Next generation sequencing facilities quantitative analysis of SHARPIN knockout or non-target LoVo cells
Colorectal cancer (CRC) is one of the most common cancers and a major cause of cancer mortality worldwide. Further improvements are needed for the treatment of CRC. The E3 ubiquitin ligase is an enzyme that plays an important role in regulating protein expression levels via posttranslational ubiquitin-mediated proteolysis, and it is reportedly involved in the progression of various cancers, making it a target of recent interest in anticancer therapy. In this study, using comprehensive expression analysis involving spatial transcriptomic analysis with single-cell RNA sequencing in clinical CRC datasets, we identified the ubiquitin-associated protein Shank-associated RH domain interactor (SHARPIN) as a putative driver gene located on amplified chromosome 8q. SHARPIN was overexpressed in tumor cells, and high expression of SHARPIN in tumor tissues was positively correlated with lymphatic invasion and was independently predictive of a poor prognosis in CRC patients. In vitro and in vivo analyses using SHARPIN-overexpressing or -knockout CRC cells revealed that SHARPIN upregulated MDM2, resulting in subsequent downregulation of p53, which inhibits tumor cell apoptosis and promotes tumor growth in CRC. Furthermore, SHARPIN overexpression and significant effects on survival were observed in various cancers. In conclusion, SHARPIN is a novel driver gene that potentially promotes tumor growth following apoptosis inhibition in part by inhibiting p53 expression via MDM2 upregulation; therefore, SHARPIN represents a potential therapeutic target for CRC. Overall design: To assess potential gene expression changes by SHARPIN individual knockout in LoVo cells, we performed a transcriptomic analysis using high throughput RNA sequencing (RNA-seq).Total RNA extracted from SHARPIN knockout or non-target LoVo cells .The sequencing libraries generated from the RNA were analyzed by DNBSEQ-G400. The sequencing reads were trimmed for adaptor sequence, and low-complexity or low-quality reads were removed. Subsequently, the reads were aligned to the human reference sequence and the gene annotations (UCSC hg19) using Tophat2 v2.1.1. STAR v2.7.10b was used to calculate fragments per kilobase of transcript per million fragments mapped (FPKM) values.