Analysis Of Differentially Expressed Profiling Of LncRNAs And MRNAs With Associated CeRNA Networks In Epidermal Stem Cells

Objective:Long noncoding RNAs（lncRNAs）have important roles in various biological processes by regulating the expression of genes.However,their potential role in epidermal stem cells（ESCs）self-renewal and differentiation remains poorly understood,and whether it act as ceRNA in ESCs needs investigation.Methods:（1）The epidermis was isolated from freshly discarded prepuce surgical samples with Trypsin.Rapid adhesion to collagen IV was used to isolate human ESCs and differentiated keratinocytes（DK）.Inverted phase contrast microscope and immunostaining of integrinsβ1,CK19,CK1 and CK10 were used for cell identification.Use Trizol to extract total RNA from samples;purify it after quality inspection qualified;then to the procedures of sample labeling and hybrization.Now hybrid images can be obtained through scanning the washed microarray slides by Agilent Scanner.Analyze these images and extract the data by Agilent Feature Extraction software;then normalize and analyze the data by Agilent GeneSpring software.The significant differential expressions of representative mRNAs and lncRNAs were further confirmed using qRT-PCR.Subsequently,we acquired associated pathways and gene ontology items by GO and KEGG analyses.（2）According to the positional relationship between lncRNA and the adjacent mRNA in the same chromosome,we classified lncRNAs as natural antisense,enhancer-like lncRNAs,intergenic and exon sense-overlap.Then,using BLAST analyses lncRNA relative to and selected two exon lncRNAs to explore their regulatory mechanisms.So,TargetScan 6.2 software.which was used to predict the target miRNAs of the similar sequence.We build the LncRNA-mRNA co-expression network of four differential lncRNAs,including NR₀73046,NR₀45013,and carry out KEGG and GO analysis of the lncRNAs–Coexpressed mRNAs.Results:（1）ESCs uniformly distributed on collagen IV and formed clones after cultured for 3 days,which were positive expression of CK19 and integrin β1.DKs were non-adherent cells and without clones after cultured for 3 days,which were positive expression of CK1 and CK10.The extracted total RNA exhibited high levelsof purity and quality.We identified 3720 lncRNAs and 4069 m RNAs to be differentially expressed in ESCs compared with DKs,in which 2292 lncRNAs and1248 mRNAs were upregulated,1428 lncRNAs and 2821 mRNAs were downregulated.The qRT-PCR results validated that expression of lncRNAs and mRNAs were consistent with the data of microarray.GO and KEGG analysis indicated that differentially expressed mRNAs participate in controlling ESC state,including regulation of developmental growth,cellular process involved in cell reproduction,death and so on.（2）Furthermore,we found 293 differentially expressed enhancer-like lncRNAs,165 natural antisense lncRNAs,768 lincRNAs and 759 exon sense-overlapping lncRNAs.16 exon sense-overlapping lncRNAs showed high sequence similarity to the exons of mRNA,in which NR₀45013 and NR₀73046 were astonishing similarity to LMO3 and DEDD2,respectively.CeRNA network inferred that NR₀45013 acted as a competing endogenous RNA,bound to miR-141-3p/200a-3p,then regulated the expression of the miRNA targets LMO3.Similarly,NR₀73046acted as a competing endogenous RNA,bound to miR-204-5P/211-5p,then regulated the expression of the miRNA targets DEDD2.Moreover,Co-expression analysis showed lncRNAs NR₀73046 and NR₀45013 expression were correlated with mRNAs DEDD2 and LMO3,respectively,which participate in apoptosis,proliferation and differentiation etc.Conclusion:（1）Differentially expressed lncRNA and mRNA detected between human ESCs and DKs in vitro,which may play an important role in ESCs self-renewal and differentiation.（2）NR₀45013 and NR₀73046 may compete with the miRNA pool to regulate the expression of LMO3 and DEDD2,respectively.