The Study On The Role And Regulatory Mechanism Of IGFBP3in Gastric Cancer

Gastric cancer is the fourth most common malignant tumor in the world, the incidence in our country is much higher than the global average level. Being lacking of specific early symptoms, half of the gastric cancer patients have already progressed to advanced stage on diagnosis and the prognosis is poor. Activation of oncogenes and inactivation of tumor suppressors are involved in the development of gastric cancer. Screening of these genes and study on the molecular mechanisms are of great interest in the early diagnosis and therapy of gastric cancer.Insulin-like growth factor binding protein-3(IGFBP3) is a major IGFBP species in circulation. Binding with type I insulin-like growth factor (IGF-I) is the main way to exert functions. Previous studies showed that IGFBP3was dysregulated in a list of tumors and affect the proliferation and apoptosis of tumor cells. Our previous cDNA microarray revealed that multiple genes including IGFBP3might be regulated by HoxD10in gastric cancer cells, which induced us to believe that IGFBP3is a downstream target of HoxD10and influence the biological behaviors of gastric cancer.To prove this hypothesis, in this study, we immunostained tissue microarray containing gastric tumor and adjacent tumor free tissues with IGFBP3antibody. The expression of IGFBP3between tumor and adjacent tumor free tissues was analyzed. Patients’ TNM stages, pathological grades and survival time were also correlated with the expression of IGFBP3. Migration and invasion abilities of gastric cancer cells were evaluated by wound-healing test and transwell assays after silencing the expression of IGFBP3. We also detected invasion-related factors to explore the potential mechanisms. Functional HoxD10binding sites at the promoter region of IGFBP3was identified by chromatin immunoprecipitation and dual-luciferase report assays.Methods:1.The relationship between the expressions of IGFBP3in gastric cancer tissues and clinical characteristics(1) Tissue microarray containing gastric tumor and adjacent tumor free tissues were immunostained with IGFBP3antibody. The expressions of IGFBP3between the tumor and adjacent tumor free tissues were compared.(2) Patients’ TNM stages, pathological grades and survival time were correlated with the expression of IGFBP3in gastric cancer tissues.2.The effect of IGFBP3on the migration and invasion of gastric cancer cells and the possible mechanisms(1) Migration ability of BGC823and SGC7901was evaluated by wound-healing test and transwell assays after IGFBP3siRNA was transfected.(2) Invasion ability of BGC823and SGC7901was evaluated in transwell chambers pre-coated with matrigel after IGFBP3siRNA was transfected.(3) Invasion-related factors were measured by RT-PCR and Western blotting after IGFBP3siRNA was transfected.3. Transcriptional regulation of HoxD10on IGFBP3in gastric cancer cells(1) RT-PCR, Western blotting and dual-luciferase report assays were applied to detect the mRNA, protein and promoter transcription activity of IGFBP3after ectopic expression of HoxD10in BGC823and SGC7901cells.(2) Potential HoxD10binding sites at the promoter region of IGFBP3were predicted using a promoter binding site prediction software. Chromatin immunoprecipitation was employed to screen the sites which could recruit HoxD10.(3) Dual-luciferase report assays was used to validate the functional HoxD10binding sites. Whether the change of luciferase activity would be silenced was also detected after the core binding sequences were altered.Results:1. The expression of IGFBP3in gastric cancer tissues was related to lymph node metastasis and prognosis.(1) The expression level of IGFBP3in tumor tissues was significantly lower than that in adjacent tumor-free tissues (p<0.01).(2) Patients with high expression of IGFBP3are less likely to be in stage N2+N3(p<0.05). Positive rate of lymph node was significantly lower in those patients (p<0.01).(3) The4.5-year survival rate in patients with high expression of IGFBP3was strikingly higher than that in low-IGFBP3patients.2. IGFBP3inhibited the migration and invasion of gastric cancer cells.(1) In a panel of wild-type gastric cancer cells, HGC27had the highest expression of IGFBP3, followed by BGC823, SGC7901and GES-1, and only mild expression of IGFBP3was detected in AGS, MKN28, MKN45, NCI-N87and MGC803cells.(2) The wound of BGC823and SGC7901cells at the bottom of plate healed faster after interfering the expression of IGFBP3with siRNA, migration and invasion rates at12h and24h were increased significantly (p<0.01).(3) An increased number of cells migrating through the transwell membrane was observed after interfering the expression of IGFBP3with siRNA (p<0.01).(4) More cells invaded the tranwell membrane coated with matrigel after interfering the expression of IGFBP3with siRNA. OD value was improved markedly in dye elution from cells attached on the outer membrane (p<0.01).(5) mRNA expression of MMP14, uPA and uPAR was upregulated significantly after interfering the expression of IGFBP3with siRNA.3. HoxD10regulates the expression of IGFBP3in gastric cancer cells transcriptionally.(1) Transfection of pcDNA3.1-HoxD10upregulated the expression of HoxD10, with concomitant overexpression of IGFBP3at mRNA and protein levels.(2) The luciferase activity of IGFBP3promoter report gene was markedly increased after HoxD10plasmid was transfected (p<0.01). Five potential HoxD10binding sites (HBS1-5) at the2.3kb upstream region of IGFBP3gene were predicted by PROMO software.(3) HBS3, HBS4and HBS5could amplify the chromatin precipitated by HoxD10antibody. Ectopic expression of HoxD10could increase the luciferase activities of HBS3, HBS4and HBS5(p<0.01), while mutant HBS3, HBS4and HBS5had no such effect.Conclusions:1. Gastric cancer patients with high expression of IGFBP3have less metastatic lymph nodes and a more favorable prognosis.2. IGFBP3inhibits the migration and invasion of gastric cancer cells, at least in part, through the MMP14and uPA/uPAR pathways.3. HoxD10binds with HBS3, HBS4and HBS5at the promoter region of IGFBP3gene and upregulates its expression at the transcription level.