Journal of Molecular Histology ( IF 3.2 ) Pub Date : 2020-10-15 , DOI: 10.1007/s10735-020-09918-0 Simin Luo 1 , Qiping Shi 2 , Wuji Li 1 , Wenrui Wu 1 , Zhengang Zha 1
Adipose-derived mesenchymal stem cell (ADSC) with a high capacity of chondrogenic differentiation was a promising candidate for cartilage defect treatment. This study’s objective is to study the roles of integrin β1 (ITGB1) in regulating ADSC chondrogenic differentiations as well as the underlying mechanisms. The identity of ADSC was confirmed by flow cytometry. ITGB1 gene was overexpressed in human ADSC (hADSC) by transfection with LV003-recombinant plasmids. Gene mRNA and protein levels were examined using quantitative RT-PCR and western blotting, respectively. Differentially expressed mRNAs and proteins were characterized by next-generation RNA sequencing and label-free quantitative proteomics, respectively. ERK signaling and AKT signaling in hADSCs were inhibited by treating with SCH772984 and GSK690693, respectively. ITGB1 gene overexpression substantially increased collagen type II alpha 1 chain (COL2A1), aggrecan (ACAN), and SRY-box transcription factor 9 (SOX9) expression but suppressed collagen type I alpha 1 chain (COL1A1) expression in hADSCs. Next-generation RNA sequencing identified a total of 246 genes differentially expressed in hADSCs by ITGB1 overexpression, such as 183 upregulated and 63 downregulated genes. Label-free proteomics characterized 34 proteins differentially expressed in ITGB1-overexpressing hADSCs. Differentially expressed genes and proteins were enriched by different biological processes such as cell adhesion and differentiation and numerous signaling pathways such as the ERK signaling pathway. ERK inhibitor treatment caused substantially enhanced chondrogenic differentiation in ITGB1-overexpressing hADSCs. ITGB1 promoted the chondrogenic differentiation of human ADSCs via the activation of the ERK signaling pathway.
中文翻译:
ITGB1通过激活ERK信号传导促进人脂肪来源的间充质干细胞的软骨分化
具有高软骨分化能力的脂肪来源的间充质干细胞(ADSC)是治疗软骨缺损的有前途的候选者。本研究的目的是研究整联蛋白β1(ITGB1)在调节ADSC软骨形成分化中的作用及其潜在机制。通过流式细胞术确认ADSC的身份。通过用LV003重组质粒转染,ITGB1基因在人ADSC(hADSC)中过表达。分别使用定量RT-PCR和Western blotting检测基因mRNA和蛋白水平。分别通过下一代RNA测序和无标记定量蛋白质组学来表征差异表达的mRNA和蛋白质。通过分别用SCH772984和GSK690693处理,可以抑制hADSCs中的ERK信号传导和AKT信号传导。ITGB1基因过表达实质上增加了hADSCs中II型胶原1α链(COL2A1),聚集蛋白聚糖(ACAN)和SRY-box转录因子9(SOX9)的表达,但抑制了IAD 1型胶原1链(COL1A1)的表达。下一代RNA测序鉴定了通过ITGB1过表达在hADSC中差异表达的总共246个基因,例如183个上调的基因和63个下调的基因。无标记蛋白质组学表征了在过表达ITGB1的hADSC中差异表达的34种蛋白质。差异表达的基因和蛋白质通过不同的生物学过程(例如细胞粘附和分化)和许多信号通路(例如ERK信号通路)进行富集。ERK抑制剂治疗在过表达ITGB1的hADSC中导致软骨分化明显增强。