Study On The Mechanism Of Hypoxia Promoting The Proliferation Of Dermal Papilla Cells Through MicroRNA-Hippo Signaling Pathway

Inner Mongolia Albas Cashmere Goat is a fine breed of goat having an excellent cashmere and meat production in Inner Mongolia Autonomous Region,especially cashmere is known as"soft gold".The hair follicle（HF）is a kind of skin accessory organ.In cashmere goats,HF can be divided into primary and secondary hair follicles,in which the primary hair follicles can grow into wool and the secondary hair follicles can grow into cashmere.Dermal papilla cells（DPC）locate at the bottom of HF as one of the most important cell types in hair follicles and have the function of regulating hair cycle and maintaining hair growth.Hypoxia,as a microenvironment widely existes in animals,has been shown to promote cell proliferation,delay cell senescence and improve the regeneration potential.Hippo signaling pathway is a highly active pathway in stem cells,which plays a key role in regulating the size of animal organs,limiting cell proliferation and inducing apoptosis.MicroRNA（miRNA）is a highly conserved,short sequence non-coding RNA consisting of 18-25 nucleotides and is widely found in animals.It has been reported that miRNA is involved in the regulatory network of the hypoxic microenvironment and the Hippo signaling pathway,but the regulatory mechanism of the miRNA-Hippo signaling pathway in the hypoxic microenvironment is still unclear.In this paper,primary dermal papilla cells（PHF-DPCs）and secondary dermal papilla cells（SHF-DPCs）of Inner Mongolia Albas Cashmere Goats were used as experimental materials.The following researches were carried out after the four treatment methods of normoxia,hypoxia,chemical hypoxia mimic 25μM DFO,and chemical hypoxia mimic150μM CoCl₂:1、Screening of treatment durationIn this study,PHF-DPCs and SHF-DPCs were treated by the above four treatment methods for 24h,48h,and 72h respectively,and then tested with CCK-8 reagent.The results showed that the best cell activity of PHF-DPCs and SHF-DPCs treated with the above four treatment methods for 48h were observed.2、Identification of hair papillary cell types and hypoxic environmentIn this study,DPC specific surface marker proteins CD133 andα-SMA were detected by immunofluorescence,the results showed that PHF-DPCs and SHF-DPCs were positive after the four treatments,which proved that the cells used in this experiment were PHF-DPCs and SHF-DPCs,and hypoxia treatment did not change the cell type.To determine whether the hypoxic environment was successfully established,Western Blot was used to detect the expression of hypoxia-inducible factor HIF-1αat the protein level.The results showed that the expression of HIF-1αin PHF-DPCs and SHF-DPCs treated with physical hypoxia,chemical hypoxia mimic 25μM DFO,and chemical hypoxia mimic 150μM CoCl₂ were increased compared to that of normoxia.This results proved that the hypoxic environment had been successfully established.3、Cell apoptosis detectionIn order to understand the effects of above four treatment methods on apoptosis of PHF-DPCs and SHF-DPCs,flow cytometry was used to detect apoptosis.The results showed that the proportion of late apoptosis and dead cells in PHF-DPCs and SHF-DPCs could be reduced after physical hypoxia,chemical hypoxia mimic 25μMDFO,chemical hypoxia mimic 150μM CoCl₂ treatment for 48h compared with normoxia,but the proportion of early apoptosis was increased.This proved that hypoxia could effectively delay cell apoptosis.4、Detection of apoptosis related genes and cell marker genesIn this study,the above four treatment methods were used to treat PHF-DPCs and SHF-DPCs for 24h,48h,and 72h respectively.First,real-time quantitative PCR was used to detect the apoptosis genes p53,Bcl-2,and Bax.The results showed that the expression of p53 was lower,and the ratio of Bcl-2/Bax was higher after 48h treatment than that of 24h and 72h treatments.The expression of p53 in physical hypoxia,chemical hypoxia mimic 25μM DFO,and chemical hypoxia mimic 150μM CoCl₂treatments were lower than that of normoxia,and the Bcl-2/Bax ratio was higher.Western Blot was used to detect the apoptosis-related genes in PHF-DPCs and SHF-DPCs treated by the above four treatment methods for 48h,the results were basically consistent with the real-time quantitative PCR results.Subsequently,real-time quantitative PCR was used to detect the cell marker genes Sox2,ALP,α-SMA,the results showed that after 48 hours of treatment,the expression levels of Sox2,ALP,andα-SMA were all higher than that of 24 and 72 hours.The expression of Sox2,ALP,α-SMA in physical hypoxia,chemical hypoxia mimic 25μM DFO,and chemical hypoxia mimic 150μM CoCl₂ treatments were higher than that of normoxia.Western Blot was used to detect the cell marker genes in PHF-DPCs and SHF-DPCs treated by the above four treatment methods for 48h,the results are basically consistent with the real-time quantitative PCR results.The results proved that hypoxia could promote the proliferation of PHF-DPCs and SHF-DPCs.5、Detection of the key genes in Hippo signaling pathwayPHF-DPCs and SHF-DPCs were treated with the above four treatment methods for 48h,the expression of the key genes Lats2 and YAP of Hippo signaling pathway were detected by Western Blot.The results showed that compared with normoxia,the expression level of YAP was increased in physical hypoxia,chemical hypoxia mimic25μM DFO and chemical hypoxia mimic 150μM CoCl₂,while the expression level of Lats2 was decreased.This results proved that the Hippo signaling pathway played a role in promoting the proliferation of PHF-DPCs and SHF-DPCs by hypoxia.6、Screening and verification MicroRNAFirst the possible miRNA targeting of Lats2 was predicted through the website,and verified in the PHF-DPC and SHF-DPC treated with the above four treatment methods for 48h by real-time quantitative PCR.The results showed that the expression levels of miR-25-3p and miR-107-3p were increased after 48 hours of physical hypoxia,chemical hypoxia mimic 25μM DFO,and chemical hypoxia mimic 150μM CoCl₂treatments compared with normoxia.Then the pmirGLO Dual-Luciferase miRNA Target Expression Vector recombinant plasmid vector containing the 3’UTR of the Lats2 gene was built,and the targeted correlation of miR-25-3p and miR-107-3p with Lats2 was preliminary judged by dual luciferase reporting system.Finally the Western Blot test results showed that the expression levels of miR-25-3p mimics and miR-107-3p mimics groups were lower than those of NC mimics groups.This results proved that miR-25-3p and miR-107-3p were both targeted miRNAs of Lats2.In conclusion:Hypoxia promoted the growth of PHF-DPCs and SHF-DPCs through miR-25-3p and miR-107-3p inhibiting Lats2.In this study it was discovered that DPC proliferation could be promoted by the pathways of miRNA and Hippo signaling pathways in hypoxia,which provided an experimental basis for the continuous deepening of related research in the future.This results were of great significance for the study of hair follicle regeneration biology and understanding the mechanism of hypoxia.