Lentivirus-Mediated Knock-Down Of OLFM4Gene Inhibits Invasion And Liver Metastasis In Gastric Cancaer And Its Relative Mechanism

Objective:Gastric cancer(GC) is the common gastrointestinal tumor in China, and hepatic metastasis is the main death cause of late-stage patients. Up to now, however, the therapeutic efficacy for anti-metastasis is not so satisfaction.Human Olfactomedin4(OLFM4), also known as GW112, is highly expression in many types of tumors and plays an important role in the occurrence and development of tumors. The present studies suggest that expression of OLFM4, specific high expression in gastric cancer, was remarkably associated with invasion, metastasis and differentiation in gastric carcinoma. Overexpression of OLFM4was shown to enhance tumor growth and to resist cell apoptosis. Many researches indicated that NF-κB was tightly correlative with tumorigenesis, proliferation, differentiation, invasion and metastasis in tumor cells. The aim of this study is to investigate the function of OLFM4gene in Invasion and liver metastasis in GC and its relative mechanism by which OLFM4can regulate the metastasis processing in GC cells.Methods:To stably establish OLFM4-knockdown cell models in gastric cancer MKN-45cell line which was shown high OLFM4expression as our previous study,OLFM4-siRNA lentiviral vectors (OLFM4-RNAi-LV) and scrambled siRNA control lentiviral vector (NC-GFP-LV) were constructed and verified by DNA sequencing. The recombinant plasmids were transfected into human embryonic kidney293T cell line to generate high titer lentivirus. After viral infection by OLFM4-siOLFM4and NC-GFP lentivirus in MKN-45cells, GFP-positive MKN-45cells were sorted by FCM to obtain high purity GFP+cell populations (MKN-45-siOLFM4and MKN-45-NC-GFP control). To detect efficiency of OLFM4knockdown, the expression of OLFM4at mRNA and protein levels were determined by Real-time PCR and Western blotting respectively.To evaluate invasion in vitro and metastasis in vivo in MKN-45cells after OLFM4knockdown, the migration and invasion abilities of gastric cancer cells were detected by Trans-well assay in vitro, and liver metastases were investigated in nude mice model in vivo. The high purity cells (MKN-45-NC-GFP and MKN-45-Si-OLFM4) were injected into the abdominal cavity and tail vein of nude mice respectively to establish the animal models of abdominal cavity and liver metastasis in vivo. The number of the mesenteric nodules and liver surface nodules were measured to analyze its ability of metastasis in abdominal cavity and liver, and the metastatic tumor tissues were confirmed by H&E staining.To explore the possible mechanisms by which knockdown of OLFM4inhibit or promote metastasis in MKN-45cells, the protein expression involved in NF-κB signal pathway were determined by western blotting and NF-κB DNA binding activity were measured by EMSA. To further study the gene expression profiles, human Metastasis Real-time PCR microarry was used to compare the gene transcriptional differences between MKN-45-Si-OLFM4and MKN-45-NC-GFP cells.Results:Lentivirus OLFM4-RNAi-LV and negative control NC-GFP-LV lentivirus were successfully constructed. The proportion of GFP+cells selected by FCM was as high as90%. The expressions of OLFM4mRNA and protein were significantly down-regulated in MKN-45-Si-OLFM4cells compared to MKN-45-NC-GFP control cells determined by Real-time PCR and Western blot. Transwell assay showed that lentivirus-mediated OLFM4knockdown significantly reduced the ability of migration and invasion in MKN-45cells were in vitro. The mice animal models showed that knock-down OLFM4obviously inhibited the ability of the mesenteric and liver metastasis in MKN-45cells by counting the metastatic tumor nodules on mice mesentery and liver surface (P<0.01). The levels of the NF-κB signal pathway-associated proteins were remarkably attenuated in MKN-45-Si-OLFM4cells compared with MKN-45-NC-GFP cells. EMSA assay showed that OLFM4knockdown significantly repressed NF-κB DNA binding activity in MKN-45cells. Human Metastasis Real-time PCR Assay showed that OLFM4knockdown significantly changed expression of the metastasis-related genes such as CCL7, CDH1, MGAT5, KISS1, MTSS1and PTEN in MKN-45cells.Conclusion:Our study show that lentivirus-mediated deleption of OLFM4significantly suppress migration and invasion the abilities in vitro and the ability of the mesenteric and liver metastasis in vivo, suggesting that OLFM4plays an important role in progression of GC. Our data also find that knock-down OLFM4significantly inhibits NF-κB DNA binding activity and reduces relative protein expression of NF-κB pathway, indicating that OLFM4can regulate NF-κB signal pathway via feedback mechanism and consequently regulates the prosess of liver metastasis.More over, the metastasis-related genes such as CCL7, CDH1, MGAT5, KISS1, MTSS1and PTEN et al were probably also involved in the processing of invasion and metastasis in gastric cancer. In conclusion, deleption of OLFM4will be potential therapeutic strategy to prevent invasion and liver metastasis of gastric carcinoma.