| BackgroundTumor metastasis is an important biological feature of malignant tumors. Metastasis, a consecutive process including local invasion, intravasation, survival in circulatory system, extravasation, and colonization in a secondary site, is the most lethal attribute of cancer. Specifically in colorectal cancer,90%of patients finally die in metastasis. Formation of new blood vessels is indispensable for expansion of metastases after disseminated tumor cell localized. Therefore, targeting tumor angiogenesis has been acknowledged as a pivotal anti-metastasis strategy.Vascular endothelial growth factor (VEGF), found in1989as a kind of glucoprotein separated from cattle pituitary gland follicle stellate cells, is the most prominent angiogenic factor to promote tumor development. Especially in colorectal cancer (CRC), VEGF system involved neovascularity represents a key mediator of cancer initiation and dissemination and the first target of antiangiogenic therapy. Anti-VEGF treatments, including VEGF monoclonal antibodies and VEGF receptor tyrosine kinase inhibitors, combined with conventional cytotoxic chemotherapy, have been clearly proven anti-metastatic effective. Bioinformatic algorithms assess that all the human miRNAs may regulate up to30%of human genes which represents the majority of genetic pathways. The overall miRNAs expression profiles in tumor have been observed distinct from that in normal tissues. Recent years, growing evidence shows miRNAs are involved in pathogenesis of various tumors, function as oncogenes or tumor suppressors depending on what products the mRNA targets encode. For instance, decreased expression of miR-143is responsible for dysregulation of KRAS in CRC. miR-135promotes WNT signaling pathway via repressing tumor suppressor APC gene.miRNAs have been reported to regulate VEGF involving tumor angiogenesis. miR-126could positively promote embryonic angiogenesis by suppressing negative regulator SPRED1of VEGF signaling. VEGF dependent expression of miR-296inhibits HGS gene to promote neovascularity by increasing affinity of VEGFR2to VEGF. These findings indicate the potential therapeutic utility of miRNAs in anti-angiogenesis strategy.In silico prediction showed VEGF was a potential target of miR-126. The aim of this study is to investigate miR-126/VEGF link and the potential mechanism for VEGF dysregulation and contribution to angiogenesis and metastasis in CRC.Methods and Results1. miR-126is frequently lost in CRC tissues and cell linesTaqman real-time PCR was used to detect endogenous miR-126level in colorectal tumor tissues and cell lines. It showed that,12selected tumor samples had significantly lower miR-126expression relative to their matched adjacent normal colonic epithelia. In6human CRC cell lines, It also showed a notable loss of miR-126, whereas the control normal colon mucosa pooled from3individuals expressed a strong level of it.2. Restoration of miR-126inhibits VEGF expression LoVo and SW620cells were transfected with miR-126precursors. Western blot showed that the enhanced miR-126in LoVo cells significantly repressed VEGF protein expression, but with no apparent alteration on VEGF mRNA detected by RT-PCR. It indicated this inhibitory effect occurred in the process of translation.3. The direct and specific interaction of miR-126with3’UTR of VEGF mRNABioinformatic data predicted that miR-126may target VEGF through binding to3’UTR of VEGF mRNA. A luciferase reporter vector with the putative VEGF3’UTR binding site for miR-126and its mutant version by site direct mutagenesis were constructed. We transfected luciferase reporter vector alone or together with pre-miR-126or scramble control into HEK293cells. The significantly decreased luciferase activity was noted when VEGF3’UTR vector was co-transfected with pre-miR-126, compared to the mutant vector. The miR-126-mediated suppression of luciferase activity was abolished in mutant VEGF3’UTR vector. These results highlighted a direct and specific interaction of miR-126with VEGF3’UTR.4. Restoration of miR-126impairs migration and invasion ability of CRC, but has no effect on its proliferationLoVo and SW620cells, transfected with pre-miR-126, were harvested at24h post-transfection and subjected to transwell assay in order to evaluate migration and invasion ability. Briefly, transfected cells were resuspended in serum free RPMI-1640medium at a density of1.0X10/ml.300μl prepared transfected cell suspension in serum free medium and500μl medium containing20%FBS were respectively added in each insert (Matrigel pre-coated) and the matched lower chamber. After48h, non-invading cells were removed using a cotton swab, then the underside of the insert was stained. For migration assay, the procedures were similar with invasion assay, except that200μl cell suspension was cultured in transwell insterts for48h. It showed that miR-126-transfected cells, compared to the blank and scramble controls, exhibited a significant decrease of migratory ability (P<0.05). The corresponding effect on invasive ability was also observed in parallel invasion assay (P<0.05).In alarmar blue proliferation assay, LoVo and SW620cells were seeded in96-well plates at0.5X104/well for24h, then transfected with miR-126precursor or scramble control. The transefected cells were incubated for24h,48h and72h respectively and subjected to cell viability detection. Within72hours, ectopic miR-126expression had no significant influence on proliferation of the transfected cells,compared to the blank and scramble controls (P>0.05).5.Restoration of miR-126expression inhibits tumor angiogenesis through targeting VEGF secretionVEGF secretion in the conditioned medium (CM) harvested from pre-miR-126transfected LoVo cells was validated using ELISA assay. At the different pre-miR-126concentrations of10nM,30nM and50nM, the VEGF secretions in CM was significantly decreased in comparison with blank and scramble controls (P<0.05). In HMVECs’migration assay, when co-cultured with pre-miR-126transfected LoVo cells in transwell system, the migration ability of HMVECs was significantly attenuated (P<0.05). In endothelial tube formation assay, CM from pre-miR-126transfected LoVo cells was used to suspend HMVECs.0.5×104HMVECs were seeded into each well of96-well plate pre-coated with Matrigel.Reduced spontaneous ability to form capillary of HMVECs in presence of CM obtained from pre-miR-126transfected LoVo cells was observed, versus if in presence of CM from the blank and scramble controls (P<0.05). For further validation of the anti-angiogenic effect of miR-126in vivo, chick embryo chorioallantoic membrane model (CAM) was performed using gelatin sponge loaded with different CM mentioned. It showed that the neo-vessel formation on the chorioallantoic membrane was significantly inhibited in presence of CM from pre-miR-126transfected LoVo cells (P<0.05).6. Promoter methylation is required for the loss of miR-126in CRCThe methylation status of miR-126CpG islands in62CRC tissues and6CRC cell lines was tested using MSP.50out of62CRC tissues, as well as all the6CRC cell lines, were identified to be methylated within promoter region of miR-126gene. Correspondingly, high frequencies of methylated CG dinucleotid within promoter region of miR-126gene were detected in all the6CRC cell lines. Further, the same panel of CRC cell lines was treated with5-aza-CdR, a specific inhibitor of DNA methyltransferase Ⅰ, and then subjected to analyz the expression of miR-126using Taqman real time-PCR and VEGF expression using western blot. Compared to the blank control cells, the demethylated counterparts showed restored expression of miR-126and expectedly decreased expression of VEGF protein. These findings suggested epigenetic silence of miR-126may partly lead to dysregulation of VEGF expression in CRC.ConclusionIn our study, we identified that miR-126could target VEGF at translational level through binding to3’UTR of VEGF mRNA. Expression of miR-126is commonly decreased in CRC.Methylation within promoter region is required for miR-126decline in CRC. Restoration of miR-126could inhibits migration and invasion ability of CRC cells, and suppresses VEGF-induced tumor angiogenesis. Collectively, miR-126may be a potential therapeutic target for anti-metastatic strategy in CRC treatment. |
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