The Role Of MiR-494-3p In Regulating Keloid Fibroblasts Biological Functions Through HTRA1 And Its Underlying Mechanism

Objective: Scar has always been a difficult problem in the field of plastic surgery.Excessive repair after trauma will lead to the formation of pathological scars,which mainly include keloid and hypertrophic scars.TGF-β1 is a potent growth factor in wound healing and is considered to be a key regulator of keloids and other fibrotic diseases.TGF-β1 binds to the TGF-β receptor(TGF-βR)and transmits activation signals to the nucleus.Following activation,TGF-β1 regulate the proliferation process of fibroblasts through a variety of signal transduction pathways.HTRA1 is a heat shock protein and belongs to the HTRA family.The study found that HTRA1 could cleavage TGF-β1 precursor and inhibit TGF-β1 signaling pathway in tumor cells.Given the fact that HTRA1 is a suppressor of TGF-β1,and the abnormal expression of HTRA1 is associated with many tumors,although there are no malignant signs of keloids,some of its characteristics resemble tumor cells including beyond the original wound range,persistent growth,and recurrence.Therefore,this study aimed to investigate the expression level,biological function and potential mechanism of action of HTRA1 in keloids.Then,based on the previous study,we will further explore the effect of miRNA targeting HTRA1 on the biological function of keloid fibroblasts.Method: Firstly,Keloids and normal skin tissues were collected and the expression of HTRA1 and TGF-β1 were detected by real time PCR,western blot and immunohistochemistry;Cell co-culture system and luciferase reporter gene assay were applied to study the regulation mechanism of HTRA1 on TGF-β1 activation during keloid formation;HTRA1 or TGF-β1 were silenced to explore the role of HTRA1 on keloids pathogenesis in a TGF-β1-independent manner;Keloids cell senescence was evaluated to explore the role of HTRA1 on the pathogenesis of keloids in a TGF-β1-independent manner.Base on the before study,we looked for the upstream regulation mechanism of HTRA1 in miRNAs.The online bioinformatics software(TargetScan,Pictar and MicroRNA)was used to predict the miRNA targeting HTRA1,and then the luciferase reporter system was used to verify that HTRA1 is the target gene of miR-494-3p.The keloid and normal skin tissues were collected and the expressionlevel of miR-494-3p was detected by real time PCR.Primary keloid-derived fibroblasts were cultured and transfected with miR-494-3p mimic and miR-494-3p inhibitor using Lipofectamine 2000 to overexpress or decrease the expression of miR-494-3p.MTT assay was used to detect the changes of fibroblast viability at different expression levels of miR-494-3p.Flow cytometry was used to detect the apoptosis and cell cycle of fibroblasts with different expression levels of miR-494-3p.Transwell invasion assay was used to detect the invasive ability of fibroblasts with different expression levels of miR-494-3p.Results: Real time PCR showed that HTRA1 and TGF-β1 were expressed in normal tissues and keloids,but the mRNA expression levels of HTRA1 and TGF-β1 were significantly higher in keloid than those in the normal skin(P<0.05).Western blot and immunohistochemical staining both detected the expression of HTRA1 and active TGF-β1 in keloids and normal skin,but the expression of HTRA1 and active TGF-β1was significantly higher in keloid than those in the normal skin(P<0.05).In addition,total Smad2 protein and phosphorylated Smad2 were detected in normal skin and keloids;compared with normal tissues,the total protein contents of Smad2 did not change significantly in keloids,and the level of phosphorylated Smad2(p-Smad2)was significantly elevated compared to the normal tissue(P<0.05).The luciferase reporter assay showed that active TGF-β1 specifically activated PAI-1 promoter-mediated luciferase gene expression,while TGF-β1 didn’t.Smad2 phosphorylation level and active TGF-β1 levels were significantly increased in fibroblasts exposed to recombinant human HTRA1(rhHTRA1)or inactive TGF-β1 treatment.RhHTRA1 and latent TGF-β1co-treatment further enhanced the phosphorylation of Smad2 and the content of active TGF-β1 in fibroblasts.Interfering with HTRA1 or TGF-β1 significantly inhibited the proliferation of fibroblasts.HTRA1 knockout significantly enhanced β-galactosidase activity,and overexpression of S328 A mutant HTRA1 in HTRA1-knockout fibroblasts failed to reverse senescence.Through bioinformatics analysis,we found that HTRA1 was a potential target gene of miR-494-3p in fibroblasts.The results of luciferase activity assay showed that miR-494-3p can bind to HTRA1 3’ UTR and inhibit the fluorescence signals.miR-494-3p was expressed in normal skin and keloids,and the expression in keloidtissues was significantly decreased(P<0.05).Treatment of fibroblasts with miR-494-3p mimic or miR-494-3p inhibitor revealed that there was no significant change in keloid cell viability after 24 h,whereas overexpression of miR-494-3p significantly inhibited cell proliferation at 48 h and 72 h after culture,and suppression of miR-494-3p promoted cell viability(P<0.05).Overexpression of miR-494-3p significantly induced fibroblast apoptosis,and inhibition of miR-494-3p reduced the apoptosis rate of fibroblasts(P<0.05).Cell cycle analysis showed that overexpression of miR-494-3p caused a decrease in the proportion of cells in G1 phase and S phase,and an increase in the proportion of cells in G2 phase;while inhibition of miR-494-3p resulted in an increase in the proportion of cells in G1 phase and reduced cells in G2 phase.Cell invasion assays showed that miR-494-3p overexpression significantly reduced the number of transmembrane cells,while interference with miR-494-3p expression increased the number of transmembrane fibroblasts.In addition,miR-494-3p overexpression significantly decreased the expression levels of collagen I and III,while interference with miR-494-3p expression enhanced the expression of these two proteins.Conclusion: HTRA1 expression was increased in keloids.This elevation of HTRA1,on the one hand,promoted fibroblast proliferation and collagen synthesis in a TGF-β1dependent manner.On the other hand,HTRA1 elevation inhibited fibroblast senescence independent of TGF-β1.The expression of miR-494-3p in keloids is decreased,and elevation of miR-494-3p inhibited fibroblast activity,induced apoptosis and cell cycle arrest by targeting HTRA1 expression.miR-494-3p may be a potential target in keloid therapy.