Expression of Reg IV and SOX9 and their correlation in human gastric cancer

Abstract

Background: Reg IV is a member of the regenerating gene family and has been demonstrated to be overexpressed in gastric cancer. However, the functional mechanism of Reg IV in gastric cancer is still unclear.

Methods: Expression of Reg IV and SOX9 were investigated by immunohistochemistry (IHC) and real-time PCR, and the correlation between the expression of Reg IV and SOX9 was analyzed in gastric cancer tissues. Reg IV expression vectors and a siRNA of Reg IV and SOX9 were transfected into human gastric cancer cells and the protein and mRNA levels of Reg IV and SOX9 were investigated by western blot and real-time PCR. The invasion and migration ability of gastric cancer cells with overexpressed Reg IV and with gene silence of Reg IV and SOX9 were examined by transwell chambers and wound healing assay.

Results: The Reg IV and SOX9 protein expression levels were both significantly higher in gastric cancer tissues compared with adjacent tissues (p = 0.022, p = 0.003). Reg IV protein expression significantly correlated with tumor invasion depth (p < 0.001), but had no significant correlations with age, clinical stage or lymph node metastasis. SOX9 protein expression also had no significant correlations with age, clinical stage, tumor invasion depth or lymph node metastasis. Reg IV transcript expression demonstrated a significant correlation with invasion depth and lymph node metastasis (p = 0.005, p < 0.001) and no significant correlations with age, clinical stage, tumor tissue differentiation or tumor size. SOX9 transcript expression demonstrated a significant correlation with invasion depth and tumor tissue differentiation (p = 0.044, p = 0.007) and no significant correlations with age, clinical stage or tumor size. The Reg IV expression showed a positive correlation with the SOX9 expression (p < 0.000, p = 0.008). Overexpression of Reg IV could upregulate SOX9 expression and promote invasiveness and migration of tumor cells, and silencing of Reg IV could downregulate SOX9 and inhibit invasiveness and migration of tumor cells in MKN-45 and AGS cells. On the other hand, silencing of SOX9 could upregulate Reg IV protein expression.

Conclusions: Our study demonstrated that Reg IV positively regulates the expression of SOX9 and is involved in tumor cell invasion and migration in gastric cancer.

Keywords: Correlation; Gastric cancer; Reg IV; SOX9.

Fig. 1

Representative immunohistochemical staining images of Reg IV and SOX9 in gastric cancer tissues. a Negative staining of Reg IV; (b, c) Cytoplasmic staining of Reg IV; (d) Negative staining of SOX9; (e, f) Nuclear staining of SOX9. Scale bar =100 μm

Fig. 2

Effects of Reg IV and SOX9 on the invasion and migration abilities in MKN-45 cells. a Reg IV overexpression vectors, control empty vectors (PEGFP) and transfection reagents control (Mock) were added to the media. Cells were seeded on the 8 μm-filter chambers after 48 h, and harvested after 24 h. The invasive cells and migrated cells were photographed (top) and counted (bottom); (b) effects of Reg IV siRNA (siR-Reg IV) on invasion and migration of MKN-45 cells compared with Mock and control scramble siRNA (siR-NC); (d) in wound healing assay, cells were transfected with Reg IV overexpression vectors, control empty vectors (PEGFP), and transfection reagents control (Mock). After creating a confluent cell monolayer, cells were scraped in a straight and homogeneous line. The migration status were recorded every 12 h (top) and calculated (bottom); (e) effects of siR-Reg IV on migration of MKN-45 cells compared with Mock and control scramble siRNA; (f) effects of siR-SOX9 on migration of MKN-45 cells compared with Mock and control scramble siRNA. The results are shown as Mean ± SEM of 3 independent experiments. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001, N.S. = not significant

Fig. 3

Regulatory relationship of Reg IV and SOX9 in MKN-45 cells. a Reg IV overexpression vectors (PEGFP/Reg IV) and control empty vectors (PEGFP) were added to the cells. Cells were harvested after 48 h, total RNA were extracted and converted to cDNA. Real-time PCR was performed to examine the mRNA level of Reg IV and SOX9 in PEGFP/Reg IV and PEGFP treated cells. Relative expression values of mRNA levels were normalized by GAPDH mRNA expression; (b) after transfection with PEGFP/Reg IV and PEGFP, the cells and medium were collected. Western blot analysis was done using anti-Reg IV antibody and SOX9 antibody, and bands were visualized (left) and the gray intensity was analyzed (right). β-actin expression level was used as an internal control; (c) compared with control scramble siRNA (siR-NC), the mRNA levels of three Reg IV siRNAs (siR-R1, siR-R2, siR-R3) were examined by real-time PCR and siR-R3 showed higher silencing efficiency. d the mRNA level of SOX9 was examined in siR-R3-treated cells and the negative control (siR-NC) by real-time PCR; (e) after transfection with siR-NC and siR-R3, western blot analysis was done using anti-Reg IV antibody and SOX9 antibody, and bands were visualized (left) and the gray intensity was analyzed (right); (f) compared with control scramble siRNA (siR-NC), the mRNA levels of threeSOX9 siRNAs (siR-S1, siR-S2, siR-S3) were examined by real-time PCR and siR-S1 showed higher silencing efficiency; (g) the mRNA level of Reg IV was examined in siR-S1-treated cells and the negative control (siR-NC) by real-time PCR; (h) After transfection with siR-NC and siR-S1, western blot analysis was done, and bands were visualized (left) and the gray intensity was analyzed (right). The results are shown as Mean ± SEM, n = 3. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001, N.S. = not significant