Microrna-145Regulates N-cadherin To Inhibit Invasion And Metastasis In Gastric Cancer

Objective:Invasion and metastasis are two major characteristics of malignant tumor. More and more reports suggested that microRNA-145(miR-145) played an important role in the process of invasion and metastasis in malignant tumor. In the present study, the expression level of miR-145in gastric carcinoma and cancer cell lines were determined by RT-qPCR. Transfection experiments with miR-145precursor were performed in vitro and vivo respectively. The target gene of miR-145was identified and the mechanism of miR-145to promote the decades of invasion and metastasis in gastric cancer was discussd.Methods:Reverse transcriptase real time quantitative PCR (RT-qPCR) was used to determine the levels of miR-145in136cases of human gastric cancer tissues,17cases of nontumorous gastric mucosa and4gastric cancer cell lines. MiR-145precursor, anti-miR-145inhibitor, as well as negative control were transfected in gastric cancer cell lines BGC823and SGC7901, and cell proliferation assay, migration assay and invasion assay were performed. Then gastric cancer cell SGC7901transfected with lenti-virus expressing vector pEZX-miR145were injected in mice to observe miR-145’s effect on tumor local invasion and metastasis in vivo. The detection of luciferase assay and western blot were further performed to identify the target gene of miR-145and its downstream effector.Results:(1). The expression level of miR-145of136cases of gastric cancer was32.35-fold lower than that of17cases of nontumorous mucosa by RT-qPCR. A further2.62-fold lower of miR-145expression was observed in the loci of secondary metastases compared with primary gastric cancers. Meanwhile, the level of miR-145expression was2-31fold lower in4gastric cancer cell lines comparing to nontumorous mucosa (P<0.05).(2). By transfection with miR-145precursor in cell lines BGC823and SGC7901, we proved a significant suppressive effect of miR-145on cancer cell migration and invasion compared with negative control (P<0.05). Moreover, this suppression phenomenon could be reversed by transfecting with anti-miR-145inhibitor (P<0.01). These data suggest that miR-145could suppress invasion and metastasis of gastric cancer cells in vitro. But miR-145had no ability to inhibit cell growth and proliferation (P>0.05).(3). Invasion experiment in vivo revealed that the xenograft tumors with pEZX-miR145transfection were noninvasive or well-encapsulated, as shown by their intact fibrotic capsules. However, the negative control group displayed local invasion around tumors, which showed islands of cancer cells invaded the stroma or muscle tissues. For experimental metastasis assayexperiment in vivo, both of the two groups had lung metastasis. However, the miR-145-expressing cells developed significantly fewer lung metastasis than the control group. These results demonstrated that miR-145could suppress invasion and metastasis of gastric cancer cells in vivo.(4). Constructed pmirGLO-cad vector and miR-145precursor were co-transfected in gastric cell lines BGC823and SGC7901. The luciferase assay showed that miR-145transfected group significantly reduced the luciferase activity of pmirGLO-cad compared to the negative control group (P<0.05). Western blot showed that N-Cadherin protein was downregulated followed by the overexpression of miR-145compared with the negative control group. These data corroborate that N-Cadherin is a target gene of miR-145.(5). Western blot analysis further showed matrix metallopeptidase9(MMP9) protein was also downregulated when miR-145was overexpressed or when N-Cadherin protein expression was inhibited by transfecting with N-Cadherin siRNA. And this phenomenon was further comformed by cell immunohistochemistry. All these data suggest that miR-145suppresses tumor metastasis by inhibiting protein translation of its direct target gene N-cadherin, and then indirectly downregulates its downstream effector MMP9.Conclusions:MiR-145was downragulated in gastirc cancer tissues and cancer cells, which played a suppressor role in the process of invasion and metastasis. MiR-145could suppress tumor invasion and metastasis by suppression its direct target gene N-Cadherin and downstream effector MMP-9.