The Role Of LncRNA5322 In HFSCs Proliferation,differentiation And Skin Injury Repair

BackgroundAs the largest tissue and organ,the skin contacts most extensively with the outside world,which thus working as the primary barrier against exterior hazards.Since the skin is the outermost layer throughout human body,skin lesions caused by various causes are quite common,especially from burns,scalds and chronic ulcers on body surface.Large areas of wounds need to be covered with new surface.However,the largest defect of clinical skin grafts is the lack of skin appendages currently.Hair follicle is one of the common types of skin appendages.Although the structure is very tiny,the biological function of follicles is complicated,among which the most essential part is hair follicle stem cells(HFSCs).HFSCs have the potentials to differentiate into different directions,including hair follicles,sebaceous glands and epidermis,which plays important roles in hair regeneration and hair color phenotype.Besides,HFSCs can work as seed cells,which makes HFSCs the research focus in the tissue engineering.Long-chain non-coding RNAs(LncRNAs)are non-coding RNAs longer than 200 nucleotides.LncRNAs are defined as non-protein-coding transcripts and play important roles in the proliferation and differentiation of stem cells.The expression and function aberration of LncRNAs have been demonstrated to be closely related with various diseases,including cancer,neurodegenerative diseases.The dysfunction mainly consists of the abnormalities of the sequence and spatial structure,expression levels,and its interaction with binding proteins,etc.LncRNA AK015322 is mainly enriched in testicular tissues and has been found to promote the proliferation of spermatogonial stem cells(C18-4)by trapping microRNA-19b-3P.As endogenous non-coding small RNA,microRNA(miRNA)can target the corresponding mRNA,thereby inducing its degradation or inhibiting the translation process,which plays important roles in regulating gene expression.miR-21 is one of the earliest studied miRNA and it has been widely proved with high expression levels in almost all the solid tumors.miR-21 regulates the development of pancreatic cancer via targeting different genes,and participates in a series of biological processes including the proliferation,differentiation,migration,invasion,and drug resistance of pancreatic cancer stem cells.PI3K/AKT is a relatively common signal pathway and it's involved in various diseases such as tumors,metabolic disorders,kidney diseases,and mental disorders.PI3K/AKT has been discovered to affect different biological behaviors such as cell proliferation,differentiation,apoptosis,and migration.Therefore,it is of vital significance to explore the effects of LncRNA5322 on the proliferation and differentiation of HFSCs and its potential roles in skin injury repair.Part 1 LncRNA5322 promotes the proliferation and differentiation of HFSCs Objective The overexpression vector of LncRNA5322 was constructed to explore the effects of LncRNA5322 on the proliferation and differentiation of HFSCs.Methods The LncRNA AK015322 overexpression vector was constructed using plasmid pCDNA3.1.After successful construction,it was transfected into HFSCs,which was named as LncRNA AK015322 overexpression group.In addition,the control group was transfected with the empty vector pCDNA3.1.The proliferation and differentiation of the two cell lines were detected by oil red O staining and CCK-8 kits.The OD value was detected by a microplate reader and the influence of LncRNA5322 on the proliferation and differentiation of HFSCs was explored based on the OD value.Results1.The constructed plasmid was transfected into cultured HFSCs,and the transfection effect was detected by real-time quantitative PCR.It was found that the transfection in the LncRNA AK015322 overexpression group did increase the expression of lncRNA5322 in HFSCs compared with the control group.2.Compared with the control group,lncRNA5322 transfection stimulated the proliferation of HFSCs(P<0.05).Besides,lncRNA5322 transfection promoted the differentiation of HFSCs(P<0.05).Conclusion:LncRNA5322 promotes the proliferation and differentiation of HFSCs.Part 2 LncRNA5322 regulates the proliferation and differentiation of HFSCs through PI3K/AKT signaling pathwayObjective To investigate the potential signaling pathways analyzing the mechanisms of the involvement of LncRNA5322 in HFSCs.Methods The LncRNA AK015322 overexpression vector constructed in the first part was used to firstly predict the correlation between LncRNA AK015322 and miR-21 through bioinformatics analysis,and then the expression levels of miR-21 in the overexpression and control cell lines were detected by real-time quantitative PCR.Flow cytometry was used to investigate the cell cycle of HFSCs.Real-time quantitative PCR and Western blot were applied to verify the expression of cyclin-dependent kinase CDK1 and CDK2 mRNA and proteins.The expression of pAKT/AKT-related pathways was reversed.On the contrary,using the miR-21 agonist(agomir-21),Western blot was again used to verify the expression of pAKT/AKT-related pathways from the opposite direction.Results1.Through the online microRNA-target tools,the target site of miRNA was predicted,and the common miRNAs related to LncRNA5322 related binding sites were identified,namely miR-21 and miR-19b-3p.Among them,miR-19b-3p has been reported to be involved in promoting the proliferation of spermatogonial stem cells C18-4.LncRNA5322 could up-regulate miR-21(P<0.05).2.LncRNA5322 transfection promoted CDK1 and CDK2 mRNA and protein expression in HFSCs.3.LncRNA5322 up-regulated the expression levels and the phosphorylation of PI3K and AKT in HFSCs(P<0.05).4.Agomir-21 eliminated the increased expression levels and the phosphorylation of PI3K and AKT in LncRNA5322 overexpressed HFSCs(P<0.05).Conclusion:LncRNA5322 regulates the proliferation and differentiation of HFSCs through PI3K/AKT signaling pathway.Part 3 The role of LncRNA5322 in skin lesion repairObjectiveA nude mouse skin wound model was constructed to explore the role of LncRNA5322 in skin lesion repair.MethodsThe nude mice were used to establish a skin injury model and were randomly divided into Sham group,trauma model group and LncRNA5322 treated wound model group.After the wound was formed,it was not treated with bandaging or medication.The mice were placed in a sterile laboratory and were sterilized daily.In addition,the LncRNA5322 group was treated with antagonist LncRNA5322 to inoculate wounds after surgical blade cutting.The wounds were photographed on the first day,the third day,and the sixth day after wound formation,and the effect of the wound healing ability of LncRNA5322 was observed via immunohistochemistry and leukocyte count.Results1.On the 1st,3rd,and 6th day after wound formation,the area of wounds treated with HFSCs transfected with plasmid expressing LncRNA5322 were reduced to 87.6%,70%,and 50.6%separately.On the 1st,3rd and 6th day,the area of wounds inoculated with conventional HFSCs was reduced to 90.9%,79.1%,and 61.5%of the original lesions.The wound area of the control group without any treatment was reduced to 95%,90.5%,and 85%.Under light microscope,neutropenia was found,a large number of mononuclear cells were increased,and fibroblasts were also increased significantly.2.LncRNA5322 promotes the regeneration of epidermis and the accessory organs.On the 14th day after wound formation,epidermis wounded treated with HFSCs transfected with plasmids expressing LncRNA5322 basically healed and began to grow hair,and wound epidermis of conventional HFSCs was inoculated.Without complete healing,the wound area of the control group without any treatment was reduced to 78.5%of the original wound area.On the 20th day after wound formation,the epidermis of the wound treated with HFSCs transfected with plasmids expressing LncRNA5322 was completely healed and the growing hair reached 2 mm.The wound epidermis inoculated with conventional HFSCs basically healed and began to grow hair.The wound area of any treated control group was reduced to 51.2%of the original wound area.Conclusion LncRNA5322 plays an important role in skin injury repair and provides a new theoretical basis for the repair of skin lesions in clinical cases.