MiR-145 Negatively Regulated Catenin-δ1 To Inhibit Tumor Invasion In Gastric Cancer

BackgroundGastric cancer is one of the most common malignant carcinomas worldwide, and the incidence and mortality rate is higher in developing countries. After the prevalence of Helicobacter pylori infection, the mortality rates of stomach cancer have declined via diet adjustment and health habits change. With the development of gastroscopy, stomach cancer could be discovered and diagnosed in the early stage. And considerable improvements in surgical and chemotherapeutic treatment, the life quality of patients in the early stage are improved. However, patients in advanced stage with lymph node and distant metastasis still have a poor prognosis. Thus, identification of tumor biomarkers which can predict prognosis has important clinical significance. Moreover, to investigate the molecular mechanism of governing metastatic dissemination can provide a theoretical basis for clinical treatment.In the previous research about N-cadherin with gastric cancer metastasis, we found that catenin delta-1 (CTNND1), as a member of the cadherin-catenin complex, had important roles in tumor development and progression. CTNND1 directly binds to the cytoplasmic tail of E-cadherin via the former’s conserved juxtamembrane domain, and localized in the inside of cell membrane to maintain the stability of cell polarity. When E-cadherin lost from cell surface, most CTNND1 stably localized in the cytoplasm and the other is interacted with N-cadherin. CTNND1 promotes migration and invasion by RhoA GTPase inhibition and Rac1/Cdc42 activation. CTNND1 was reported in various cancers. Reduced CTNND1 expression has been reported in colon cancer, bladder cancer, and breast cancer; CTNND1 gene amplification was described by Castillo et al. in non-small cell lung cancer, implicating its different roles in different tumors. The pro-metastasis role of CTNND1 has been reported by Liu et al. and Mann et al. They found that cytoplasmic expression of CTNND1 correlated with tumor metastasis in lung cancer and pancreatic adenocarcinoma, which results also suggested that its function may be associated with its localization. To date, the expression pattern of CTNND1 and its role in gastric cancer initiation and progression are still obscure. The present study aims to detect the expression level and cellular localization of CTNND1 in gastric cancer tissues, to analyze the clinical significance and to further investigate the function of CTNND1.At present, the reason of CTNND1 abnormal expression and the mechanism of CTNND1 deregulation in gastric cancer remain unknown. MicroRNAs (miRNAs), small non-coding RNAs, about 22 nucleotides in length, have been reported to play crucial roles in tumor development and progression. It was reported that about 1/3 genes were targeted by 650 human miRNAs, which were also involved in many signal pathways. And about 50% miRNAs were reported to localize in human chromosomes regions which associated with tumor. Mature miRNAs bind onto the RNA-induced silencing complex (RISC) and target specific mRNAs via 3’untranslated regions (3’-UTRs). miRNAs negatively regulates gene expression by mRNA degradation or translational repression, depending on perfect or partial complementarity between its seed region (nucleotide positions 2-8) and the target mRNA. Considering the extensive functions of miRNAs in physiological (including cell growth, proliferation, differentiation and apoptosis) and pathological (tumor cell growth, proliferation, invasion, metastasis) progresses, it would be interesting to determine whether or not CTNND1 expression could be regulated by a specific miRNA in gastric cancer. Through three predictive softwares Targetscan, PicTar and miRanda, the seed sequences of miR-145, miR-96, miR-143, miR-197 are complementary to the 3’-UTR of CTNND1. The activity of the Luciferase reporter gene was significantly reduced by miR-145, but not the other three miRNAs. So we speculate that CTNND1 may be regulated by miR-145. In this study, we want to verify whether one of these miRNAs regulated the expression and localization of CTNND1. And whether there exist an interaction between CTNND1 and cadherin family, and a novel molecular mechanism to regulate CTNNDl expression or localization is also explored. In addition, Nude mice xenograft models were used to observe the relationship between the expression and localization of CTNND1 with gastric cancer invasion in vivo.Methods1. The expression level and cellular localization of CTNND1 were determined by immunohistochemistry in 126 human gastric cancer tissues and 50 nontumorous tissues. The association between the expression level and cellular localization of CTNND1 with clinicopathological parameters were also analyzed.2. The survival curves were plotted using the Kaplan-Meier method and the differences in overall or disease-free survival were assessed with the log-rank test.3. Univariate and multivariate survival data was analyzed using a Cox regression model, which also was used to identify whether cytoplasmic CTNND1 could be an independent prognostic factor.4. CTNND1 overexpression and siRNA plasmid were constructed to conform the biological function of CTNND1. Cell migration and invasion assays were performed to assess the effect of CTNND1 cDNA or CTNND1 siRNA transfection on gastric cancer cells. Cell proliferation and apoptosis were also examined after gastric cancer cells transfected with CTNND1 siRNA or mock control. 