NANOGP8 is the key regulator of stemness, EMT, Wnt pathway, chemoresistance, and other malignant phenotypes in gastric cancer cells.

Accumulating evidence demonstrated that NANOG1, the key transcription factor for embryonic stem cells, is associated with human cancers. NANOGP8, one of the pseudogenes in NANOG gene family, contains an intact open reading frame and also said to be expressed in cancer tissues. Therefore, a systematic study is greatly needed to address the following questions: among NANOG1 and NANOGP8, which gene is the main contributor for NANOG expression in cancer cells and which one is the key regulator responsible for stemness, epithelial-mesenchymal transition (EMT), metastasis, chemoresistance and other malignant phenotypes. Here we try to explore these issues with gastric adenocarcinoma cell lines in vitro using variety of molecular and cellular techniques.Special primers were designed to distinguish PCR products from NANOG1 and NANOGP8. Sphere-forming cells were cultured with serum-free and selective medium. A stable cell line was established with infection of lentivirus containing NANOGP8. qPCR was performed to measure NANOGP8 expression and its association with stemness, EMT and CSC markers in adherent cells and sphere-forming cells. Western blot analysis was deployed to confirm results of the transcript analysis. Experiments of cell proliferation, migration, invasion, clonogenic assay, sphere cell growth assays, cell cycle analysis, β-catenin accumulation and translocation in nucleus, and drug resistance were conducted to measure the impact of NANOGP8 on malignant statuses of gastric cancer cells. Immunofluorescence staining was used to analyze cell subpopulations with different markers.NANOGP8 is mainly responsible for NANOG expression in sphere-forming (stem cell-like) cells derived from gastric cancer cell lines regardless their differentiation status. Ectopic expression of NANOGP8 significantly up-regulates stemness transcription factors, EMT inducers, and cancer stem cell markers (CSC) including Lgr5. NANOGP8 also promotes expression of the signature genes vimentin and N-caderin for mesenchymal cells and down-regulates the signature gene E-caderin for epithelial cells whereby confer the cells with mesenchymal cell phenotype. In NANOGP8 over-expressed adherent and sphere-forming cells, Lgr5+ cells are significantly increased. Ectopic expression of NANOGP8 endows gastric cells with enhanced proliferation, migration, invasion, sphere-forming and clonogenic capacity, and chemoresistance. NANOGP8 expression also enhances β-catenin accumulation in nucleus and strengthens Wnt signal transduction.NANOGP8 is the main regulator of gastric cancer stem cells. It is closely associated with EMT, stemness, and CSC marker as well as Wnt signal pathway. NANOGP8 is correlated with cell proliferation, migration, invasion, clonogenic capacity, β-catenin accumulation in nucleus, and chemoresistance in gastric cancer. NANOGP8 is a promising molecular target for clinical intervention of gastric cancer

Detection of <i>NANOG-1</i> and <i>NANOGP8</i> expression by qPCR.

<p><b>A.</b> Diagram of the gene structures of NANOG-1 and NANOGP8, note that the most 5’ of the first exon in NANOG1 gene contains a stretch of 21nt unique sequences; <b>B.</b> Diagram of the sequence locations of the primers designed to distinguish PCR products amplified from NANOG1 and NANOGP8. NANOG1/P8 stands for common primers shared by NANOG1 and NANOGP8 that can amplify DNA from both NANOG1 and NANOGP8 cDNA, and NANOG1-s stands for NANOG1-specific primers that only can amplify DNA from NANOG1 cDNA; <b>C.</b> Expression of NANOG1 and NANOG1/NANOGP8 in sphere-forming cells versus parental adherent cells derived from different cell lines. **<i>p</i><0.01.</p

NANOGP8 over-expression enhances EMT and CSC Markers.

