| Glioma, one of the most common primary brain tumors, presents particularly aggressive activities and highly fatal prognosis. Although accumulated advanced techniques contribute to the diagnosis and treatment of glioma, the median survival still comes to ranging from 9 to 12 months. Given the high incidence, refractoriness and poor prognosis, novel therapeutic strategies based on molecular interaction networks must be achieved for human glioma.Fms-related tyrosine kinase 1(Flt1), also named VEGFR-1, is a full-length tyrosine kinase receptor characterized by extracellular domain, which is usually thought to be located in the vascular endothelial cells. Flt1 involves in important process like vessel formatting, collateral circulation establishing and wound healing via blinding with VEGF. Meanwhile Flt1 is also one of receptors of PIGF, which is involved in embryonic development. Due to the significant function in vessel formatting, the important role of Flt1 in tumor development process was more and more attentioned by researchers. And the investigation of anti-VEGF/VEGFRs therapeutic regimen has performed into phase ? clinical trial. Along with the accumulated study, Flt1 was found to express in many solid tumor cells, which was verified to take significant effect on not only the vessel formatting, but also the proliferation, apoptosis, migration and invasion of tumor cells. However, the detailed mechanism of how Flt1 in glioma progression are still barely known. The Wnt/?-catenin signaling pathway is a conservative pathway, which has been observed to act as a vital role in many cancers containing glioma. The activation of Wnt1 induced the GSK-3? phosphorylatied into p-GSK-3?, which resulting in the accumulation of ?-catenin in the cytoplasm. Then ?-catenin was transported into the nucleus and interacted with the transcription factor TCF/Lef-1, which was next to regulate the expression of the downstream related target genes and subsequently involved in important process of tumor growth, metastasis and angiogenesis. While some studies have reported that the activation of Flt1 might be a basis of Wnt/?-catenin activity in malignant glioma progression.Recently, microRNAs(miRNA) have been the topic that participated in the biological process of tumor initiation and progression. These small noncoding single-stranded RNAs serve as tumor-suppressors or oncogenes by completely or incompletely binding the 3'-untranslated region(UTR) of its target gene to result in either the degradation of messenger RNA(mRNA) or the reduction of protein expression in numerous cancers. Under-expression of certain miRNA is intently correlated with varieties of tumorigenic processes. Among these, miR-139-5p has been validated to decrease in several tumors and restoration of miR-139-5p contributes to explicitly inhibitory impact on tumor malignancies. So in the present study, we explored the effect of miR-139-5p on the malignance of glioma cells and sought for the possible mechanism to offer a potential supplementary therapeutic strategy of glioma in the future.Our present study was divided into four parts:In the first part of this study, we examined the levels of miR-139-5p in 26 glioma tissues, 12 normal brain tissues and 5 glioma cell lines via qRT-PCR. The results were declared that miR-139-5p was distinctly downregualted in glioma tissues and glioma cells compared with normal tissues.In the second part of this study, we focused on the inhibitory effect of miR-139-5p on the malignant phenotypes of glioma cells. We transfected miR-139-5p mimics into U87 and LN229 cells. We then determined the proliferation ability of glioma cells via MTT assay and colony-formation assay. Flow cytometry was used to examine the cell cycle. Transwell assay was performed to evaluate the migration and invasion abilities of glioma cells. Vasculogenic mimicry formation assay(VM) was used to estimate the ability of tube-formation of U87 cells. Then western blot was performed to determine the levels of EMT-associated protein. And the results we got showed that miR-139-5p suppressed the proliferation ability of glioma cells and blocked the cell cycle in G0/G1 phrase and inhibited the growth of cells in vivo and vitro. Over-expression of miR-139-5p significantly inhibited the migration/invasion abilities. Meanwhile, miR-139-5p reduced VM and EMT of U87 cells.In the third part, we examined the target gene of miR-139-5p to explore the inhibitory mechanism of miR-139-5p in malignant phenotypes. Considerate analysis of 2 bioinformatics algorithms was performed to predict a putative target gene. Then Flt1 caters our criteria. Next we detected Flt1 expression levels in cells treated with miR-139-5p mimics and found the reduction expression of Flt1 in mRNA and in protein levels. Then IHC was performed and reduced expression of Flt1 was observed in tissues treated with miR-139-5p removed from nude mice. Luciferase activity indicated a significant decrease in cells co-transfected with miR-139-5p and PGL3-Flt1-3'UTR-wild plasmid, which indicated that Flt1 is the direct target gene of miR-139-5p. Then q RT-PCR was used to examine the levels of Flt1 mRNA in 26 glioma tissues, 12 normal brain tissues and 5 glioma cell lines, and the high-expression of Flt1 indicate that Flt1 might work as an oncogene in glioma. We futher explored the effect of Flt1 in the malignant phenotypes of glioma cells through MTT assay, colony-formation assay, flow cytometry, Transwell assay, VM assay and western blot. Subsequently out results indicated the inhibitory influence of Flt1-siRNA on the phenotypes glioma cells.To futher investigated whether the effect of miR-139-5p on glioma cells was mediated through its inhibitory impression on Flt1 expression, we constructed an Flt1 expression plasmid without its 3'UTR(GV230-Flt1) and cotransfected it with miR-139-5p mimics. We further executed the rescue experiment in different phenotypes of transfected glioma cells. As our results manifested, the restoration of Flt1 could validly neutralize the suppressive influence of miR-139-5p on the malignant phenotypes of glioma cells.In the forth part, we further explored the regulatory effect of miR-139-5p on Wnt/?-catenin pathway in glioma cells through western blot. Firstly we proved that knockdown of Flt1 reduced the expression of Wnt1, ?-catenin and the phosphorylated GSK-3? and increased the level of GSK-3? in glioma cells. As Flt1 is the target gene of miR-139-5p, we speculated that miR-139-5p executed inhibitory effect on Wnt/?-catenin pathway, which was subsequently confirmed via western blot. To further verify the influence of miR-139-5p on regulating the Wnt/?-catenin signaling pathway is mediated by Flt1, miR-139-5p mimics and Flt1 expression plasmid without 3'UTR were cotransfected in U87 cells. We got the results that Flt1 can rescue the inhibition of Wnt/?-catenin pathway induced by miR-139-5p. All the data indicate that miR-139-5p may down-regulate the Wnt/?-catenin pathway through its target Flt1.Conclusively, our results found that miR-139-5p was dysregulated and conduced to the abrogation of the malignant phenotypes of glioma cells by directly pairing with the 3'-UTR of Flt1. Furthermore, miR-139-5p suppressed Flt1-mediated Wnt/?-catenin signaling pathway in the progression of glioma. Together, our finding may provide a potential supplementary therapeutic strategy concerning the molecular mechanism of glioma in the future. |
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