MicroRNA-126 inhibits SOX2 expression and contributes to gastric carcinogenesis

Abstract

Background: SRY (sex-determining region Y)-box 2 (SOX2) is a crucial transcription factor for the maintenance of embryonic stem cell pluripotency and the determination of cell fate. Previously, we demonstrated that SOX2 plays important roles in growth inhibition through cell cycle arrest and apoptosis, and that SOX2 expression is frequently down-regulated in gastric cancers. However, the mechanisms underlying loss of SOX2 expression and its target genes involved in gastric carcinogenesis remain largely unknown. Here, we assessed whether microRNAs (miRNAs) regulate SOX2 expression in gastric cancers. Furthermore, we attempted to find downstream target genes of SOX2 contributing to gastric carcinogenesis.

Methodology/principal findings: We performed in silico analysis and focused on miRNA-126 (miR-126) as a potential SOX2 regulator. Gain- and loss-of function experiments and luciferase assays revealed that miR-126 inhibited SOX2 expression by targeting two binding sites in the 3'-untranslated region (3'-UTR) of SOX2 mRNA in multiple cell lines. In addition, miR-126 was highly expressed in some cultured and primary gastric cancer cells with low SOX2 protein levels. Furthermore, exogenous miR-126 over-expression as well as siRNA-mediated knockdown of SOX2 significantly enhanced the anchorage-dependent and -independent growth of gastric cancer cell lines. We next performed microarray analysis after SOX2 over-expression in a gastric cancer cell line, and found that expression of the placenta-specific 1 (PLAC1) gene was significantly down-regulated by SOX2 over-expression. siRNA- and miR-126-mediated SOX2 knockdown experiments revealed that miR-126 positively regulated PLAC1 expression through suppression of SOX2 expression in gastric cancer cells.

Conclusions: Taken together, our results indicate that miR-126 is a novel miRNA that targets SOX2, and PLAC1 may be a novel downstream target gene of SOX2 in gastric cancer cells. These findings suggest that aberrant over-expression of miR-126 and consequent SOX2 down-regulation may contribute to gastric carcinogenesis.

Figure 1. Schematic diagrams of predicted target sites of miR-126 and miR-522 in the SOX2 3′-UTR.

The predicted binding sites of miR-126 and miR-522 are indicated (arrowheads) in the SOX2 3′-UTR (1119 bp). The first nucleotide after the stop codon of SOX2 is defined as “1”, and the start- and end-positions of the complementary sequence between SOX2 and miRNAs are indicated above or beneath the arrowheads. *The horizontal bar below the SOX2 3′-UTR indicates the region targeted by the SOX2 siRNA. Sequence alignments of miR-126 and miR-522 with their corresponding potential target sites in the SOX2 3′-UTR are presented in each rectangle. The conservation status among species of the predicted binding sites is also indicated in each rectangle.

Figure 2. Effects of miR-126 and miR-522 on SOX2 expression.

(A) Western blot analysis of SOX2 protein expression after transfection of a negative control oligonucleotide (NC), Pre-miR-126 (126), Pre-miR-522 (522), SOX2 siRNA (siR), and Anti-miR-126 (A126) in the indicated gastric cancer cell lines and mouse ES cells. The final concentrations were 50 nM for Pre-miRNAs and siRNA, and 100 nM for Anti-miR-126, respectively. α-tubulin expression was used as a protein loading control. (B) Quantitative real-time RT-PCR analysis of SOX2 mRNA expression after transfection of the negative control, Pre-miR-126 and SOX2 siRNA into HSC43 cells. The expression levels were normalized against internal GAPDH expression. The assays were performed in triplicate, and the bars indicate s.d. *P<0.05.

Figure 3. Interaction between miR-126 and its binding sites in the SOX2 3′-UTR.

(A) Dual luciferase assay with the pGL4-SOX2 3′-UTR (1050 bp) reporter vector (Wt). 30 nM Pre-miRNAs or SOX2 siRNA, which targets the SOX2 3′-UTR, was co-transfected with 10 ng of the indicated reporter vector into HEK293T cells. (B) Dual luciferase assay with cotransfection of 10 ng of the reporter vectors containing the wild type SOX2 3′-UTR (Wt), single deletion mutant A (Del-A), single deletion mutant B (Del-B), or double deletion mutant AB (Del-AB), and 30 nM negative control or Pre-miR-126 in HEK293T cells. The assays were performed in triplicate, and the bars indicate s.d. *P<0.05; **P<0.01; n.s., not significant.

Figure 4. Expression of SOX2 and miR-126 in human gastric cancer tissues.

(A) Representative immunohistochemical staining of SOX2 protein in non-cancerous mucosa (Non-Ca) and gastric cancers (FG2 Ca and FG21 Ca). Original magnification: ×400. (B) Quantitative TaqMan real-time PCR analysis for miR-126 was carried out by using the 15 human gastric cancer tissues (filled bars) and paired non-cancerous tissues (open bars). The expression levels of cancer tissues were independently compared to those of paired non-cancerous tissues, which are normalized to 1, and the bars indicate s.d. *P<0.05. The intensities of SOX2 expression were indicated beneath each case by x-axis. The expression levels were determined by the following criteria: “++” for 10% or more cancer cells were strongly stained; “+” for 10% or more cancer cells were stained; “w+” for less than 10% cancer cells were weakly stained; “–” for almost all cells were negatively stained.

Figure 5. Effects of miR-126 expression on anchorage-dependent and -independent cell growth.

(A) In vitro cell proliferation assays after SOX2 knockdown by Pre-miR-126 or siRNA in gastric cancer cell lines. The number of viable cells was determined with a Cell Counting Kit-8 on days 1, 3, 5 and 7 after plating. (B) Representative phase contrast microphotographs of the colonies of MKN45 cells in soft agar at 9 days after transfection of the negative control, Pre-miR-126 or SOX2 siRNA. Original magnification: ×100. (C) Soft agar colony formation assays for measurement of the anchorage-independent growth of gastric cancer cell lines. The vertical axis (Absorbance) indicates the relative number of colony-forming cells, which was determined by the colorimetric assay method. The assays were performed in quadruplicate, and the bars indicate s.d. *P<0.05; **P<0.01.

Figure 6. Expression changes of predicted SOX2 target genes.

(A) Changes in gene expression after adenovirus-mediated ectopic SOX2 over-expression in NUGC3 cells. RT-PCR analysis was performed to validate the cDNA microarray results for Ad-GFP-infected (G) and Ad-SOX2-infected NUGC3 cells (S). GAPDH expression was used as an internal loading control. (B) Quantitative real-time RT-PCR analysis of the KRT6A and PLAC1 mRNA expression levels after SOX2 knockdown by Pre-miR-126 or siRNA in HSC43 cells. The expression levels were normalized against internal GAPDH expression. The assays were performed in triplicate, and the bars indicate s.d. **P<0.01.