Gene expression profile analyze the molecular mechanism of CXCR7 regulating papillary thyroid carcinoma growth and metastasis

Abstract

Background: To detect genetic expression profile alterations after papillary thyroid carcinoma (PTC) cells transfected with chemokine receptor CXCR7 gene by gene microarray, and gain insights into molecular mechanisms of how CXCR7 regulating PTC growth and metastasis.

Methods: The Human OneArray microarray was used for a complete genome-wide transcript profiling of CXCR7 transfected PTCs (K1-CXCR7 cells), defined as experimental group. Non CXCR7 transfected PTCs (K1 cells) were used as control group. Differential analysis for per gene was performed with a random variance model and t test, p values were adjusted to control the false discovery rate. Gene ontology (GO) on differentially expressed genes to identify the biological processes in modulating the progression of papillary thyroid carcinoma. Pathway analysis was used to evaluate the signaling pathway that differentially expressed genes were involved in. In addition, quantitative real-time polymerase chain reaction (q-PCR) and Western blot were used to verify the top differentially expression genes.

Results: Comparative analysis revealed that the expression level of 1149 genes was changed in response to CXCR7 transfection. After unsupervised hierarchical clustering analysis, 270 differentially expressed genes were filtered, of them 156 genes were up-regulated whereas 114 genes were down-regulated in K1-CXCR7 cells. GO enrichment analysis revealed the differentially expressed genes were mainly involved in biopolymer metabolic process, signal transduction and protein metabolism. Pathway enrichment analysis revealed differentially expressed genes were mainly involved in ECM-receptor interaction, Focal adhesion, MAPK signaling pathway and Cytokine-cytokine receptor interaction pathway. More importantly, the expression level of genes closely associated with tumor growth and metastasis was altered significantly in K1-CXCR7 cells, including up-regulated genes FN1, COL1A1, COL4A1, PDGFRB, LTB, CXCL12, MMP-11, MT1-MMP and down-regulated genes ITGA7, and Notch-1.

Conclusions: Gene expression profiling analysis of papillary thyroid carcinoma can further delineate the mechanistic insights on how CXCR7 regulating papillary thyroid carcinoma growth and metastasis. CXCR7 may regulate growth and metastasis of papillary thyroid carcinoma via the activation of PI3K/AKT pathway and its downstream NF-κB signaling, as well as the down-regulation of Notch signaling.

Figure 1

Histograms with fold change between the experimental and control groups (O vs N). Fold change represents logarithm of fluorescence signal intensity ratios for differentially expressed genes. And log2 ratios ≥ 1.0 or log2 ratios ≤ −1.0 means two Fold change. The histogram plot shows fold change distribution of all probes excluding control and flagged probes. |Fold change| ≥1 means genes differentially expressed.

Figure 2

Volcano plot of distribution of differentially expressed genes between the experimental and control groups. The dotted line in red and green represent the cut-off, a measurement of gene expression fold-change on the X-axis versus a measure of statistical significance [−Log10 (P-value)] on the Y-axis. Differentially expressed genes are established at |Fold change| ≥1 and P-value < 0.05 (Blue dots in Figure 2).

Figure 3

Hierarchical clustering of differentially expressed genes in the experimental and control groups. A hierarchical clustering tree indicates the gene expression patterns similarity of the 270 genes between the experimental and control groups. The expression levels of the 270 genes are showed by different color lump: red, high (up-regulated); black, medium; and green, low (down-regulated).

Figure 4

mRNA and protein expression of differentially expression genes in K1 cell and K1-CXCR7 cell. (A) Relative mRNA expression level of differentially expression genes (FN1, COL1A1, COL4A1, PDGFRB, LTB, CXCL12, MMP-11, MT1-MMP, ITGA7 and Notch-1). (B) Upper panel: protein electrophoregram of differentially expression genes. GAPDH protein was used as a loading control. Lower panel: relative protein levels of differentially expression genes. *p < 0.05.