MiR-124-5p Inhibits The Growth Of High-grade Gliomas Through Posttranscriptional Regulation Of LAMB1

Gliomas is the most common malignant brain tumors, the incidence is about35.26～60.96%(average44.69%) of all intracranial primary brain tumors. Generally, the center of gliomas is a hemorrhagic necrosis, while the surrounding is a high density of tumor cell area. Glioma cells are characterized by unlimited proliferation and strong aggression. Glioma is easy to invade into the surrounding normal brain tissue. Gliomas grow rapidly and with a characteristic of invasive growth, therefore, glioma is difficult to be completely removed by surgery. In addition, glioma is insensitive to radiotherapy and chemotherapy, so glioma has a high recurrence rate and poor prognosis. Despite given a variety of treatments, the effect of treatment is not satisfactory. Survival of glioblastoma patients is only12-15months. Malignant glioma is not easily being cured largely depended by its strong invasion. Therefore, it is necessary to investigate invasiveness of glioma and find more effective treatments.Commonly, the process of tumor invasion is complex. Tumor cells shed from a primary tumor foci and invade into basement membrane, then have an interaction with extracellular matrix (ECM). Firstly, tumor cells adhere to endothelial cells and basement membrane, and secrete proteolytic enzymes and promote angiogenesis. All malignant glioma cells will invade into the surrounding tissue. Glioma cells often invade into the surrounding tissue along with myelinated fibers and blood vessels. Therefore, invasion of glioma is related to compositions of the basement membrane and extracellular matrix. Compositions of the basement membrane and extracellular matrix have been confirmed overexpressed in human gliomas, such as fibronectin, laminin, III and IV collagen, vitronectin and tenascin. These substances are closely linked to migration and invasion of glioma cell.Laminin (laminin, LN) is a large family of extracellular heterologous glycoprotein trimers and mainly constituted by three different α, β, γ chain. Laminin plays an important role in many fundamental biological processes, including cell adhesion and migration, embryonic development, tumor invasion, angiogenesis, tissue differentiation and wound healing. Laminin is a major functional component of the vascular basement membrane extracellular matrix (ECM). Moreover, Laminin is main component of microvascular BMs of normal brain tissue. Glioma cells can synthesize and secrete a variety of LN involved in the vascular basement membrane and vascular basement membrane. A large number of integrin receptors are expressed on the glioma cell surfaces. Studies have found that overexpression of α4chain laminin is observed in the low-level or high-level glioma and normal brain tissue adjacent to GBM. The majority of GBMs had increased expression of laminin-8(α4β1γ1) chains in blood vessel walls, whereas low-grade tumors overexpressed laminin-9(α4β2γ1) chains.Laminin-8(a4b1g1) is the predominant isoform expressed in vascular endothelial cell BMs. Along with its receptor integrin a6β1, laminin-8has been shown to be important for BM function and the maintenance of the blood-brain barrier. Laminin-8also promote adhesion and migration of glioma cell through interactions with α3β1and α6β1integrin. GBMs that predominantly expressed laminin-8had a shorter time to recurrence than did GBMs that predominantly expressed laminin-9.GBM is characterized by endothelial cell proliferation and prominent vascularization arising from a combination of blood vessel co-option and tumor angiogenesis, the latter being a fundamental aspect of tumor growth, invasion, and metastasis. a4chain laminin itself can promote tumor angiogenesis. Above illustrate that laminin-8plays an important role in the invasion and angiogenesis of gliomas to promote tumor recurrence. During progression of human gliomas, the expression of capillary basement membrane(BM) laminins containing a4chain switched from the predominant laminin-9(α4β2γ1) to laminin-8(α4β1γ1). Laminin-8is distinguished to laminin-9by having αβ1subunit. The factors regulating the switch to laminin β1expression in gliomas are still unknown. Therefore, it is very meaningful to study the mechanisms of gene regulation about laminin-8.MicroRNAs are an abundant class of endogenous small RNA molecules,20-25nucleotides in length. The role of miRNAs as key regulatory molecules that control a wide variety of fundamental cellular processes, such as proliferation, death, differentiation, motility, invasiveness, etc. Because miRNA genes are frequently located at the chromosomal fragile sites of cancer genomes, miRNAs have been considered as novel classes of oncogenes and tumor suppressors.Abnormal expression levels of microRNAs were found in most solid tumors and hematologic malignancies. These abnormal expression of microRNAs can also inhibit tumor cell proliferation and induce tumor cell apoptosis and inhibit tumor cell migration, reduced expression of multiple target genes, and so on.A series of microRNAs expression profiling about brain tumors also found a number of abnormal microRNAs expression. The genes of these abnormal expression microRNAs usually located upstream or downstream signaling pathways of brain tumors to regulate oncogenic phenotype of brain tumor cells. The microRNA (miRNA) miR-124is abundantly expressed in the embryonic and adult cortex in various mammalian species, suggesting an essential role in normal functioning of the brain. Indeed, expression profiling of various human brain tumors has shown that miR-124is among the most dramatically downregulated miRNAs in anaplastic astrocytoma (Ⅲ grade) and glioblastoma (IV grade), suggesting reduction of miR-124plays a key role in formation and progression of glioma. Restoring miR-124expression in gliomas may slow tumor progression by repressing various miR-124targets such as laminingl and integrin β1. MiR-124-3p (known as miR-124or miR-124a) and miR-124-5p (known as miR-124*) are both mature forms of miR-124(A