5. Real time quantitative PCR was used to detect the relative expression of miR-145.The correlation between percentage of CTNND1 positivity or cellular localization and miR-145 expression was calculated using Spearman’s correlation. The sequence of CTNND1 3’-UTR was amplified and introduced into the pmirGLO vector. Luciferase report assay and Western blot analysis were used to determine whether CTNND1 could be regulated by miR-145.6. After transfection with miR-145 or miR-control, the cellular localization of CTNND1, N-cadherin and E-cadherin of these cells were detected by immunofluorescence. The expression of CTNND1, N-cadherin and E-cadherin protein were analyzed by western blot. Furthermore, when N-cadherin siRNA was transfected into cells, the expression and localization of CTNND1, N-cadherin and E-cadherin were detected again. These assays aim to assess the role of N-cadherin in the regulation of CTNND1 mediated by miR-145, and further clarify the mechanism that miR-145 regulated cytoplasmic CTNND1 to inhibit gastric cancer invasion.7. SGC7901 cells were transfected with lentivirus expressing pEZX-miR-145 or pEZX-miR-control vector. For the tumor bearing model, the cancer cells were injected subcutaneously into the left axillary fossa of 4-week old male Nude mice. Six weeks later, the mice were euthanized and the tumor nodules were harvested and observed in hematoxylin and eosin (H&E) sections under the microscope. This study aims to further demonstrate that miR-145 regulates CTNND1 expression level and cellular localization in vivo.Results1. The immunohistochemical results showed that CTNND1 was expressed at the membrane in the nontumorous gastric mucosae. CTNND1 was upregulated in gastric cancer compared with nontumorous gastric mucosae. Both membranous and cytoplasmic localization of CTNND1 were observed in gastric caner tissues. The results demonstrate that the cytoplasmic localization of CTNND1 protein was indicative of higher clinical stage, higher T stage, positive lymph node metastasis, and poorer prognosis in gastric cancers. The expression level of CTNND1 was associated with differentiation and Lauren type, but not other parameters.2. The Kaplan-Meier survival curves showed no significant correlation between the expression level of CTNND1 and patients’overall or disease-free survival. However, the patients with cytoplasmic localization of CTNND1 displayed significantly poorer overall survival and disease-free survival than those in whom CTNND1 was localized to the membrane.3. Based on univariate analysis, CTNND1 cytoplasmic expression, clinical stage (UICC), T classification, lymph node metastasis, and distant metastasis showed a significant higher hazard ratio (HR) for poor prognosis. Furthermore, multivariate analysis revealed that cytoplasmic CTNND1 was associated with decreased overall survival and disease-free survival. The data demonstrated that the cytoplasmic localization of CTNND1 could serve as an independent prognostic factor for patients with gastric cancers.4. After 48h of transfection, the migration and invasion ability of SGC7901 cells was increased in the CTNND1 cDNA treated group compared with the mock control group, while knockdown of CTNND1 with siRNA dramatically reduced cell migration and invasion activity. However, our results showed that CTNND1 had no influence on cell proliferation or apoptosis by MTS and flow cytometry assays. CTNND1 could promote gastric cancer cell migration and invasion, with little effect on cellular proliferation and apoptosis.5. An inverse correlation was found between the relative expression of miR-145 and percentage of CTNND1 positive expression. The activity of the Luciferase reporter gene was significantly reduced by miR-145, compared with the miR-control both in MKN28 and SGC7901 cells. Western blot showed that the expression of CTNND1 protein was suppressed in miR-145-expressing MKN28 and SGC7901 cells, compared with the respective miR-controls. These results showed that CTNND1 was proved to be a direct target gene of miR-145.6. Further, an inverse correlation was found between cytoplasmic CTNND1 expression and miR-145 levels rather than membranous CTNND1 expression. These results suggested a potential link between miR-145 expression and CTNND1 cellular localization. CTNND1 was showed cytoplasmic localization in miR-control transfection group of SGC7901, while the membranous localization of CTNND1 was found in miR-145 transfected cells by immunofluorescence assay. These data showed that miR-145 may induce translocation of CTNND1 protein from cytoplasm to membrane. The cellular localizations of N-cadherin and E-cadherin were also examined. We found that miR-145-transfected cells showed membranous staining of E-cadherin and reduced N-cadherin expression, compared with the miR-control group. We further investigated whether N-cadherin expression was associated with the translocation of E-cadherin and CTNND1. Membranous localization of CTNND1 and E-cadherin was observed in N-cadherin siRNA transfection cells compared with mock control group. Western bolt analysis showed that the expression level of total CTNND1 was reduced and membranous CTNND1 was increased in miR-145 or N-cadherin siRNA transfection cells, though total E-cadherin expression was not affected.7. In vivo, we found that miR-145 showed no effect on tumor growth compared with miR-control group. The xenograft tumors of the pEZX-miR-control group displayed the sign of local invasion, including an outside-invasion of fibrotic capsule, muscle invasion and vascular invasion. However, the xenograft tumors of the pEZX-miR-145 group were well-encapsulated, showing a noninvasive phenotype. Immunohistochemistry was used to detect the expression of CTNND1 in the xenograft tumors. Our data showed that CTNND1 was expressed strongly at the cytoplasm in the pEZX-miR-control group. While weak membranous expression of CTNND1 was found in the xenograft tumors of pEZX-miR-145 group. Western blot analysis showed that CTNND1 protein was significantly downregulated in the pEZX-miR145 group, compared with the pEZX-miR-control group.Conclusion1. CTNND1 was upregulated and mainly localized at cytoplasm in gastric cancer. Cytoplasmic localization of CTNND1 protein was indicative of higher clinical stage, higher T stage, positive lymph node metastasis, and poorer prognosis in gastric cancer.2. The patients with cytoplasmic localization of CTNND1 displayed significantly poorer overall survival and disease-free survival than those in whom CTNND1 was localized to the membrane. Cytoplasmic CTNND1 could serve as an independent prognostic factor for patients with gastric cancers.3. Upregulated cytoplasmic CTNND1 could promote gastric cancer cell migration and invasion, with little effect on cellular proliferation and apoptosis. CTNND1 was expected to be a target for clinical treatment.4. miR-145 could regulate CTNND1 expression level and cellular localization. CTNND1 was upregulated in gastric cancer partly owing to the reduced expression of miR-145. CTNND1 is a direct target of miR-145. Besides suppressing cytoplasmic CTNND1 expression, miR-145 could recover the membranous localization of CTNND1.5. miR-145 inhibits invasion of gastric cancer cells not only by downregulating cytoplasmic CTNND1 expression but also by inducing the translocation of CTNNDl and E-cadherin from the cytoplasm to the cell membrane through downregulating N-cadherin.