LncRNA TRHDE-AS1 Inhibit The Scar Fbroblasts Proliferation Via MiR-181a-5p/PTEN Axis

BackgroundHypertrophic scar(HS)is a fibroproliferative disease caused by abnormal wound healing after skin injury.It is characterized by excessive deposition of extracellular matrix and aggressive growth of fibroblasts.Recent studies have shown that some non-coding RNA(nc RNA)such as long noncoding RNA(lncRNA)and micro RNA(miRNA)are involved in the formation of HS,but the mechanism is still unclear.It is reported in the literature that lncRNA TRHDE-AS1 is down-regulated in HS tissue and hypertrophic scar fibroblast(HSF),which may be related to the formation of scars.In order to understand the role of lncRNA TRHDE-AS1 mediated HSF proliferation and apoptosis,we used Lnc Base(http://carolina.imis.athena-innovation.gr/diana＿tools)to predict miRNA targets,and bioinformatics analysis showed that lncRNA TRHDE-AS1 contains a conserved target site of miR-181a-5p.No.10 chromosome lost tensing protein homologous phosphates and tensing homologue deleted on chromosome 10(PTEN)is a tumor suppressor with key functions in cell cycle,apoptosis,proliferation and migration.It has been reported in the literature that the lack of PTEN is related to the pathogenesis of HS,and its down-regulation leads to the activation of the PTEN/protein kinase B(AKT)pathway.We predicted the target genes of miR-181a-5p by using Target Scan(http://www.targetscan.org).It is predicted that PTEN contains the binding sequence of miR-181a-5p.Therefore,we speculate that lncRNA TRHDE-AS1 is down-regulated in hypertrophic scar tissue,and the down-regulated lncRNA TRHDE-AS1 negatively regulates miR-181a-5p,which makes the level of miR-181a-5p increase,and the increased miR-181a-5p pair Over-regulation of the PTEN gene leads to a decrease in PTEN,and its down-regulation leads to the activation of the PTEN/AKT pathway.If the level of lncRNA TRHDE-AS1 in local tissues is increased,it will be possible to inhibit the formation of scars and provide a new target for the clinical treatment of HS.Hypertrophic scar(HS)is a fibroproliferative disease caused by abnormal wound healing after skin injury.It is characterized by excessive deposition of extracellular matrix and aggressive growth of fibroblasts.Recent studies have shown that some non-coding RNA(nc RNA)such as long noncoding RNA(lncRNA)and micro RNA(miRNA)are involved in the formation of HS,but the mechanism is still unclear.No.10 chromosome missing tensing protein homologous phosphates and tensing homologue deleted on chromosome 10(PTEN)is a tumor suppressor,and PTEN is also involved in the formation and development of HS.It has been reported in the literature that lncRNA TRHDE-AS1 is down-regulated in HS tissues and hypertrophic scar fibroblast(HSF),which may be related to the formation of scars.In order to explore the role and mechanism of lncRNA TRHDE-AS1 in the formation of HS,the research of this subject was carried out.Objective1.Clarify the role of lncRNA TRHDE-AS1 in the formation of HS.2.Confirm the existence of lncRNA TRHDE-AS1 /miR-181a-5p /PTEN regulatory pathway in HS formation.Methods1.Collect 30 cases of HS patients with scar tissues and adjacent normal skin(Normal Skin,NS)tissues,q RT-PCR method to detect the expression of lncRNA TRHDE-AS1.2.Regulate the effect of lncRNA TRHDE-AS1 expression on HSF proliferation and apoptosis: use pc DNA3.1,pc DNA3.1-TRHDE-AS1,NC si RNA and TRHDE-AS1 si RNA to transfect HSF,CCK-8 assay,flow cytometry detection The effect on cell proliferation and apoptosis.3.To study the relationship between lncRNA TRHDE-AS1 and miR-181a-5p and its effect on HSF proliferation and apoptosis: use Lnc Base(http://carolina.imis.athena-innovation.gr/diana＿tools)to predict lncRNA