Study Of MiR-21-5p On Proliferation In Dermal Papilla Cells Of Cashmere Goat By Regulating SPRY2 Expression

Hair follicle morphogenesis of cashmere goats originates from the interaction between epithelial cells and dermal cells,and mainly goes through three stages: induction,organogenesis and cytodifferentiation.Hair follicle morphogenesis plays an important role in the development of hair follicles.Therefore,it is of great significance to study the molecular mechanism of hair follicle morphogenesis for improving the yield and quality of cashmere.The preliminary transcriptomic sequencing results of our research group and further tests showed that the expression level of SPRY2 in the skin tissue of cashmere goats at 65 d was significantly higher than that at 120 d,but its effect on hair follicle development in cashmere goats has not been studied.Dermal papilla cells continue to send out induction signals throughout the hair follicle growth process to stimulate the hair follicle to continue to grow.Therefore,this study took Shannorthern cashmere goat dermal papilla cells as the research object.By constructing adenovirus vector with SPRY2 overexpression,to reveal its effects on the viability and proliferation of dermal papilla cells of cashmere goats,and to study its upstream regulatory mechanism,the following main results were obtained:1.SPRY2 overexpressed adenovirus vector was constructed to transfect dermal papilla cells.The function of SPRY2 in cashmere goat dermal papilla cells was studied by CCK8 and Ed U.The results of CCK8 cell viability test showed that the activity of dermal papilla cells was significantly increased after overexpression of SPRY2.The Ed U cell proliferation test results showed that the proportion of Ed U positive cells after overexpression of SPRY2 was significantly higher than that in the control group.After overexpression of SPRY2,the m RNA and protein expressions of CDK4,MKI67,PCNA and C-Myc were significantly increased,which further indicated that SPRY2 could promote the proliferation of dermal papilla cells.2.Biological information analysis software was used to predict miR-21-5p targeting SPRY2,and dual luciferase assay was used to verify the targeting relationship between miR-21-5p and SPRY2.The results showed that after overexpression of miR-21-5p,the relative enzyme activity ratio of SPRY2 gene was significantly lower than that of the control group.After inhibiting its expression,the ratio increased significantly.However,there was no significant difference in the relative enzyme activity ratio of SPRY2 after mutation of the targeted binding site.The results of RT-q PCR and WB showed that the expression level of SPRY2 gene was significantly decreased after overexpression of miR-21-5p,while the expression level of SPRY2 was significantly increased after inhibition of miR-21-5p expression,indicating that miR-21-5p regulates its expression through targeted binding to SPRY2 3’UTR region.3.To further study the function of miR-21-5p in dermal papilla cells,mimics/nc and inhibitor/nc of miR-21-5p were transfected into dermal papilla cells,respectively.CCK8 and Ed U tests showed that the viability and proliferation of dermal papilla cells were significantly inhibited after overexpression of miR-21-5p.However,inhibition of miR-21-5p can increase the activity of dermal papilla cells and promote their proliferation.The results of RT-q PCR and WB further showed that the expression of proliferation-related genes was significantly decreased after overexpression of miR-21-5p,while the expression of proliferation-related genes was significantly increased after inhibition of miR-21-5p,indicating that miR-21-5p could inhibit the proliferation of dermal papilla cells.SPRY2 overexpressed adenovirus and miR-21-5p mimics/nc were further co-transmised into dermal papilla cells for recovery tests.The results showed that overexpression of SPRY2 could antagonize the inhibitory effect of miR-21-5p on its endogenous expression.The results indicated that miR-21-5p could inhibit the proliferation of cashmere goat dermal papilla cells by negatively regulating the expression of SPRY2 gene.This study revealed the role of SPRY2 gene in the proliferation of cashmere dermal papilla cells,and providing a theoretical basis for exploring the hair follicle development in cashmere goats.