The Mechanisms Of MiR-218-regulated Angiogenesis In Gastric Cancer

Gastric cancer is a common gastrointestinal malignancy.In China,though incidence and mortality are decreasing,gastric cancer remains the third leading cause of cancer related deaths.It is well known that tumor growth and metastasis are closely related to angiogenesis.Therefore,vascular targeting therapy has received increasing attentions in recent years.As a result,a variety of vascular molecular targeting drugs,including bevacizumab,have been approved for clinical use.Among them in the gastric cancer,ramucirumab,a monoclonal antibody VEGFR-2 antagonist was approved for the treatment of advanced gastric cancer and became the first vascular targeted drug for gastric cancer treatment.However,despite of fruitful results,vascular targeted therapy are also faced with some urgent problems,such as a suboptimal response to therapy in some cases,drug resistance and side effects.Thus,to solve these problems is of great importance so that more patients can benefit from AIAs.MicroRNA(microRNA)is a class of endogenous small RNA.In tumor,microRNA involves a variety of tumor malignancies,such as tumor metastasis and angiogenesis.As we all know,angiogenesis is orchestrated by complex signaling pathways,thus the single target treatment may not be efficient and can be easily by-passed.While miRNAs can regulate multiple signaling pathways through complementary binding to the seed sequence located at the 3'-UTR of target genes.Besides,miRNAs have been reported to be transmittable between cancer and host cells,remodeling the cancer microenvironment.These characteristics make miRNAs favorable candidates for novel AIAs with lower risk of drug resistance.miR-218,located in the introns of SLIT2 and SLIT3,directly targets ROBO1,which is in turn a receptor of SLIT2 and SLIT3.We first demonstrated that the miR-218 oriented activation of SLITs-ROBO1 pathway was involved in GC metastasis.However,the SLIT-ROBO pathway was originally identified in the central nervous system.During neural development,the N-terminal fragment of SLIT2 stimulates the formation of axon branches by binding to ROBO1 and ROBO2.Angiogenesis has been shown to share many similarities with axonal guidance,as indicated by the concomitant relationship and molecular cues between nerves and blood vessels.Thus,we hypothesized that miR-218 also contributed to tumor angiogenesis through the SLIT/ROBO pathway.In this study,we systematically examined the role of miR-218 in tumor angiogenesis.Our results showed that mi R-218 disrupted the overall structure of vessels in vitro.miR-218 treated xenograft GC tumors showed a reduced blood vessel density and thus a repressed growth rate in vivo.Furthermore,mechanistic analysis demonstrated that the effect of miR-218 was partly mediated by ROBO1.These discovery identified a novel role of miR-218 in GC and indicated it could potentially be used in anti-angiogenic therapy.Aims:1.To determine the expression pattern of mi R-218 in GC vascular endothelium.2.To explore the relationship between miR-218 level and various clinicopathological variables in GC.3.To observe the regulation effect of miR-218 on angiogenesis in vitro and in vivo.4.To explore the potential molecular mechanism of miR-218 in the regulation of angiogenesis in gastric cancer.Method:1.The expression of mi R-218 in GC and normal tissues was detected by ISH in a gastric cancer tissue microarray.Also,ISH staining of CD31 were performed in a series of microarray sections to display vessels.And combined with the CD31 IHC results,the expression of miR-218 in blood vessels of gastric cancer and normal gastric epithelium was analyzed and quantified.2.The effect of miR-218 on endothelial cells was detected by in vitro scratch healing assay,tube formation assay as well as an optimized fibrin gel bead assay.3.The regulation of mi R-218 on ROBO1 in vascular endothelial cells was verified by both qRT-PCR and western blot.4.siRNAs and lentivirus that respectively carry the antisense or CDS region of ROBO1 were constructed to manipulate the expression of ROBO1 in HUVEC-2C,followed by Transwell migration assay and tube formation assay to explore the role of ROBO1 in mi R-218-mediated inhibited angiogenesis.5.A subcutaneous xenotransplant tumor model was established.On this basis,terminally modified mature miR-218 or vehicle control was injected into subcutaneous GC xenografts respectively.Two weeks after the first injection,the tumor mass was removed.Tumor volume and MVD in each group was measured to reflect the effect of miR-218 on tumor angiogenesis.Results:1.miR-218 level in GC was significantly lower than that in adjacent normal tissues.And statistics results showed that low level of mi R-218 was associated with lymph node metastasis and advanced stage in GC.Moreover,Kaplan–Meier analysis of the high and low miR-218-expressing patient groups revealed that low miR-218 level was related to a poor prognosis in GC2.Tumor tissues were apparently richer in blood vessels.And combined with IHC staining of CD31,we observed a significantly lower expression of miR-218 in tumor vessels compared with that in vessels from adjacent tissues3.miR-218 inhibit the migration of primary umbilical vein endothelial cells(HUVECs)in vitro.In tube formation assay,the length and number of HUVEC-forming tubules after overexpression of miR-218 were dramatically decreased.Consistently,with miR-218 inhibited,the migration and tube formation ability of HUVEC was significantly enhanced.In addition,an increased sprouting of HUVEC in 3D culture environment after overexpression of miR-218 was observed.Similarly,miR-218 inhibited cell migration and planar tube formation in an immortalized umbilical vein endothelial cell lines(HUVEC-2C).4.RT-PCR and western blot results confirmed that mi R-218 could regulate the expression of ROBO1 in endothelial cells.The number of migrating cells in endothelial cells was significantly decreased when ROBO1 was knocked down by siRNA.The length and number of tubules were also lower than those of the control group.Subsequently,ROBO1 was stably overexpressed using lentiviral vectors in HUVEC-2C-miR-218 and we found that overexpression of ROBO1 restored the migration and tube formation ability reduced by mi R-218.Overall,the above results indicate that miR-218 can regulate the expression of ROBO1 in endothelial cells,and ROBO1 mediated the inhibitory effect of miR-218 on angiogenesis.5.In vivo,intratumoral injection of miR-218 significantly inhibited the growth rate of transplanted tumors.And CD31-labeled tumor microvessel density was distinctly lower than that in the control group.Conclusion:miR-218 was down-regulated in GC tissues and was closely related to poor prognosis.Compared with normal blood vessels,a decreased expression of miR-218 was found in GC endothelium.Restoration of mi R-218 played important roles in GC angiogenesis in vitro.Furthermore,we demonstrated a potential therapeutic role of miR-218 in xenografted tumor by reducing the vessel plexus.In conclusion,our finding enriched the understanding of the mechanism of gastric cancer angiogenesis from a microRNA-target perspective.And delivery of miR-218 may be a new therapeutic strategy in combating GC angiogenesis.