HIF-1α-induced HOXC6 Promotes The Proliferation And Invasion Of Keloid Fibroblasts Through The ERK Signaling Pathway

Background and AimsKeloid is a common post-traumatic pathological scar,characterized by abnormal proliferation of fibroblasts and excessive deposition of extracellular collagen matrix[1],causing severe pain to the patients[2].The biological behavior of abnormal proliferation and invasion of keloids exhibits the "tumor-like" feature[3].The recurrence rate of keloid is high,and postoperative proliferation and invasiveness become stronger.There is no radical cure for keloids.Preventing abnormal proliferation and invasion of keloids is an urgent problem in the medical field.The molecular mechanism of keloid is still poorly understood.Therefore,the exploration of the mechanism of keloid and the search for new therapeutic targets are key to facilitate the treatment of keloids.Homeobox(HOX)family are major genes involved in embryonic development and cell differentiation,playing an important biological role in the occurrence and development of tumors by encoding transcription factors.In recent years,HOX genes have become a new research focus in the fields of nervous system tumors,squamous cell carcinoma,lung cancer and leukemia[4,5,6,7,8].Meanwhile,some products of HOX genes also perform an important role in the development of keloids.Studies have shown that antisense IncRNA HOXA11 promotes keloid progression through miR-124-3p sponge-mediated Smad5 pathway[9].Therefore,it is of great significance for the prevention and treatment of keloids to explore homeobox genes and upstream and downstream mechanisms in keloids.Methods1.Datasets of keloid fibroblasts(KF)and normal skin fibroblasts(NF)were obtained from the Gene Expression Omnibus(GEO)database using bioinformatics methods,and the common differentially expressed genes of the two datasets were identified.The expressions of core genes were verified in keloid tissues and KF.2.The down-expressed HOXC6 group(si-HOXC6-KF),the control KF group(si-NC-KF)and the control NF group(si-NC-NF)were constructed.The proliferation of fibroblasts was detected by CCK-8 method.The apoptosis of fibroblasts was detected by flow cytometry.The migration of fibroblasts was observed by Transwell method.The mRNA and protein expression of collagen Ⅰ and α-SMA were detected by RT-qPCR and Western blot.3.The KF and NF datasets were uploaded to the gene set enrichment analysis(GSEA)software.The gene set enrichment analysis method was used to predict the related signaling pathway of HOXC6 in KF.The si-HOXC6-KF group and the si-NC-KF group were constructed for transcriptome sequencing to predict the downstream signaling pathway of HOXC6 in KF.4.The si-HIF-lα-KF group,the si-NC-KF group and the si-NC-NF group were constructed.The protein expressions of HIF-1α,HOXC6,ERK and p-ERK were detected using Western blot.5.The si-HOXC6-KF group,the si-NC-KF group and the si-NC-NF group were constructed.The protein expressions of HIF-1α,HOXC6,ERK and p-ERK were detected using Western blot.6.The ERK inhibitor(SCH772984)was added to the KF group(KF+SCH772984 group).The NF group and the KF group were used as the control groups.The proliferation activities of fibroblasts were detected by CCK-8 method.The apoptosis of fibroblasts was detected by flow cytometry.The migration of fibroblasts was observed by Transwell method.The protein expressions of collagen I and α-SMA were detected by Western blot.Results1.By analyzing the gene expression matrix of KF and NF in the GEO database,we identified HOXC6 as the core differential gene of KF and NF datasets.Meanwhile,we confirmed that HOXC6 was significantly up-regulated in both keloid skin specimens and KF extracted from the primary keloid tissues.2.Down-expression of HOXC6 prevented the proliferation,migration and extracellular matrix(ECM)deposition of KF,and promoted KF apoptosis.3.GSEA analysis predicted that hypoxia signaling pathway was involved in the pathogenesis of HOXC6 in KF.Transcriptome sequencing suggested that the ERK signaling pathway was one of the downstream pathways of HOXC6 in KF.4.Compared with the si-NC-KF group,down-expression of HIF-la suppressed the proteins expressions of HOXC6 and p-ERK in KF.5.Compared with the si-NC-KF group,down-expression of HOXC6 attenuated the protein expressions of p-ERK in KF,without significant difference in the protein expression of HIF-1α.6.Compared with the KF group,down regulation of p-ERK inhibited the proliferation,migration and ECM deposition of KF,and promoted the apoptosis of KF.There is no significant difference in the protein expression of HIF-1α and HOXC6 between two groups.ConclusionsOur findings first demonstrated that HOXC6 acts as an oncogenic driver in the molecular mechanisms of fibroblasts in keloids.HIF-1α/HOXC6/ERK axis promotes the proliferation,migration,and ECM accumulation in KFs,contributing to the progression of keloids.Taken together,HOXC6 may serve as a promising novel therapeutic target and new focus for research designed to understand the pathogenesis of keloids.