<p><b>A.</b> Comparison of EMT genes in NANOGP8-transfected SGC7901 cells (SGC7901-NANOGP8) and mock-transfected SGC7901 cells (SGC7901-NC). **<i>p</i><0.01. <b>B.</b> Expression of CSC marker genes in NANOGP8-transfected SGC7901 cells and mock-transfected SGC7901 cells. **<i>p</i><0.01. <b>C.</b> Expression of ABCG2 and MSI1 genes in NANOGP8-transfected SGC7901 cells and mock-transfected SGC7901 cells. **p<0.01. <b>D.</b> Protein detection of EMT and CSC markers in NANOGP8-transfected SGC7901 cells (NANOGP8) and mock-transfected SGC7901 cells (NC). <b>D1.</b>Comparison of N-cad protein expression in NANOGP8-transfected SGC7901 cells (NANOGP8) and mock-transfected SGC7901 cells (NC); <b>D2.</b> Comparison of NANOGP8 protein expression in NANOGP8-transfected SGC7901 cells (NANOGP8) and mock-transfected SGC7901 cells (NC); <b>D3.</b> Comparison of Lgr5 protein expression in NANOGP8-transfected SGC7901 cells (NANOGP8) and mock-transfected SGC7901 cells (NC); <b>D4.</b> Comparison of vimentin protein expression in NANOGP8-transfected SGC7901 cells (NANOGP8) and mock-transfected SGC7901 cells (NC); <b>D5.</b> Comparison of CD44 protein expression in NANOGP8-transfected SGC7901 cells (NANOGP8) and mock-transfected SGC7901 cells (NC); <b>D6.</b> Comparison of E-cad protein expression in NANOGP8-transfected SGC7901 cells (NANOGP8) and mock-transfected SGC7901 cells (NC).</p

Immunofluorescence staining of LGR5 and NANOGP8 in SGC7901 cells and spheres.

<p><b>A.</b> SGC7901 adherent cells; <b>B.</b> SGC7901 sphere-forming cells; <b>C.</b> NANOGP8-transfected SGC7901 adherent cells; <b>D.</b> NANOGP8-transfected SGC7901 sphere-forming cells. Note the localization and expression of NANOGP8 and Lgr5 were visualized. All the images are 200× magnified.</p

Impact of NANOGP8 on malignant phenotypes of gastric cancer cells.

<p>NANOGP8 stands for the NANOGP8 transfected SGC7901 cells, and mock stands for the mock transfected SGC7901 cells. All the experiments were repeated at least 3 times, and the results were statistically analyzed by a paired t-test for two groups using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered a statistically significant difference. <b>A.</b> Result of wound healing assay (cell migration). The cell wounds were visualized at 0 h, 12 h, 24 h, 48h, and 72 h; <b>B</b>. Histogram of wound healing assay at different time points. *<i>p</i><0.05; **<i>p</i><0.01. <b>C</b>. Result of transwell Boyden chamber assay. Note that the cell number passing the chamber membrane from NANOGP8-transfected cells is much more than that of mock-transfected cells; <b>D.</b> Histogram of the transwell Boyden chamber assay; **<i>p</i><0.01. <b>E.</b> Result of cell proliferation assay *<i>p</i><0.05; **<i>p</i><0.01. <b>F.</b> Result of clonogenic capacity assay. The images show the Giemsa staining of clones at 14d; <b>G.</b> Histogram of clonogenic capacity assay, **<i>p</i><0.01.</p

The impact of NANOGP8 on cell cycle in NANOGP8-transfected, mock and SGC7901 cells.

<p><b>(%, mean ±</b> SD).</p

Flow cytometry analysis of cell cycle in NANOGP8-transfected, mock-transfected, and SGC7901 cells.

<p><b>A.</b> Cell cycle in NANOGP8-transfected SGC7901 cells; <b>B.</b> Cell cycle in mock-transfected cells; <b>C.</b> Cell cycle in SGC7901 cells.</p

The fluorescence images of nucleus-located β-catenin in NANOGP8-transfected and mock cells.

<p><b>A.</b> Accumulated β-catenin in nucleus of the NANOGP8-transfected cells (SGC7901-NANOGP8); <b>B.</b> Very weak or undetectable β-catenin in nucleus of the mock-cells (SGC7901-MOCK) without NANOGP8 transfection.</p

Primers used in this study.

<p>Primers used in this study.</p