mol/L concentration was added to the basic medium.The effects of vitamin B6 on the proliferation,cell cycle,ocu-miR-205-5p and expression of genes related to hair follicle development were observed for 72 hours.MTT result shown that the optical density(OD)value of vitamin B6 added 160 ?mol/L in culture medium was significantly lower than other groups(P<0.05),and the cell cycle of vitamin B6 group was significantly different from control group.The cell ratio in G0/G1 phase was significantly lower than control group(P<0.05),and the cell ratio in G2/M phase was significantly higher than control group(P<0.05),but there was no significant effect on S phase cell ratio(P<0.05).The appropriate concentration of vitamin B6(10 and 20 ?mol/L)could significantly inhibit cell apoptosis(P<0.05),but high concentration of vitamin B6(80and 160 ?mol/L)could significantly promote cell apoptosis(P<0.05).Vitamin B6 could significantly affect the expression of ocu-miR-205-5p(P<0.05).The expression of ocu-miR-205-5p decreased significantly with the increase of vitamin B6 concentration in culture medium(P<0.05).Vitamin B6 could promote the expression of Inppl1,Inpp4 b and Phlda3 in PI3K/Akt signaling pathway(P<0.05),Wnt10 b,Fzd4,Lrp6,CTNNB1,APC and Lef1 in Wnt signaling pathway,and inhibit DKK1 expression(P<0.05).Notch1,Jagged1,Hes1 and Hes5 in Notch signaling pathway were promoted by vitamin B6(P<0.05),BMP2,BMP4 and TGF-?1 expression in BMP signaling pathway had no significant affected by vitamin B6(P>0.05).The gene expression levels of IGF1,EGF,FGF5,SHH and TNF were significantly affected by vitamin B6(P<0.05).Vitamin B6 could promote the protein phosphorylation of CTNNB1,Akt and the protein expression of NOG,inhibiting of GSK-3?phosphorylation level(P<0.05).6.Effects of vitamin B6 on the growth of hair follicles of Rex rabbits30-day-old Rex rabbits were selected.After slaughter,the skin of the tentacle with complete tentacle hair follicles was carefully cut and destained with 75% ethanol and cut into2cm*5cm strips before digesting with 0.2 mg/ mL Collagenase D overnight at 37 ? and the free hair follicles were separated.The tentacle hair follicles were divided into six groups,10 in each group.The extracted hair follicles were placed in 24-well plate.Vitamin B6 concentration was 0,10,20,40,80 and 160 ?mol/L William E basic culture medium,and suspended culture was carried out at 31 ? and 5% CO2.Growing length of wool trunk was counted every 24 hours after 6 days of incubation.The result shown that the hair follicle of Rex rabbit tentacles cultured in vitro grew at an average growth ratio of 3.89 ?m/d for 144 hours,and the growth ratio was faster in the early stage than in the late stage.The growth ratio of hair stem was promoted by adding vitamin B6 in the culture medium.The growth length of hair follicle of Rex Rabbit tentacles cultured at 144 hours reached 36.15 ?m in the80 ?mol/L supplementation group,with the fastest growth ratio being 6.03 ?m/d,which was significantly higher than that of the control group(P<0.05).Therefore,vitamin B6 can promote hair growth and delay hair follicle degeneration and death.7.Vitamin B6 on production performance and hair follicle development of Rex rabbitsIn this study,200 3-month-old Rex rabbits were randomly assigned to one of the 5 diets,the following 5 different concentrations were vitamin B6 supplemented into the diets of the study animals: 0,5,10,20,and 40 mg/kg.The 60-day feeding trial included a 7-day adjustment period and a 54-day experimental period.The result shown that,dietary vitamin B6 had significant influence on average daily gain(ADG)and average daily feed intake(ADFI)(P<0.05),with the vitamin B6 level increasing,the feed/gain(F/G)was firstly decreased and then increased,and it had the lowest value when vitamin B6 supplemental levelwas 20 mg/kg.Dietary vitamin B6 level had significant influences on fur weight and fur area(P<0.05),and the fur weight or fur area in 20 mg/kg group were higher than 0 mg/kg group(P<0.05).Dietary vitamin B6 had significant effects on total follicle density,secondary follicle density and S/P(P<0.05).With the increasing of supplemental level,the follicle density first increased and then decreased,and reached the maximum at 20 mg/kg.Vitamin B6 could significantly affect the expression of ocu-miR-205-5p(P<0.05),and the expression of ocu-miR-205-5p decreased with the increasing of vitamin B6 level in diet(P<0.05).Dietary vitamin B6 level could increase the expression of Inppl1 in PI3K/Akt signaling pathway.Wnt10 b,Fzd4,CTNNB1 and APC in Wnt signaling pathway were increased,and GSK-3? in Wnt signaling pathway were decreased by vitamin B6(P<0.05);Vitamin B6 can promote the expression of Notch1,Hes1 and Hes5 in Notch signaling pathway(P<0.05);There were no significant effect of BMP2,BMP4 and TGF-?1 expression in BMP signaling pathway(P>0.05).The expression of hair follicle development related factors IGF1,IGF1 R,EGF,FGF5 and SHH was significantly affected by vitamin B6(P<0.05).Vitamin B6 could promote the protein phosphorylation level of CTNNB1 and Akt,inhibit the protein phosphorylation level of GSK-3?(P<0.05).In conclusion,the molecular mechanism of vitamin B6 promoting hair follicle density in Rex rabbit through inhibiting the expression of ocu-miR-205-5p,activating the expression of related gene and proteins in PI3K/Akt,Wnt and Notch signaling pathways,prolonging hair follicle growth and delaying the arrival of telogen.