Stemness-related transcriptional factors and homing gene expression profiles in hepatic differentiation and cancer

Abstract

Stem cell transcriptional signature activation is an essential event in the development of cancer. This study aimed to investigate the differential expression profile of three pluripotency-associated genes (OCT4, NANOG, and SOX2), G-protein-coupled chemokine receptor 4 (CXCR4) and the ligand (CXCL2), and alpha feto-protein (AFP) in hepatogenic differentiated stem cells and in sera of hepatitis C virus (HCV) and HCV-induced hepatocellular carcinoma (HCC) patients. Mesenchymal stem cells derived from umbilical cord blood were differentiated using hepatogenic differentiation media. Serum specimens were collected from 96 patients (32 cirrhotic HCV, 32 early HCC, and 32 late HCC) and 96 controls. Real-time quantitative reverse transcription polymerase chain reaction was performed for relative quantification of the 6 target genes using LIVAC method. In silico network analysis was also executed to explore the pluripotency and tumorigenic regulatory circuits in liver cancer. The expression levels of all genes declined gradually during the stages of stem cell differentiation. On univariate and multivariate analyses, NANOG, CXCR4 and AFP were significantly up-regulated in HCC patients with late clinical stage. In contrast, SOX2 and CXCL2 were markedly over-expressed in cirrhotic patients and could be used for clear demarcation between cirrhotic and HCC patients in our cases. In conclusion, our data highlight the potential role of SOX2 stem cell marker and CXCL2 chemokine in liver cell degeneration and fibrogenesis in HCV-induced hepatic cirrhosis in our sample of the Egyptian population. In addition, the significant association of NANOG and CXCR4 high-expression with late HCC, could contribute to the acquisition of stem cell-like properties in hepatic cancer and dissemination in late stages, respectively. Taken together, our results could have a potential application in HCC prognosis and treatment.

Keywords: Real-time qPCR; chemokines; hepatocellular carcinoma; liver cirrhosis; pluoripotent genes.

Figure 1.

Gene expression levels in cultured stem cells and hepatic patients. Data are presented as means. ANOVA test was used, followed by Newman-Keuls multiple comparison test at 0.05 significance levels. UDSC, undifferentiated stem cells; DSC, differentiated stem cells; HCC, hepatocellular carcinoma.

Figure 2.

Association between gene expression and clinical characteristics. Child-Turcotte-Pugh classification was used for prognostic scoring in patients with liver disease. Mann Whitney U test was used. Statistical significance at p < 0.05.

Figure 3.

Multivariate analysis by nonmetric multidimensional scaling (NMS) ordination analysis in patients. NMS joint plot showing the separation of three distinct groups of patients along axes 1 and 2 based upon the data collected from gene expression. Different genes’ direction and power of correlation with axis 1 and 2 are demonstrated. Both CXCL2 and AFP might be considered as potential genes responsible for the groups’ separation. CXCL2 increased dramatically toward the noncancer group, while much less expression was shown in cancer patients. AFP increased dramatically toward the patients with late hepatic carcinoma and much less expression was shown with early cancer patients.

Figure 4.

Interactive functional association network of the gene set. The network nodes are proteins. Predicted functional links between the proteins are indicated by edges in color coded lines. GeneMANIA was used (http://www.genemania.org).