Characterization of normal fibroblast transcriptomes upon transient silencing of LTBP2 by RNAseq. Characterization of normal fibroblast transcriptomes upon transient silencing of LTBP2 by RNAseq

Purpose: Our single-cell RNAseq data show that LTBP2 is a marker of a cancer associated fibroblast subpopulation in colorectal cancer liver metastases. Moreover, MTT assays of cultured fibroblasts treated with anti-LTBP2 antibodies suggest that LTBP2 is essential for their survival. The goal of this study is to understand the role of LTBP2 in fibroblasts. Methods: Normal olon fibroblasts (CCD18Co) were transfected 48h with siRNA containing an anti-LTBP2 or a non target sequence. Cell monolayer total RNA of 3 biological replicates was then extracted and submitted to Illumina RNAseq using NovaSeq6000. Results: Transcriptomics revealed massive changes in gene expression when LTBP2 is knocked down: 496 genes were differentially expressed between the colon fibroblasts treated with siRNA anti-LTBP2 and NT. Those genes are implicated in diverse biological processes such as extracellular matrix organization, angiogenesis or cell adhesion. Conclusions: Our study explored LTBP2 functions in normal fibroblasts. RNAseq data support its important role. After 48h, LTBP2 silencing induces changes in gene expression of numerous genes involved in fibroblast functions. Overall design: Total RNA profiles of normal colon fibroblasts (CCD18Co) 48 hours after transfection with siRNA anti-LTBP2 or non target sequence (siNT)

研究目的：
本研究的单细胞RNA测序（single-cell RNAseq）数据显示，LTBP2是结直肠癌肝转移灶中癌相关成纤维细胞亚群的标志物。此外，对经抗LTBP2抗体处理的培养成纤维细胞进行的MTT实验表明，LTBP2对成纤维细胞的存活至关重要。本研究旨在阐明LTBP2在成纤维细胞中的作用。

研究方法：
将正常结肠成纤维细胞（CCD18Co）分别用靶向LTBP2的小干扰RNA（siRNA）或非靶向序列的siRNA转染48小时。收集3次生物学重复的细胞单层总RNA，提取后使用NovaSeq6000平台开展Illumina RNA测序。

研究结果：
转录组学分析显示，敲低LTBP2后基因表达发生显著改变：靶向LTBP2的siRNA处理组与非靶向对照组相比，共有496个基因呈现差异表达。这些基因参与细胞外基质组织、血管生成、细胞黏附等多种生物学过程。

研究结论：
本研究探索了LTBP2在正常结肠成纤维细胞中的功能，RNA测序数据证实了其重要作用。转染48小时后，LTBP2基因沉默可诱导大量参与成纤维细胞功能的基因表达发生变化。

整体实验设计：
本实验检测了正常结肠成纤维细胞（CCD18Co）在分别用靶向LTBP2的siRNA或非靶向序列siRNA（siNT）转染48小时后的总RNA表达谱。