| Hair follicles are important appendages of the skin and play a key role in appearance modification,wound repair,and remodeling of the skin microenvironment.Nevus sebaceous(NS),is a benign hamartoma of the skin,characterized by hyperplasia of the epidermis,immature hair follicles,normal or hyperplastic sebaceous glands.The development of hair follicles in nevus sebaceous is arrested in early stage mimicing hair germ-like structures which provides a good clinical model for the study of hair follicle dysplasia.The exact mechanisms of folliculo-sebaceous-apocrine defects are unknown in NS.Long non-coding RNAs(lncRNAs)have been implicated in various important biological processes and regulate inflammatory diseases and tumors.In order to study the cause of hair folliclesebaceous gland dysplasia in sebaceous nevus,we performed RNA-seq on NS and normal scalp tissue to obtain differentially expressed mRNAs and LncRNAs.We selected and verified a downregulated gene CDKN2AIP by qPCR and western blot and constructed a ceRNA regulatory network between CDKN2AIP and differentially expressed LncRNAs.Finally,we confirmed that lncRNA linc3556,was downregulated in nevus sebaceous.In order to explore the regulatory relationship between linc3556 and CDKN2AIP genes and their effects on hair follicle development and keratinocyte,we further investigated their roles from cellular and molecular levels.Part Ⅰ:Genome wide differential expression profiles in nevus sebaceousObjective:To identify NS-associated mRNA and lncRNA profiles and predict their potential roles in the development of the folliculo-sebaceous unit.Methods:RNA-seq was used to identify NS-associated mRNAs and lncRNAs.Analysis software Illumina NovaSeq 6000 was used to analyze the sequences and real-time PCR and western blot were used to validate the differentially expressed genes.Competing endogenous RNAs(ceRNA)networks were constructed by prediction software TargetScan&miRanda.Results:Many mRNAs were significantly differentially expressed between nevus sebaceous and adjacent normal scalp tissues.Among them,72 were upregulated and 18 were downregulated.KEGG pathway analysis further revealed that 32 functional pathways were associated with the upregulated mRNAs,while only 1 pathway was associated with the downregulated mRNAs.Verification by real-time PCR and western blot indicated CDKN2AIP gene was downregulated consistently in NS tissue compared to normal scalp skin.Additionally,7 upregulated and 10 downregulated significantly differentially expressed lncRNAs were detected between NS and adjacent normal scalp tissues.Three downregulated lncRNAs including AL355607.2,RP5-1024G6.8 and AC007780.1 were predicted to consistently associate with CDKN2AIP expression by competing endogenous RNAs(ceRNA)construction.AL355607.2(linc3556)was verified down expressed in NS by qPCR.Conclusion:The mRNA and lncRNA expression profiles in sebaceous nevus were constructed,and the down-regulated gene CDKN2AIP in sebaceous nevus was screened,and the(ceRNA)regulatory network was constructed to verify the downregulated linc3556,which provided a data source for subsequent research.Part II:To investigate the biological function of CDKN2AIP in human dermal papilla cell and human immortalized keratinocyteObjective:To study the effect of CDKN2AIP gene on the proliferation,apoptosis,cell cycle and dermal papilla cytokines of hDPC and HaCat.Methods:The expression of CDKN2AIP gene in hDPC and HaCat cell was detected by qPCR and western blot.CDKN2AIP gene was silenced in hDPC and HaCat using short hairpin(shRNA)technology,and the efficiency of transcriptional and translational level was detected by qPCR and western blot,respectively.The effect of CDKN2AIP gene on the proliferation of hDPC and HaCat cells were detected by CCK-8 and Edu,cell cycle and apoptosis were detected by PI and AnnexinVAPC/7AAD staining,respectively.The expression of cytokines ALP,IGF-1,VEGF,wnt-10b,β-catenin,lef-1 and c-myc in hDPC were detected by qPCR.Results:The transcription and translation levels of CDKN2AIP gene between hDPC and HaCat were nonspecific;The CDKN2AIP gene silenced in hDPC had significantly decreased proliferation(P<0.05),increased apoptosis(P<0.05)and did not change the proportion of cell cycle GO/G1,S,G2/M(P>0.05).Besides,the secretion of cytokines ALP,IGF-1,VEGF,wnt-10b,β-catenin,lef-1 and c-myc was inhibited(P<0.05).While,CDKN2AIP gene silenced in HaCat significantly decreased proliferation(P<0.05),increased apoptosis(P<0.05),cell cycle GO/G1 had no significant difference(P>0.05),S phase was decreased(P<0.05),and G2/M phase was increased(P<0.05),respectively.Conclusion:CDKN2AIP can promote the proliferation of hDPC,inhibit apoptosis,and promote the ability of DPC to induce hair follicle reconstruction.While,it has no influence on hDPC cell cycle.In HaCat cell,CDKN2AIP can promote proliferation,inhibit apoptosis,and promote cell from entering S phase.Part III:To investigate the regulatory effect between linc3556 and CDKN2AIP,and biological function of linc3556 in human dermal papilla cell and human immortalized keratinocyteObjective:To study the regulatory effect of linc3556 on CDKN2AIP,and its effect on proliferation,apoptosis,and cell cycle in hDPC and HaCat.Methods:The expression of linc3556 in hDPC and HaCat was detected by qPCR.The localization of linc3556 in hDPC and HaCat was detected by fluorescence in situ hybridization(FISH)and nucleocytoplasmic separation.Oligonucleotide(ASO)technology was used to interfered the expression of linc3556 in hDPC and HaCat,then interfering efficiency and CDKN2AIP expression were detected by qPCR.The effect of linc3556 on proliferation were detected by CCK-8 and Edu methods.Cell cycle and apoptosis were detected by PI and AnnexinV-APC/7AAD staining,respectively.The expression of cytokines ALP,IGF-1,VEGF and wnt-10b,β-catenin,lef-1 and c-myc in hDPC were detected by qPCR.Results:There was no significant difference in the transcriptome expression of linc3556 between hDPC and HaCat(P>0.05).FISH and nucleocytoplasmic separation technique indicated that linc3556 was mainly expressed in the cytoplasm.The expression level of linc3556 and CDKN2AIP expression were significantly inhibited(P<0.05)after interfering linc3556 in hDPC and HaCat.Interfering with linc3556 in hDPC significantly inhibited cell proliferation(P<0.05),increased apoptosis(P<0.05),inhibited expressions of ALP,IGF-1,VEGF,wnt-10b,β-catenin,lef-1,and c-myc,while had no significant difference in the ratio of GO/G1,S,G2/M phases of cell cycle(P>0.05).HaCat interfered with linc3556 had significantly decreased proliferation(P<0.05),increased apoptosis(P<0.05),decreased G2/M phase(P<0.05),while had no significant difference in GO/G1 and S phase in cell cycle.Conclusion:Linc3556 located mainly in the cytoplasm and has transcriptional regulation on CDKN2AIP.In hDPC,it promotes proliferation,inhibits apoptosis,promotes the ability of dermal papilla cells to induce hair follicle reconstruction,but does not change the cell cycle.In HaCat,Linc3556 can promote cell proliferation,inhibit apoptosis,and promote cells to enter G2/M phase. |
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