Let-7b-5p Regulates Proliferation And Apoptosis By Targeting ΔNp63 In HaCaT Cells

Objective: Wound healing has long been an insurmountable clinical problem,recent study identified the presence of the “incognito healing” phenomenon on fetal skin,at the same time,p63 has a crucial role in regulating epidermal development and other keratinized epithelial’s formation and maintenance.Micro RNAs(miRNAs)has been involved in molecular regulation of epidermal development.This study thus aims to explore the potential function of miRNA targeting to ΔNp63 in human epidermal development,with the goal of guiding better designs for wound therapies.Methods:Expression profiles of miRNAs were obtained by small RNA sequencing of fetal skins samples,and the differentially expressed miRNAs(DEmiRNAs)between the skin samples associated with phase of gestation were identified by Network Analyst website.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses were used to functionally analyze the target m RNAs of the DEmiRNAs.Thereafter,the DEmiRNAs were subjected to a miRNA set enrichment analysis using the TAM 2.0(http: //www.lirmed.com/tam2/)tool,aiming to screen out miRNAs that may play a role in skin development.RNAbridy were used to predict the binding of miRNAs with ΔNp63.Finally,let-7b-5p which can bind to ΔNp63 was screened out to further biological function and underlying mechnism research.Ha Ca T cells were transfected with let-7b-5p mimic or let-7b-5p inhibitor.Cell proliferation testing was detected by Cell Counting Kit-8(CCK-8),and cell apoptosis testing was taken by flow cytometry.At the same time,quantitative real time polymerase chain reaction(q RT-PCR)was used to detect the relative expression levels of apoptosis-related genes.To further determine the relationship between miRNA let-7b-5p and ΔNp63,Dual-luciferase reporter assay(DLR)used for reporter bindings between them.We tested the proliferation and apoptosis of Ha Ca T cells after stably infected with ΔNp63 overexpressing lentivirus.Western-blot was used to detect the changes of the expression of PI3K/AKT signaling pathway.Results: 1.Bioinformatics analysis: There were 375 DEmiRNAs in the fetal skin samples.GO and KEGG analyses showed that the target m RNAs were mainly enriched in "positive regulation of cell proliferation" ","translation initiation factor binding"," PI3K/AKT signaling pathway" and "cancer pathway".Next,the miRNA set enrichment analysis using TAM 2.0(http: //www.lirmed.com/tam2/)showed that differentially expressed up-regulated miRNAs are mainly enriched in the let-7 family and are mainly involved in“ cell-related differentiation”,“cell death” and other functions;differentially expressed down-regulated miRNAs are mainly enriched in the mi R-3180 family,and are mainly involved in cell adhesion,cell senescence and other functions.2.Function of let-7b-5p on Ha Ca T cells: we selected let-7b-5p for cell-level verification based on the predicted binding relationship between let-7 family and ΔNp63.CCK-8 showed that let-7b-5p overexpression can inhibit the proliferation of Ha Ca T cells(P <0.05).When let-7b-5p expression is inhibited,it can promote the proliferation of Ha Ca T cells(P<0.05).Flow cytometry and quantitative real-time polymerase chain reaction show that upregulation of let-7b-5p could promote apoptosis in Ha Ca T cells,the expression BAK,BAX,BIM apoptosis-related molecules is up,the expression of BCL-XL is down,(P<0.05);while downregulation of let-7b-5p levels could inhibit apoptosis of Ha Ca T cell,the expression of BAK,BAX,BIM is down regulated,the expression of BCL-XL is up regulated,(P<0.05).3.Mechanism of let-7b-5p affecting proliferation and apoptosis in Ha Ca T Cells:Double-Fluorase reporter gene assay show that let-7b-5p combining with the ΔNp63 3’-UTR zone,affecting ΔNp63 transcription and thus inhibiting ΔNp63 expression.The quantitative real-time PCR show that the expression level of ΔNp63 can be down regulated in Ha Ca T cells after let-7b-5p mimic transfection,the expression level ofΔNp63 can be up regulated after let-7b-5p inhibitor transfection.CCK-8 results showed that ΔNp63 over-expression promotes Ha Ca T cell proliferation(P < 0.05).Flow cytometry and quantitative real-time PCR showed that ΔNp63 overexpression inhibits Ha Ca T cell apoptosis,the expression of BAK,BAX,BIM is down regulated,the expression of BCL-XL is up regulated(P<0.05).Additionally,Western-blot showed that let-7b-5p mimic transfection could increase PI3 K and AKT protein levels in PI3K/AKT pathways.Conclusions: The study successfully established miRNA expression profile of human embryo skin and found that let-7 family plays an important role in skin development.In the let-7 family,let-7b-5p can bind to ΔNp63,let-7b-5p can negatively regulate the expression of ΔNp63,inhibit the proliferation and promote the apoptosis of Ha Ca T cells,it changes the PI3K/AKT signal pathway,which shows that let-7b-5p plays a key role in skin development and provides important reference for the diagnosis and treatment of skin development related diseases.