TRHDE-AS1 and miR-The binding target of 181a-5p was detected by q RT-PCR to regulate the expression level of miR-181a-5p in lncRNA TRHDE-AS1 expressing cells.The dual luciferase reporter gene experiment confirmed the relationship between lncRNA TRHDE-AS1 and miR-181a-5p.Use miR-181a-5p mimic to reverse the effect of overexpression of lncRNA TRHDE-AS1 on cell proliferation and apoptosis..4.The establishment of miR-181a-5p target gene PTEN and the study of its regulatory pathway for hypertrophic scars: Using Target Scan(http://www.targetscan.org)to predict the binding site of miR-181a-5p and PTEN 3’-UTR.The PTEN 3’-UTR wild-type and mutant reporter gene vectors were constructed,and the dual-luciferase reporter experiment was used to determine the interaction between miR-181a-5p and PTEN.Western blot method was used to detect the effect of miR-181a-5p on PTEN protein expression.Use miR-181a-5p mimic to reverse the effect of overexpression of lncRNA TRHDE-AS1 on PTEN protein expression.5.To verify whether the pathways of lncRNA TRHDE-AS1 and miR-181a-5p in regulating HSF proliferation and apoptosis pass through PTEN: use CCK-8 and flow cytometry to detect the inhibition of PTEN to reverse lncRNA TRHDE-AS1,miR-181 aThe effect of 5p on cell proliferation and apoptosis.Results1.Compared with the corresponding normal tissues in 30 cases of HS,the results of q RT-PCR showed that LncRNA TRHDE-AS1 was significantly down-regulated in hypertrophic scar tissues,and the difference was statistically significant(P <0.05).2.CCK-8 results showed that overexpression of lncRNA TRHDE-AS1 significantly inhibited the proliferation of HSF(P <0.05),and silencing lncRNA TRHDE-AS1 significantly promoted the proliferation of HSF(P <0.05).Flow cytometry results showed that overexpression of lncRNA TRHDE-AS1 significantly promoted HSF apoptosis(P<0.05),while silencing lncRNA TRHDE-AS1 significantly inhibited HSF apoptosis(P<0.05).3.Bioinformatics analysis showed that lncRNA TRHDE-AS1 contains a binding site for miR-181a-5p.The results of the dual luciferase report experiment show that lncRNA TRHDE-AS1 can bind to miR-181a-5p.The q RT-PCR results showed that when lncRNA TRHDE-AS1 was overexpressed,the expression of miR-181a-5p was inhibited,and when lncRNA TRHDE-AS1 was knocked down,the expression of miR-181a-5p was up-regulated.The CCK-8 method showed that overexpression of miR-181a-5p reversed the effect of lncRNA TRHDE-AS1 on inhibiting HSF proliferation(P <0.05).The results of flow cytometry showed that overexpression of miR-181a-5p reversed the effect of lncRNA TRHDE-AS1 in promoting HSF apoptosis(P <0.05).4.Bioinformatics predictions show that PTEN 3’-UTR contains miR-181a-5p binding sites.The results of dual luciferase reporter gene showed that PTEN is one of the target genes of miR-181a-5p.Western blot results showed that overexpression of miR-181a-5p reduced the expression level of PTEN(P <0.05),and inhibition of miR-181a-5p increased the expression of PTEN(P <0.05).miR-181a-5p mimic can partially reverse the increase in PTEN expression caused by the overexpression of lncRNA TRHDE-AS1.5.CCK-8 analysis showed that inhibition of PTEN can reverse the inhibitory effect of lncRNA TRHDE-AS1 overexpression or miR-181a-5p knockdown on cell proliferation.Flow cytometry results showed that inhibition of PTEN could reverse the overexpression of lncRNA TRHDE-AS1 or the knockdown of miR-181a-5p to promote HSF apoptosis(P<0.05).Conclusions1.In hypertrophic scar tissue,LncRNA TRHDE-AS1 showed low expression,while miR-181a-5p expression increased,and the level of lncRNA TRHDE-AS1 was negatively correlated with the level of miR-181a-5p.2.LncRNA TRHDE-AS1 participates in the formation and development of HS through the miR-181a-5p/PTEN pathway.