摘要
目的利用转录组测序技术,筛选重组人甲状旁腺素(1-34)[rhPTH(1-34)]影响角质形成细胞(KC)内表皮生长因子受体(EGFR)表达的差异转录因子,以探讨rhPTH(1-34)治疗银屑病的机制。方法慢病毒转染培养的HaCaT细胞,使之过表达EGFR。rhPTH(1-34)组加入0.01μmol/L rhPTH(1-34),空白组加入等量DMEM溶液。抽提两组细胞RNA,用紫外吸收测定法和变性琼脂糖凝胶电泳法进行RNA产量和质量检测。合成cDNA,实时定量PCR扩增,对文库有效浓度进行准确定量。上机测序,对所得数据进行处理和分析。用DESeq2软件分析两组数据之间的差异,以|log_2foldchange|≥1且P<0.05作为筛选条件对测序结果进行分析。结果rhPTH(1-34)组和空白组共表达的基因数目12455个,rhPTH(1-34)组单独表达421个,空白组单独表达461个。rhPTH(1-34)组与空白组比较,差异基因共185个,其中108个上调,77个下调。下调中,|log_2foldchange|最高的5个差异转录因子为ZNF525、ZNF774、ZNF576、ZBTB42和POU4F1(P<0.05)。其中ZNF774、POU4F1主要涉及Notch2、MAPK信号通路的调节。结论rhPTH(1-34)抑制KC增殖可能与下调ZNF525、ZNF774、ZNF576、ZBTB42、POU4F1表达有关。其中ZNF774、POU4F1可能与KC增殖相关。
Objective Using transcriptome sequencing,we screened differentially expressed transcription factors(TFs)in keratinocyte(KC)with overexpression of the epidermal growth factor receptor(EGFR)induced by recombinant human parathyroid hormone(1-34)in order to investigate the mechanism of rhPTH(1-34)in the treatment of psoriasis.Methods Overexpression of EGFR in HaCaT cells was achieved by lentiviral transfection.Cells in rhPTH(1-34)group were incubated with 0.01μmol/L of rhPTH(1-34),while the control group were treated with DMEM solution alone.After extraction of RNA,the quantity and quality of RNA were assessed using UV absorbance assay and denaturing agarose gel electrophoresis,respectively.The cDNA was synthesized and amplified by real-time quantitative PCR for accurate quantification of the effective concentration of the library.DESeq2 software was used to analyze the differentially expressed genes,and significance was defined as|log2foldchange|≥1 and P<0.05.Results A total of 12455 genes were detected in the rhPTH(1-34)and control groups,with 421 genes in the rhPTH(1-34)group and 461 genes only in the control group.Of 185 differentially expressed genes between the rhPTH(1-34)-treated cells and the control group,108 genes were up-regulated and 77 genes were down-regulated.Among the down-regulated genes,top five differentially expressed transcription factors were ZNF525,ZNF774,ZNF576,ZBTB42 and POU4F1(P<0.05).ZNF774 and POU4F1 were mainly involved in the regulation of Notch2 and MAPK signaling pathways,possibly regulating KC proliferation.Conclusions Inhibition of KC proliferation by rhPTH(1-34)may be associated with down-regulation of ZNF525,ZNF774,ZNF576,ZBTB42,and POU4F1 expression.ZNF774 and POU4F1 may be associated with KC proliferation.
作者
赵文琪
邬思远
范娟
张玲玲
卜晓琳
ZHAO Wenqi;WU Siyuan;FAN Juan;ZHANG Lingling;BU Xiaolin(Ningxia Medical University,Postgraduate Training Base in Shanghai Gongli Hospital;Shanghai Pudong New Area Gongli Hospital,Shanghai 200135,China)
出处
《皮肤性病诊疗学杂志》
2023年第4期314-321,共8页
Journal of Diagnosis and Therapy on Dermato-venereology
基金
国家自然科学基金(81703151)
上海市卫生健康委员会青年科研项目(20214Y0404)
浦东新区卫生系统学科建设计划特色专病(PWZzb2022-01)
公利医院青年基金资助计划(2020YQNJJ-12)。
