MicroRNA 135a suppresses lymph node metastasis through down-regulation of ROCK1 in early gastric cancer

Abstract

MicroRNAs (miRNAs) play a critical role in gastric cancer progression and metastasis. This study investigated the role of miRNA-135a in early gastric cancer (EGC) including lymph node (LN) metastasis. We examined the correlation between miRNA-135a expression and clinical outcomes in 59 patients who underwent surgery for EGC. Using gastric cancer cell lines, we performed functional and target gene analyses. miRNA-135a expression was down-regulated in 33.9% of patients. These patients showed a significantly more advanced stage (TNM stage ≥ IB, 35.0% vs. 12.8%, p = 0.045) and higher rate of LN metastasis (30.0% vs. 5.1%, p = 0.014) than those with up-regulation of miRNA-135a expression. In a multivariate analysis, down-regulation of miRNA-135a was an independent risk factor for LN metastasis (adjusted odds ratio, 8.04; 95% confidence interval, 1.08-59.81; p = 0.042). Functional analyses using gastric cancer cell lines showed that miRNA-135a suppressed cell viability, epithelial-mesenchymal transition, cell invasion, and migration. ROCK1 was a target of miRNA-135a and its expression was inversely correlated to that of miRNA-135a. ROCK1 expression was significantly increased in EGC patients with LN metastasis than in those without LN metastasis. Our results confirm the tumor-suppressive role of miRNA-135a, and demonstrate its role in LN metastasis in EGC. miRNA-135a and its target gene ROCK1 may be novel therapeutic and prognostic targets for EGC.

Figure 1. miRNA-135a expression patterns in gastric cancer cell lines and human early gastric cancer tissues.

(A) miRNA-135a expression, examined by real-time PCR analysis, is down-regulated in gastric cancer cells compared with normal gastric mucosa cells (HFE 145 cells). (B) Relative miRNA-135a expression of tumor tissue in reference to normal counterparts by real-time PCR analysis according to patient stage.^* Patients with more advanced cancer stage (stage IB or greater) show significantly down-regulated miRNA-135a expression levels in tumor tissues compared to levels in patients with less advanced stage (stage IA) cancer (mean 9.97 vs. 2.30, p = 0.009). (C) Relative miRNA-135a expression of tumor tissue in reference to normal counterparts in real-time PCR analysis according to LN metastasis status. Patients with LN metastasis show significantly down-regulated miRNA-135a expression levels in tumor tissues (when compared to corresponding normal tissues) than patients without LN metastasis (mean 9.63 vs. 0.61, p = 0.002). *7^th American Joint Committee on Cancer TNM staging.

Figure 2. miRNA-135a suppresses cell viability and inhibits EMT factors, cell invasion, and migration in gastric cancer cells.

(A) Up-regulation of miRNA-135a induced by miRNA-135a mimics suppresses gastric cancer cell viability in SNU668 cell line, a cell line which rarely expresses miRNA-135a (p = 0.012), whereas miRNA-135a inhibitors rarely affect cell viability in YCC2 cell line, a cell line which substantially expresses miRNA-135a. (B) Up-regulation of miRNA-135a by miRNA-135a mimics increases the subdiploid fraction of DNA, as determined by flow cytometry analysis, suggesting increased apoptosis in SNU668 cells. (C) In the presence of miRNA-135a mimics, RT-PCR analysis shows that SNU668 cells have increased mRNA expression of E-cadherin and suppress mRNA expression of both N-cadherin and Slug. Conversely, in the presence of miRNA-135a inhibitors, YCC2 cells show decreased mRNA expression of E-cadherin and increased mRNA expression of both N-cadherin and Slug in RT-PCR analysis. (D) Overexpression of miRNA-135a using miRNA-135a mimics significantly suppresses migration and invasion of SNU668 cells. (E) Inhibition of miRNA-135a using miRNA-135a inhibitors significantly increases migratory and invasive cells of the YCC2 cells. Results shown are the means ± SD of at least three experiments. NC, negative control.

Figure 3. ROCK1 is a direct target of miRNA-135a in gastric cancer cells.

(A) The 3′ UTR sequence of ROCK1 has one possible binding site for miRNA-135a, as determined by TargetScan analysis. The 3′ UTR constructs for ROCK1 WT and MUT are shown. (B) In the presence of miRNA-135a inhibitors, relative luciferase activity of ROCK1 3′ UTR WT is significantly increased compared to ROCK1 3′ UTR MUT in SNU668 cells (p = 0.035). (C) On the contrary, in the presence of miRNA-135a mimics, the relative luciferase activity of ROCK1 3′ UTR WT is significantly decreased (p = 0.043), whereas the relative luciferase activity of ROCK1 3′ UTR MUT is not decreased in the YCC2 cell line. (D) Western blot analysis shows that overexpression of miRNA-135a using miRNA-135a mimics suppresses ROCK1 protein expression in MKN28 and SNU688 gastric cancer cells, which are cell lines that rarely express miRNA-135a. (E) Inhibition of miRNA-135a associated with increased ROCK1 protein expression in YCC2 and KATO III gastric cancer cells, which are cell lines that substantially express miRNA-135a. Results shown are the means ± SD of at least three experiments. WT, wild type; MUT, mutant; NC, negative control.

Figure 4. miRNA-135a expression is inversely correlated to ROCK1 protein expression in human early gastric cancer tissue.

(A) miRNA-135a expression was measured by real-time PCR analysis. The ROCK1 protein level was evaluated using western blot analysis and quantified using densitometric method. In 59 patients with early gastric cancer, expression of ROCK1 shows a significantly negative correlation with that of miRNA-135a (r = −0.697, p<0.001). (B) Western blot analysis shows that patients with down-regulated miRNA-135a expression have significantly higher ROCK1 protein expression in tumor tissues compared to expression in corresponding normal tissues (p<0.001).

Figure 5. The association between ROCK1 expression and clinical outcomes.

(A) Western blot analysis of 59 early gastric cancer patients shows that patients with LN metastasis have significantly higher ROCK1 protein expression than patients without LN metastasis (p = 0.007). Representative microscopic images of tumor sections with immunohistochemical staining (original magnification ×100) show that (B) early gastric cancer patients without LN metastasis have no ROCK1 expression in tumor tissues, but (C) those with LN metastasis have strong ROCK1 expression within the nuclear membrane and cytoplasm of tumor tissues. (D) ROCK1 siRNA significantly suppresses migration (p<0.001) and invasion (p = 0.002) in SNU668 gastric cancer cells. (E) ROCK1 protein expression is increased by treatment with miRNA-135a inhibitors and decreased by treatment with ROCK1 siRNA, and is associated with an increased miRNA-135a level in YCC2 gastric cancer cells, suggesting that ROCK1 protein expression patterns are inversely correlated with those of miRNA-135a expression. (F) When YCC2 cells are treated with miRNA-135a inhibitors, cell invasion and migration are similar or even increased compared to controls. ROCK1 siRNA or combined ROCK1 siRNA and miRNA-135a inhibitors decrease cell migration (p = 0.003) and invasion (p = 0.017). These suggest that ROCK1 offsets the suppressive effects of both migration and invasion that is induced by miRNA-135a. Results shown are the means ± SD of at least three experiments. LN, lymph node; NC, negative control.