Roles Of MiR-128, MiR-149and MiR-181on The Proliferation, Invasion And Chemosensitivity Of Glioma Cells

BackgroundGlioma is a common type of intracranial tumor. The therapeutic outcomes of glioma are quite poor due to their aggressive growth. The carcinogenesis of glioma is a multi-stage process involving multiple factors. It is still a challenge to detect molecular biomarkers for early diagnosis and therapeutics.miRNAs are endogenous non-coding RNAs and their dysregulated expression is closely related to the development of glioma, providing new tools for diagnosis and therapeutics of glioma. We have previously constructed differentially expressed miRNA and mRNA profiles of glioma with different WHO differentiation grades. We found that miR-107, miR-124and miR-149could regulate expressions of multiple target genes, whereas Rap IB could be regulated by multiple miRNAs including miR-128a/128b, miR-149and miR-181b et al. The Pearson correlation analysis was performed, showing an inverse correlation between Rap1B and miR-128a/b, miR-149or miR-181b. It suggested that Rap1B which is regulated by these miRNAs is involved in the development of glioma. In this study, we aim to confirm that Rap1B is the direct target of miR-128、miR-149and miR-181and their differentially expression in different WHO differentiation grades. We will further investigate the roles of miR-128、miR-149and miR-181in the proliferation and invasion of glioma cells through targeting Rap1B. The sensitization of glioma cells toward temozolomide by miR-128、miR-149and miR-181will also be investigated.Rap1B is the common target gene of miR-128, miR-149and miR-181a/b/c/dTargetScan, Picta and miRDB were used to search for miRNA binding sites in the3’UTR of Rap1B, showing that miR-128, miR-149and miR-181a/b/c/d may bind to the3’UTR of Rap1B. The luciferase assay, real-time PCR and western blot were performed to confirm that Rap1B is the common target genes of miR-128, miR-149and miR-181a/b/c/d and can be transcriptionally or post-transcriptionally regulated by these miRNAs.Correlation between miR-128, miR-149, miR-181a/b/c/d and Rap1B in astrocytoma with different WHO differentiation gradesReal-time PCR was performed to measure the expressions of miR-128, miR-149and miR-181a/b/c/d and the expression of their target gene Rap1B in astrocytoma with different WHO differentiation grade. The results showed that down-regulated expressions of miR-128and miR-181family members were closely related to the initiation of astrocytoma and down-regulated expressions of miR-149and up-regulated expression of Rap1B were closely linked to the development of astrocytoma. The Pearson correlation analysis was performed, showing an inverse correlation of expressions between miR-128, miR-149, miR-181a/b/c/d and Rap1B in astrocytoma and the inverse correlations were not related to the differentiation grading of astrocytoma.Roles of miR-128,miR-149and miR-181a/b/c/d in the growth and invasion of glioma through Rap1BmiRNAs with dysregulated expression can always be oncogenes or tumor suppress genes, playing important roles in the development of cancer. Clonogenic assay, MTT and transwell assay were performed to investigate roles of miRNAs in the growth and invasion of glioma cells, indicating that overexpression of miR-128, miR-149or miR-181a/b/c/d or knockdown of Rap1B could inhibit the proliferation and invasion of glioma cells. It suggested that miR-128, miR-149and miR-181a/b/c/d could function as tumor suppressor genes.Mechanism of sensitization of glioma cells towards chemotherapy mediated by miR-128, miR-149and miR-181a/b/c/dTMZ is a newly developed chemotherapeutical drug used to treat malignant glioma or recurring glioma. Although effective rate of TMZ treating malignant glioma is up to45%, the insensitivity or the required resistance to TMZ still present major obstacles to successful treatment to glioma. miRNAs can be the potential therapeutic targets for glioma, sensitizing glioma cells towards TMZ. In this study, we found that overexpression of miR-128, miR-149and miR-181a/b/c/d sensitized glioma cells towards TMZ. Rap1B, which is the direct target genes of miR-128, miR-149and miR-181a/b/c/d, is the subunit of Ras related GTP-binding protein Rap1. Like Rho family of GTPases Rac1, RhoA and CDC42, Rap1play roles in multiple biologic behaviors such as cytoskeleton remodeling, cell adhesion and invasion. We also found that TMZ upregulated the expression miR-128, miR-149and miR-181family members, in turn inhibiting the expression of Rap1B. The inhibition of Rap1B could affect the expression cytoskeleton proteins including CDC42、RhoA and N-cadherin, regulate the expression and distribution of cytoskeleton protein F-acin, and inhibit the aggressive invasion of glioma cells. miR-128、miR-149and miR-181family members can also sensitize glioma cells towards TMZ by targeting Rap1B mediated cytoskeleton remodeling.