Expression profile of COMMD7 in patients with HCC
In order to understand the expression profile of COMMD7 in liver cancer, we performed bioinformatics analysis of COMMD7. As shown in Fig. 2, according to the 369 liver cancer tissues and 160 normal tissues included in Gene Expression Profiling Interactive Analysis (GEPIA) dataset (http://gepia2.cancer-pku.cn/), the mRNA expression level of COMMD7 in liver cancer tissues is much higher, as the transcripts per million (TPM) in tumor tissues was nearly 1.5 times higher than in normal tissues (p < 0.01; Fig. 2a). The protein relative expression level of COMMD7 in tumors and non-tumors was consistent with that of mRNA (Fig. 2b). Further, we detected the expression dynamic changes of COMMD7 during the development of HCC. The results showed that the expression of COMMD7 gradually increased from stage I to stage III (p < 0.01), indicating that COMMD7 was closely related with the early progression of HCC (Fig. 2c).
Our team also clarified that COMMD7 was highly expressed in liver cancer tissue specimens. We verified the correlation of COMMD7 with early stage of HCC by detecting the mRNA expression level of COMMD7 in 68 pairs of liver cancer and adjacent tissues. Subsequently, a self-prepared monoclonal antibody to COMMD7 was uesd to detect the protein level of COMMD7 in 7 liver cancer cell lines including HepG2, Huh7, Hep3B, HLE, HLF, SK-Hep-1 and PLC/PRF5. The results revealed that COMMD7 protein expressed totally higher in these hepatoma cells than in normal hepatocytes, especially in HepG2 and SK-Hep-1[36, 37]. For further study, we constructed a pGenesil-COMMD7-shRNA plasmid both in HepG2 and SK-Hep-1cells by means of short-hairpin RNA targeted technology. As a result, silence of COMMD7 significantly reduced cell proliferation and colony formation, while increased apoptosis and led to cell cycle arrest at S-phase in both two cell lines.
Gene mutation and co-expression analysis of COMMD7 in HCC
We analyzed COMMD7 gene alterations, expression correlation of COMMDs family members, and co-expression genes of COMMD7 by using the cBioPortal online tool for hepatocellular carcinoma (TCGA, Firehose Legacy, http://www.cbioportal.org/ ). AS a result, COMMD7 was altered in 41 samples out of 373 patients with HCC (11%) (Fig. 4a), and the altered group obtained better overall survival (median month overall survival 81.67 Vs 53.35, Fig. 4b and 4c). We also calculated the mRNA expression correlations of COMMD7 and other COMMDs family members with each other by Pearson’s correction. The results indicated that most of the COMMDs family members were expressed positively with COMMD7, however the expression of COMMD2 and COMMD10 were negatively correlated with COMMD7 (Fig. 4d).
Co-expression gene analysis results signified that the top 10 genes such as DNYLRB1, PDRG1, MANBAL, PFDN4, RALY were positively related with COMMD7 in HCC patients (p < 0.01), most of these genes were located on chromosome 20, which may be due to COMMD7 was also located on that chromosome (Table 2). The top 10 negatively related genes were ABHD18, ERN1, DDI2, USP12, METTL14, TOR1AIP2, PPTC7, MAP3K2, RSC1A1, ZNF281(p < 0.01). However, those negatively related genes distributed randomly in chromosome1, 2, 4, 12, 13, and 17 (Table 3).
Functional analysis of COMMD7 in liver cancer stem cells
We have previously knocked down COMMD7 gene in liver cancer stem cells (LCSCs) to observe the effect of COMMD7 in LCSCs. After gene-sequencing, we analyzed the function of COMMD7 by Gene Ontology (GO) in the Database for Annotation, Visualization, and Integrated Discovery (DAVID, https://david.ncifcrf. gov/ summary. jsp), the go enrichment including biological processes (BP), cellular components (CC), and molecular functions (MF) were launched. We found that GO:0055114 (oxidation-reduction process), GO:0098590 (plasma membrane region), GO:0070742 (C2H2 zinc finger domain binding) were most enriched for up-regulated genes after COMMD7 knock-down, while GO:0007399 (nervous system development), GO:0031012 (extracellular matrix), GO:0005509 (calcium ion binding) were significantly enriched for down-regulated genes(Fig. 5), indicating that COMMD7 may mainly combined with targeted domain or ions to exert its functional effect.