| The Yangtze Delta White Goat is a unique local goat breed in China.It is characterized by a special high-quality brush hair.Transcriptome sequencing revealed that the expression levels of DUSP6 differed significantly in the skin tissues of quality and non-quality pencil wool individuals.The aim of this study was to investigate the miRNAs targeting DUSP6 during the formation of quality pencil hair traits and further investigate the regulatory role of these miRNAs on the growth of HFSCs in Yangtze Delta white goats.The miRNAs interacting with DUSP6 were predicted and screened by TargetScan database.miR-133a-3p RNA oligo,miR-145-5p RNA oligo and DUSP6 dual luciferase reporter gene vectors were constructed to verify the targeting relationship between miR-133a3p,miR-145-5p and DUSP6.The targeting relationship between miR-133a-3p,miR-145-5p and DUSP6 was verified.On this basis,the changes of DUSP6 expression levels after overexpression or knockdown of miR-133a-3p and miR-145-5p,respectively,were further examined by qPCR and Western Blot(WB).To investigate the effects of miR-133a-3p and miR-145-5p on cell growth,the expression levels of proliferation-related genes(PCNA,CDK1,CCND2)and apoptosis-related genes(BAX BCl2)in HFSCs after overexpression or knockdown of miR-133a-3p and miR-145-5p were analyzed;meanwhile,the expression levels of miR-133a-3p and miR-145-5p were combined with flow cytometry and EdU.The effects of miR-133a-3p and miR-145-5p on cell cycle and apoptosis of HFSCs were also verified by combining flow cytometry and EdU.The results of this study are as follows:(1)13 miRNAs with possible targeting relationship with DUSP6 were predicted by TargetScan database,and finally miR-133a-3p and miR-145-5p with the highest binding site specificity score were selected for further study in combination with the current studies on hair follicle-related studies.(2)By comparing miRBase,TargetScan database,the mature sequences of miR-133a-3p and miR-145-5p were found to be highly conserved in different species such as bovine,murine,porcine and dog.(3)The effects of overexpression or knockdown of miR-133a-3p and miR-145-5p in HFSCs of Yangtze Delta white goats were examined by qPCR and WB on the expression levels of proliferation and apoptosis-related genes;the changes of cell proliferation ability after overexpression and knockdown of miR-133a-3p and miR-145-5p were investigated by EdU assay;the changes of cell proliferation ability after overexpression and knockdown of miR-133a-3p and miR-145-5p were examined by The changes of cell cycle and apoptosis after miR-133a-3p and miR-145-5p were detected by flow cytometry,and the results confirmed that overexpression of miR-133a-3p and miR-145-5p both promoted apoptosis and inhibited proliferation of HFSCs.(4)The DUSP6 dual luciferase reporter gene vector was constructed.The results of dual luciferase reporter gene assay,qPCR and WB experiments indicated that miR-133a-3p and miR-145-5p had a targeting relationship with DUSP6.In this study,the regulatory mechanisms of miR-133a-3p and miR-145-5p on HFSCs were investigated,i.e.,miR-133a-3p and miR-145-5p targeted to inhibit DUSP6 through MAPK signaling pathway,which in turn inhibited the proliferation of HFSCs and promoted their apoptosis.This experiment provides a theoretical basis for further elucidation of the molecular mechanism of quality pen hair traits in Yangtze River Delta white goats. |
