Target Gene Validation Of MiR-148a And MiR-10a And Their Functional Analysis In Dermal Papilla Cells Of Hu Sheep

Hu sheep,a unique sheep breed with high reproductive performance,is also well known for its rare white limbskin in China.However,in recent years,the quality of Hu sheep lambskin has been declining because people paid more attentation to meat performance and introduced many exotic sheep breeds from the outside of Hu sheep production area.Hair follicle is the subsidiary organ of skin,consisting of more than 20 kinds of cells,among which,dermal papilla cells play a key role in hair follicle growth and development.MicroRNAs(miRNAs)are a class of short single-stranded noncoding RNAs with about 18-24 nt length,which can mediate gene silencing at post-transcriptional level.Studies have shown that a large number of miRNAs are involved in the development and periodical growth of hair follicles.In this study,Hu sheep was took as the experimental object,and dermal papilla cells from Hu seep lambskin hair follicles were successfully cultured by mechanical separation and enzyme digestion.Based on the previous results of gene chips and high-throughput sequencing,BMP7,a differentially expressed gene in different kinds of patterns of Hu sheep lambskin,was considered as the research clue.Bioinformatic softwares were used to predict miRNAs which were targeted to BMP7,and then miR-148a and miR-10a were screened as a result combined with miRNA sequencing dates in different patterns of Hu sheep lambskin that previously conducted by our research group.After that,dual luciferase reporter system,Western Blot and RT-qPCR were employed to verify their target relationships.After transfecting miR-148a/miR-10a mimics and inhibitors into dermal papilla cells,we detected dermal papilla cells proliferation by CCK-8,and some genes expression,such as Smad1,Smad2,Smad3,Smad4,Smad5,Smad6 and TGF-?1 in TGF-? singling pathway,were detected by RT-qPCR with the aim to study the function of miR-148a and miR-10a in hair follicle development.The main findings are as follows:1.Hu sheep dermal papilla cells were cultured in vitro and exhibited a typical aggregative behavior in culture.Cell histochemical staining showed that dermal papilla cells were positive affected by PAS,AB-PAS and toluidine blue staining,and there were different staining responses to toluidine blue staining.Moreover,a-SMA,the specific marker of dermal papilla cells,was positively expressed by indirect immunofluorescence detection.All these results indicated that Hu sheep dermal papilla cells were successfully isolated and cultured in vitro,which would lay the groundwork for subsequent gene functional verification.2.The dual luciferase reporter system showed that miR-148a and miR-lOa could significantly reduce the activity of BMP 7 luciferase reporter(wild type vector),and preliminarily proved that BMP7 was the target gene both for miR-148a and miR-10a.In order to further verify the targeting relationship between miR-148a/miR-10a and BMP7,Western Blot and RT-qPCR were used to detect the expression of BMP 7 at the protein level and mRNA level,respectively.The result showed that both miR-148a and miR-lOa could promote the degradation of BMP7 mRNA,and then reduce the expression of BMP7 protein,which demonstrated that BMP 7 was a target gene of miR-148a and miR-10a.3.The CCK-8 detection showed that miR-148a and miR-10a could inhibit the proliferation of Hu sheep dermal papilla cells.4.After overexpression of miR-148a,the expression of Smadl,Smad2,Smad3,Smad4,Smad5 and Smad6 were all up-regulated.The expression of Smad3 and Smad6 in experimental groups were significantly higher than the control groups(P<0.05),Smad4 and Smad5 in experimental groups were significantly higher than the control group(P<0.01),however,the expression of TGF-?1 was lower than the control group.After inhibiting the endogenous miR-148a,the expression of Smad1,Smad2,Smmnad3,Smad4,Smad5,Smad6 and TGF-?1 were lower than that control groups,and the expression level of Smad3 and Smad4 were significantly lower than that in control groups(P<0.05),while the expression level of TGF-?1 was extremely significant lower(P<0.01).After overexpression of miR-10a,the expression of Smad1,Smad2,Smad3,Smad4,Smad5 and Smad6 were all down-regulated,in which the expression of Smad2 in experimental group was significantly lower than the control group(P<0.05),Smad1,Smad4 and Smad5 in experimental groups were significantly extremely lower than the control group(P<0.01),while the expression of TGF-/31 was significantly higher than the control group(P<0.05).After inhibiting the endogenous miR-10a,the expression of Smadl,Smad2,Smad3,Smad4,Smad5,Smad6 and TGF-?1 were lower than that control groups,in which the expression level of Smadl was significantly lower than that the control group(P<0.05)and the expression level of Smad2 was extremely significant lower than the control group(P<0.01).These results suggested that overexpressing or inhibiting miR-148a and miR-10a in Hu sheep dermal papilla cells could affect genes expression in TGF-?/Smads signiling pathway which plays an important role in the growth and development of hair follicles.