putative miR-124-5p binding site in the3’UTR of LAMB1was mutated by replacing the cytosine with guanine [marked by an asterisk]. MiR-124-3p is significantly downregulated in AA and GBM tumors, which is correlated with poor prognosis in glioma patients However, whether the expression of miR-124-5p has a similar result as miR-124-3p in human gliomas is still unknown.Therefore, it is very meaningful to screen out exact target genes of miR-124in the development of regulatory networks in glioma.Based on the reduced miR-124-5p expression and high levels of laminin β1protein that were observed in GBM cell lines and tissues, the present work investigated the potential regulation of laminin β1by miR-124-5p.Therefore, the potential regulatory mechanisms of miR-124-5p this were discussed. The expression levels of LAMB1and miR-124-5p were examined in glioma cell lines (U87and U251) and GBM tissue samples by quantitative PCR and Western blotting. Regulation of miR-124-5p on LAMB1(protein) expression is defined by dual luciferase assay system. The potential regulation of LAMB1by miR-124-5p was investigated by assessing the effects of restored miR-124-5p expression on cell proliferation, colony formation, and tumor growth and angiogenesis. Chapter I Relationships between laminin8and miR-124-5p expression in gliomasObjective:Detection of laminin-8mRNA and protein, miR-124-5p expression levels in all grade gliomas, and to analyze whether there is an association between laminin-8and miR-124-5p in glioma progression.Methods:U87and U251human glioma cell lines were cultured. Glioma tissue samples were obtained and proscessed through the Department of Neurosurgery1andDepartment of Pathologyof Zhujiang Hospita. The expression levels of LAMB1and miR-124-5p were examined in glioma cell lines (U87and U251) and GBM tissue samples by quantitative PCR and Western blotting.Results:AA and GBM tissues showed significantly higher levels of LAMB1mRNA and protein relative to normal brain tissue, and grade I/II gliomas, and GBM tissues had the highest level of LAMB1gene expression. A significant decrease in miR-124-5p expression was observed in AAs and GBMs compared with normal brains and grade Ⅰ/Ⅱ gliomas. Rank correlations calculated between miR-124-5P expression level and LAMB1mRNA or protein levels indicated that elevated LAMB1mRNA or protein expression occurred concomitantly with reduced miR-124-5p expression.Conclusions:Progressive increases in the levels of LAMB1mRNA and protein were observed with increasing glioma severity. miR-124-5p expression was significantly downregulated during glioma progression. miR-124-5p and laminin-8can act as biomarkers of glioma. Reduction of miR-124-5p and overexpression of LAMB1is associated with progression of gliomas. There is a negative correlation between miR-124-5p and LAMB1. Chapter Ⅱ MiR-124-5p Regulates LAMB1Expression at the Posttranscriptional LevelObjective:To verify regulation of LAMB1expression at the posttranscriptional level by miR-124-5p.Methods:An in silico analysis using MicroCosm Targets Version5test a potential miR-124-5p binding site in the3’UTR of LAMB1. Lipofectamine2000transfecte U87and U251glioma cells by miR-124-5p minic, NC, miR-124-5p inhibitor and miRNA NC inhibitor. The expression levels of LAMB1were examined in glioma cell lines (U87and U251) by quantitative PCR and Western blotting. Regulation of miR-124-5p on LAMB1(protein) expression is defined by dual luciferase assay system.Results:An in silico analysis using MicroCosm Targets Version5revealed a potential miR-124-5p binding site in the3’UTR of LAMB1. After72hours, LAMB1protein expression was reduced in cells overexpressing miR-124-5p compared with NC-transfected cells. Furthermore, suppression of miR-124-5p resulted in higher levels of LAMB1protein compared with cells transfected with NC inhibitor. Significantly, the levels of LAMB1mRNA were unaffected by miR-124-5p overexpression or inhibition. LAMB1(protein) expression is directly and negatively regulated by miR-124-5p.Conclusions:MiR-124-5p has no effect on expression LAMB1mRNA in U87and U251cells, but a direct negative regulation of LAMB1(protein) at the posttranscriptional level. LAMB1is a direct target gene of MiR-124-5p.Chapter Ⅲ Restoration of miR-124-5p expression inhibits tumor growth and microvessel density in glioma xenograftsObjective:To assess the effects of restored miR-124-5p expression on cell proliferation, cell cycle, cell migration as well as tumor growth and angiogenesis.Methods:1.In vitro:miR-124-5p minic and miRNA minic negative control (NC) transfect U87glioma cells by Lipofectamine2000.Cell proliferation, cell cycle and cell invasion of U87glioma cell transfected miR-124-5p minic and miRNA minic negative control were analyzed by MTT assay and Trans well cell migration assay.2. In vivo:Glioblastoma xenografts immunocompromised nu/nu mice models of U87and U251were established. When tumor volume reached150-200mm3, the mice were randomly assigned to two treatment groups:miR-124-5p minic and NC groups (n=6), the mice were respectively given miR-124-5p minic and NC. We monitored tumor volume changes for three weeks. The expression levels of LAMB1in glioma tissues were examined by Q-PCR, Western blotting and immunohistochemistry. Tumor microvessel density changes of tumor specimens is determined by immunohistochemical analysis of endothelial marker CD31.Results:miR-124-5p did not significantly inhibit U87cell proliferation, cell cycle and cell invasion. A significant growth inhibition of U87and U251xenograft tumors injected with miR-124-5p minic was observed, compared with tumors injected with the NC.LAMB1protein levels of MiR-124-5p minic group were reduced, compared with the NC group. LAMB1mRNA levels of MiR-124-5p minic group were not statistically significant to NC group. Injection of the miR-124-5p minic to restore miR-124-5p expression led to a dramatic reduction in microvessel density in U87and U251xenografts.Conclusions:Restoration of miR-124-5p expression can inhibits tumor growth and microvessel density in glioma xenografts.