Yonsei Med J.  2017 Sep;58(5):925-933. 10.3349/ymj.2017.58.5.925.

Gene Expression Profiling of Hepatocellular Carcinoma Derived Cancer Stem Like Cell under Hypoxia

• Choi SH
• Lee SW
• Ok M
• Kim KS
• Kim S
• Ahn SH

• KMID: 2418927
• DOI: http://doi.org/10.3349/ymj.2017.58.5.925

Abstract

PURPOSE
Cancer stem like cells (CSCs), with unlimited self-renewal potential and other stem cell characteristics, occur in several cancers including hepatocellular carcinoma (HCC). Although CSCs can initiate tumors, malignant proliferation, relapse and multi-drug resistance, the ways how to activate them still remain unknown. This study aims to evaluate whether CSC acquire tumorigenic characters under tumor hypoxia, analyzed by microarray analysis.
MATERIALS AND METHODS
CSCs were purified from HCC patients and Affymetrix microarray was used to investigate their gene expression profiles. The results were validated by real-time polymerase chain reaction (PCR).
RESULTS
The results of the microarray indicated that 18 genes were up-regulated and 10 genes were down-regulated in CSCs. Several genes were identified to be significantly involved in the regulation of CSCs such as HCC. Furthermore, the up-regulated genes were related with metabolism, angiogenesis and hypoxia, whereas the down-regulated genes were related with apoptosis and inflammation.
CONCLUSION
The results may help to understand the mechanisms of tumor development through CSCs which acquired their distinctive tumorogenic properties by hypoxic stimulation.

Keyword

Hypoxia; microarray; hepatocellular carcinoma; cancer stem cell

MeSH Terms

Carcinoma, Hepatocellular/*genetics/pathology
Cell Line, Tumor
*Gene Expression Profiling
*Gene Expression Regulation, Neoplastic
Humans
Hypoxia/*complications/genetics
Liver Neoplasms/*genetics/*pathology
Neoplasm Recurrence, Local/pathology
Neoplastic Stem Cells/metabolism/*pathology
Real-Time Polymerase Chain Reaction
Reproducibility of Results

Figure

• 

  Fig. 1 Identification of hepatocellular carcinoma (HCC) derived cancer stem-like cells. (A) Microscopic image of cancer stem-like cells isolated from the cancer tissue of a HCC patient, showing spinal shape of stem cell morphology. (B) For characterization of cancer stem cells (CSCs); a positive portion of CD133 expression, which functions as a marker to differentiate from adipose derived stem cells, was analyzed using flow cytometry. (C) Oct4, Nanog, and Sox2 expression portion, the stem cell marker of isolated CSCs, were measured using flow cytometry. HCC cell line (Huh7), pancreatic cancer cell line (SNU478) and mesenchymal stem cells were compared and analyzed. Oct4, a potency marker, was expressed at very low levels in Huh7 and SNU478, while Nanog which is involved in Oct4 expression and proliferation, and Sox2 which has a multipotency property were highly expressed in isolated CSCs. (D) To identify the expression of surface markers in CSCs, immunocytochemistry assay was used. CD105 and CD90 which are the common MSCs marker, CD133 which is a marker of CSCs, and beta-catenin which is a self-renewal related marker were expressed. CD13, which is known as a therapeutic-specific marker in hepatic CSCs, was also clearly expressed. Scale bar=200 µm.

• 

  Fig. 2 Characterization of hepatocellular carcinoma-derived cancer stem-like cells. (A) Spheroid formation derived from self-assembly of cancer stem cells. (B) High expression of CD73 as a positive marker of mesenchymal stem cells in spheroid formed cancer stem cells (×100). (C) The expression of E-cadherin and vimentin, the related marker of Epithelial-mesenchymal transition, was identified (×100). (D) The expression of an angiogenesis marker influenced by cancer stem cells was identified. The expressions of vascular endothelial growth factor (VEGF) and CD34 were identified in spheroid formed cells (×100). LM, light microscopy.

• 

  Fig. 3 Gene profiling of hypoxia-stimulated cancer stem-like cells by microarray analysis. The changes in gene expression were compared between hypoxia and normoxia. (A) The alteration of gene expression was measured in 419 genes of 26000 human genes. The alteration was also measured in 237 genes under hypoxia. (B) The function of altered genes was classified and analyzed according to cellular component, molecular function, biological process, and panther-chart pathway. Variable gene expressions were identified. In particular, genes involved in cell signaling and cell formation were significantly altered.

• 

  Fig. 4 Gene validation of hypoxia-stimulated cancer stem-like cells. Each gene expression was identified through a real-time qPCR. Most gene expressions were found to increase two to ten times over those under hypoxia. The expressions of NDRG1, ER01L, ALDOC, GNRH, ADM, CSPR2, BHLH, and PIM1 increased more than five times, and the expression of HIG2 increased more than 20 times. In particular, genes involved in cell signaling and cell formation showed an increase in their expression.

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