| Hu sheep is the unique sheep breed with white lambskin and known as its lambskin with a waved pattern in the world.However,in recent years,Hu sheep has been biased towards the improvement of meat traits,neglecting the original lambskin traits.In addition,unplanned crossbreeding has resulted in an increasing number of genetic confounding in Hu sheep with a dramatic decline in the quality of lambskin and a serious threat to germplasm resources.Thus,it is significant to explore the molecular mechanism of the formation of the lambskin for the conservation and utilization of germplasm resources of Hu sheep lambskin.Wool curvature is key factor to determine the quality of lambskin.According to the pattern width and the degree of wool curvature,lambskins are divided into four types,including small waves,medium waves,large waves and straight wool,of which small waves is the best quality and straight wool is the worst.Therefore,the molecular mechanism of wool curvature is important to reveal the molecular mechanism of lambskin pattern formation.Bone morphogenetic protein 7(BMP7),expressing differentially among different patterns of Hu sheep lambskin,is significantly correlated with the index of hair follicles of different patterns(number and diameter of primary and secondary hair follicles).ceRNA regulatory network is a hot spot research for gene function and mechanism.In previous,We have predicted and validated the targeting relationship between BMP7 and miR-148a,miR-10a.However,litter research has been found on circRNA in hair follicle of sheep.Therefore,it is essential to explore the formation of the lambskin pattern in Hu sheep from the perspective of ceRNA regulatory mechanism of circRNA-miR-148a/10a-BMP7.The pattern formation of Hu sheep lambskin is closely associated with wool curvature,which in turn is regulated by hair follicle.The reports ponit out that the type and number of dermal papilla cells in the hair follicle are closely associated with hair curvature,and Multiple Papillary Centres(MPC)hypothesis also explains the cytological mechanism of hair curvature.Therefore,this study will investigate the effect of BMP7 as the core of ceRNA regulatory network on Hu sheep dermal papilla cells so that it will provide a basis for the subsequent analysis of the mechanism of wool curvature and pattern information.Our findings are as follows:(1)Effect of BMP7 gene on proliferation of Hu sheep dermal papilla cells and TGF-?1/Smad signaling pathway.After overexpression and interference of BMP 7 in Hu sheep dermal papilla cells,the results showed that the cell viability and cell proliferation in the overexpression group were significantly higher than in the control group(P<0.05),and the cell cycle was significantly accelerated(P<0.05).At the same time,the expression level of Smad3,Smad4,and TGF-?1 showed a extremely significant decrease(P<0.01)and Smad6 expression level showed a significant decrease(P<0.05)compared to the control group in TGF-?1/Smad signaling pathway.When the expression level of BMP 7 was inhibited in dermal papilla cells,the cell viability and cell proliferation were significantly lower than in the control group(P<0.05),and the cell cycle progression was significantly slower(P<0.05).At the same time,the expression levels of Smad3 and Smad4 were extremely significantly higher in the interference group than in the control group(P<0.01),and the expression levels of Smad5 and TGF-?1 expressed in the interference group were significantly higher than that in the control group(P<0.05).These results suggested that BMP7 had a positive effect on the proliferation of Hu sheep dermal papilla cells and could affect the expression level of key genes of the TGF-?1/Smad signaling pathway.Additionally,the target site for BMP7 knockdown by CRISPR/Cas9 was validated in 293T cells and could be used for subsequent knockdown in animal models.(2)Effect of miR-148a and miR-10a targeting BMP7 on proliferation of Hu sheep dermal papilla cells.After overexpression and interference of miR-148a and miR-lOa in dermal papilla cells,respectively,the results showed that the cell viability and cell proliferation were significantly or extremely significantly lower in the miR-148a mimic and miR-10a mimic groups than in the control group(P<0.05 or P<0.01).The mRNA expression levels of cell proliferation-related genes CDK2,PCNA and cyclind1 were also significantly or extremely significantly lower than those of the control group(P<0.05 or P<0.01),and their protein expression trends were consistent with mRNA.The dermal papilla cell cycle progression was significantly slower(P<0.05).The mRNA expression level of BMP7 was significantly or extremely significantly lower(P<0.05 or P<0.01)than the control group,and its protein trends were consistent with mRNA.Overexpression of miR-148a and miR-10a also resulted n significant or extremely significant changes(P<0.05,P<0.01,respectively)in key genes of the TGF-?1/Smad signaling pathway.When the expression levels of miR-148a and miR-10a were inhibited in dermal papilla cells,the cell viability and cell proliferation were significantly or extremely significantly higher than the control group(P<0.05).The mRNA expression levels of CDK2,PCNA and cyclindl were also extremely significantly higher than those of the control group(P<0.01),and their protein expression trends were consistent with mRNA.The dermal papilla cell cycle progression was significantly accelerated(P<0.05).The mRNA expression level of BMP7 was significantly or extremely significantly higher(P<0.05,P<0.01,respectively)than the control group,and its protein trend was consistent with mRNA.Interference of miR-148a and miR-10a also resulted in significant changing(P<0.05)in key genes of the TGF-?1/Smad signaling pathway.Rescue experiments showed that exogenous addition of BMP7 protein could restore the inhibiting effects on dermal papilla cell viability,proliferation and cycle caused by miR-148a/miR-10a overexpression.Exogenous addition of BMP 7 interference vectors could restore the promotion effects on dermal papilla cell viability,proliferation and cycle caused by miR-148a/miR-10a inhibition.It was shown that miR-148a and miR-10a could binding to BMP7 to affect the proliferation of Hu sheep dermal papilla cells.(3)The screening of differentially expressed circRNAs between small waves and straight wool of Hu sheep lambskin.A total of 5,527 circRNAs were identified in Hu sheep hair follicle by RNA-seq,accompanying 114 differentially expressed circRNAs between small waves and straight wool groups.Enrichment analysis showed that host genes ' with partially differentially expressed circRNAs were enriched in the TGF-? signaling pathway and Notch signaling pathway.129 miRNAs were found to potentially bind to 114 differentially circRNAs,including miR-148a,miR-199a-3p,miR-200a,miR-10a,miR-let-7a,miR-143 and others using miRanda software.In addition,11 circRNAs were predicted and identified with potential coding ability.These results will provide a basis for the molecular mechanism of circRNA sponging to miR-148a and miR-10a to regulate BMP7 expression in hair follicle growth and development.(4)circFTO and circCSPP1 competitively binding miR-148a and miR-10a to regulate BMP7 expression to affect the proliferation of Hu sheep dermal papilla cells.circFTO and circCSPP1 was successfully identified by RNase R enzyme digestion as well as reverse primer amplification.Nucleoplasmic localization determined that circFTO and circCSPP1 are mainly distributed in the cytoplasm.The dual luciferase assay validated the target relationship of circFTO and miR-148a,circCSPP1 and miR-10a,respectively.Rescue experiments showed that exogenous addition of circFTO and cricCSPP1 could restore the inhibiting effects on dermal papilla cell viability,proliferation and cycle caused by miR-148a and miR-10a overexpression,respectively,suggesting that circFTO and cricCSPPl could competitively bind miR-148a and miR-10a to regulate BMP7 expression to affect the proliferation of Hu sheep dermal papilla cells. |
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