mRNA-Seq analysis of cSCC cells followed by siRNA-induced gene knockdown of CFI.. mRNA-Seq analysis of cSCC cells followed by siRNA-induced gene knockdown of CFI.

Keratinocyte carcinomas (BCC and cSCC) are the most common cancers worldwide. cSCC is considered as the most prevalent metastatic skin malignancy and its incidence is increasing globally. The complement system is a fundamental part of the host immune defense. It is composed of three distinct pathways (classical, alternative and lectin) that get activated sequentially. However, the process of complement activation is rigorously regulated by a series of soluble and membrane-bound inhibitor proteins that protect the host cells from lytic damage. One of these soluble negative regulators is complement factor I (CFI) which is an 88 kDa serine protease that hampers all three complement pathways through blockade of C3- and C5- convertases by cleaving C3b and C4b. Oligonucleotide array (Affymetrix)-based analysis of normal human epidermal keratinocytes (NHEKs; n=5), primary (Prim. cSCC; n=5) and metastatic (Met. cSCC; n=3) cSCC cell lines as well as next-generation-sequencing (SOLiD) based transcriptome profiling of NHEKs (n=4), primary (Prim. cSCC; n=5) and metastatic (Met. cSCC; n=3) cSCC cell lines revealed marked overexpression of CFI in cSCC cells compared to NHEKs (GSE66368 and GSE66412 respectively). Furthermore, we have previously shown that knockdown of CFI inhibits proliferation and migration of cSCC cells and potently impedes growth of cSCC xenograft tumors in vivo. In these respects, we intended to further investigate the functional role and molecular mechanism of CFI in cSCC progression via analyzing the mRNA expression profile of CFI in cSCC cells. Overall design: Keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is the most prevalent metastatic skin malignancy and its incidence is increasing globally. The complement system is a fundamental part of the host immune defense. It is composed of three distinct pathways (classical, alternative and lectin) that get activated sequentially. The process of complement activation is rigorously regulated by soluble and membrane-bound inhibitor proteins that protect the host cells from lytic damage. One of these soluble negative regulators is complement factor I (CFI) which is an 88 kDa serine protease that hampers all three complement pathways through blockade of C3 and C5-convertases by cleaving C3b and C4b. Oligonucleotide array (Affymetrix)-based analysis of normal human epidermal keratinocytes (NHEKs; n=5), primary (Prim. cSCC; n=5) and metastatic (Met. cSCC; n=3) cSCC cell lines, as well as next-generation-sequencing (SOLiD) based transcriptome profiling of NHEKs (n=4), primary (Prim. cSCC; n=5) and metastatic (Met. cSCC; n=3) cSCC cell lines revealed marked overexpression of CFI in cSCC cells compared to NHEKs (GSE66368 and GSE66412, respectively). Furthermore, we have previously shown that knockdown of CFI inhibits proliferation and migration of cSCC cells and potently suppresses growth of cSCC xenograft tumors in vivo (Riihilä et al., J Invest Dermatol. 2015 Feb;135(2):579-588). In this respect, we have investigated the molecular mechanism of CFI in cSCC progression via analyzing the mRNA expression profile in cSCC cells after knockdown of CFI expression. Total RNAs from negative control siRNA and CFI siRNA-treated cSCC cell lines (n=3) were extracted. The samples were sequenced using Illumina sequencing.

角质形成细胞癌（Keratinocyte carcinomas）涵盖基底细胞癌（BCC）与皮肤鳞状细胞癌（cSCC），是全球范围内最常见的恶性肿瘤。其中皮肤鳞状细胞癌（cSCC）被视为最常见的转移性皮肤恶性肿瘤，其全球发病率呈逐年上升态势。补体系统是宿主免疫防御的核心组成部分，由经典通路、替代通路与凝集素通路三条依次激活的独特通路构成。补体激活过程会被一系列可溶性及膜结合型抑制蛋白严格调控，以保护宿主细胞免受裂解损伤。其中一类可溶性负调控因子即为补体因子I（complement factor I, CFI），它是一种88 kDa的丝氨酸蛋白酶，可通过裂解C3b与C4b阻断C3及C5转化酶，从而抑制三条补体通路的激活。研究团队此前通过基于寡核苷酸芯片（Oligonucleotide array, Affymetrix）的分析，对正常人类表皮角质形成细胞（normal human epidermal keratinocytes, NHEKs；n=5）、原发性（Prim. cSCC；n=5）及转移性（Met. cSCC；n=3）cSCC细胞系进行检测；同时采用基于新一代测序（next-generation-sequencing, SOLiD）的转录组分析方法，对NHEKs（n=4）、原发性（Prim. cSCC；n=5）及转移性（Met. cSCC；n=3）cSCC细胞系进行检测。结果显示，相较于NHEKs，cSCC细胞中CFI呈现显著过表达（对应公开数据集GSE66368与GSE66412）。此外，本团队既往研究证实，敲低CFI的表达可抑制cSCC细胞的增殖与迁移，并在体内有效抑制cSCC异种移植瘤的生长。鉴于此，本研究旨在通过分析cSCC细胞的mRNA表达谱，进一步探究CFI在cSCC进展中的功能作用与分子机制。总体设计：皮肤鳞状细胞癌（cSCC）作为角质形成细胞来源的恶性肿瘤，是最常见的转移性皮肤恶性肿瘤，其全球发病率逐年上升。补体系统是宿主免疫防御的核心组分，由经典、替代及凝集素三条依次激活的通路组成。补体激活过程会被可溶性与膜结合型抑制蛋白严格调控，以保护宿主细胞免受裂解损伤。其中一类可溶性负调控因子为补体因子I（CFI），它是一种88 kDa的丝氨酸蛋白酶，可通过裂解C3b与C4b阻断C3及C5转化酶，从而抑制三条补体通路的激活。本团队此前已证实，敲低CFI的表达可抑制cSCC细胞的增殖与迁移，并在体内有效阻断cSCC异种移植瘤的生长（Riihilä等人，《皮肤病学杂志》，2015年2月；135(2):579-588）。本研究通过分析阴性对照小干扰RNA（siRNA）与CFI小干扰RNA（siRNA）处理的cSCC细胞系（n=3）的mRNA表达谱，探究CFI在cSCC进展中的分子机制。研究人员提取了上述两组处理后细胞的总RNA，并采用Illumina测序技术对样本进行测序。