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. 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.