【作者】 王琦；

【导师】 曾抗；

【作者基本信息】 南方医科大学 ， 皮肤病与性病学（专业学位）， 2022， 博士

【摘要】 研究背景及目的瘢痕疙瘩是临床上常见的一种创伤后病理性瘢痕,以过度增多的瘢痕疙瘩成纤维细胞及异常沉积的胞外基质为特征[1],可导致容貌毁损和功能障碍[2],严重影响患者的生活质量。瘢痕疙瘩异常增殖侵袭的生物学行为具有“类似肿瘤”的特性[3],临床复发率高,且术后增殖能力和侵袭性更强。临床上尚无根治瘢痕疙瘩的治疗方法。如何防治瘢痕疙瘩异常增殖、侵袭是目前医学界急需解决的关键问题。究其原因之一是目前对瘢痕疙瘩增殖侵袭的分子机制仍然知之甚少。因此,深入探究瘢痕疙瘩增殖侵袭的重要机制并寻找新的治疗靶点是治疗瘢痕疙瘩的关键。同源异形盒(Homeobox,HOX)家族基因是参与胚胎发育和细胞分化的主要基因,通过编码转录因子在肿瘤的发生发展中发挥重要的生物学作用。近年HOX基因在神经系统肿瘤、鳞状细胞癌、肺癌以及白血病等领域成为新的研究热点[4,5,6,7,8]。同时,HOX基因的某些产物也在瘢痕疙瘩的发生发展中发挥重要的作用。研究表明,反义lncRNA HOXA11通过miR-124-3p海绵介导的Smad5通路促进瘢痕疙瘩病情进展[9]。因此,研究同源异形盒基因在瘢痕疙瘩中的表达及其上下游调控机制对于瘢痕疙瘩的防治具有极其重要的意义。研究方法1、采用生物信息学方法在基因表达综合数据库(Gene Expression Omnibus,GEO)数据库获取瘢痕疙瘩成纤维细胞(Keloid fibroblast,KF)与正常皮肤成纤维细胞(Normal skin fibroblast,NF)基因数据矩阵,筛选两者间的差异表达基因,并在组织和细胞两个层面进行验证。2、构建降表达 HOXC6 的 KF 组(si-HOXC6-KF)、KF 对照组(si-NC-KF)、NF对照组(si-NC-NF)。CCK-8法检测成纤维细胞的增殖活性、流式细胞学检测成纤维细胞的凋亡、Transwell法观察成纤维细胞的迁移、RT-qPCR和Western blot 检测 collagen Ⅰ、α-SMA 的 mRNA 和蛋白表达。3、将KF和NF基因数据集上传到基因集富集分析(Gene set enrichment analysis,GSEA)软件,采用GSEA方法预测KF中HOXC6相关信号通路。构建si-HOXC6-KF组和si-NC-KF组并进行转录组测序,预测KF中HOXC6下游信号通路。4、构建降表达 HIF-1α 的 KF 组(si-HIF-1α-KF)、si-NC-KF 组和 si-NC-NF 组,采用 Western blot 检测 HIF-1α、HOXC6、ERK 和 p-ERK 蛋白表达。5、构建 si-HOXC6-KF 组、si-NC-KF 组和 si-NC-NF 组,采用 Western blot 检测HIF-1α、HOXC6、ERK 和 p-ERK 蛋白表达。6、KF组加入ERK抑制剂(SCH772984)构建KF+SCH772984组,设立NF和KF为对照组,采用CCK-8法比较成纤维细胞的增殖活性,流式细胞学检测成纤维细胞凋亡,Transwell法观察成纤维细胞迁移,Western blot检测HIF-1α、HOXC6、collagen Ⅰ、α-SMA、ERK 和 p-ERK 的蛋白表达。结果1、通过分析GEO数据库中KF和NF的基因表达矩阵,我们发现,HOXC6是KF和NF数据集的核心差异基因。同时,我们在瘢痕疙瘩皮肤组织标本和组织原代提取的KF中均证实了 HOXC6的表达存在显著上调。2、降表达HOXC6能够抑制KF增殖、迁移和细胞外基质(ECM)沉积,并促进KF凋亡。3、GSEA预测缺氧信号通路参与了 HOXC6在KF中的发病机制。转录组测序提示ERK信号通路是KF中HOXC6的下游作用机制之一。4、与si-NC-KF组相比,降表达HIF-1α能够抑制KF中HOXC6和p-ERK蛋白的表达。5、与si-NC-KF组相比,降表达HOXC6能够抑制KF中p-ERK蛋白的表达,但对HIF-1α蛋白的表达无显著性差异。6、与KF对照组相比,抑制p-ERK表达能够抑制KF的增殖、迁移和ECM沉积,同时增加KF的凋亡,但对HIF-1α和HOXC6蛋白的表达无显著性差异。结论我们的研究首次发现,HOXC6可能是KF促进瘢痕疙瘩发展的关键致病分子。HIF-1α/HOXC6/ERK轴可促进KF增殖、迁移和胶原沉积,从而进一步促进瘢痕疙瘩的恶化。综上,HOXC6有望成为瘢痕疙瘩疾病治疗的一个新的靶点,深入研究HOXC6将为探索瘢痕疙瘩发病机制提供新的思路。更多还原

【Abstract】 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.更多还原