Abstract. Malignant gliomas are the most common and deadly primary brain tumors in adults and the high proliferative ability of these cells is one of the most important causes of the poor prognosis of this cancer. Suppressing the proliferation of malignant gliomas cells by altering effector molecules can significantly improve the prognosis of a patient. microRNAs (miRNAs) are small non-coding RNA molecules ~22 nucleotides in length that are able to function as oncogenes or tumor suppressors in human cancer. In the present study, it was demonstrated that the expression level of miRNA-218 (miR-218) is markedly downregulated in glioma cell lines and human primary glioma tissues. Upregulation of miR-218 in glioma U87 cells dramatically inhibited the proliferation by inducing G 1 -S checkpoint arrest. Furthermore, it was demonstrated that ectopically expressing miR-218 in glioma U87 cells results in the downregulation of the expression of cyclin dependent kinase (CDK)6 and cyclin D1 and upregulation of the expression of p21 Cip1/Waf1 . In addition, it was identified that miR-218 inactivated the CDK6/cyclin D1/p21 Cip1/Waf1 pathway by downregulating CDK6 expression through the direct targeting of the 3'-untranslated region of CDK6. The present results suggest that miR-218 plays an important role in the prevention of the proliferation of glioma cells, and the present study also revealed a novel mechanism for miRNA-mediated direct suppression of the CDK6/cyclin D1/p21Cip1/Waf1 pathway in glioma cells. IntroductionHuman glioma is the most common primary tumor in the central nervous system and is characterized by a high proliferative and invasive ability (1). Standard therapies for glioma, including surgery, radiation and chemotherapy, are only effective in treating patients with a high-grade condition. Numerous glioma patients have already developed metastasis at the onset of clinical symptoms (2). The mechanism of glioma tumorigenesis remains unclear, and the molecular determinants of the aggressiveness of glioma have been the subject of numerous studies, but these investigations have not yet reached full fruition (3-6). Therefore, there is an acknowledged requirement for novel approaches based on increased understanding of the biological and molecular nature of these tumors. microRNAs (miRNAs) are short non-coding, single-stranded RNA molecules that are 22-25 nucleotides in length and negatively regulate gene expression by post-transcriptional silencing of target messenger RNAs (mRNAs) through complementary binding (7,8). An increasing number of studies have indicated that miRNA plays an important role in the development of various cancers, including glioma, and miRNA has been associated with tumor suppressor and oncogenic activities (9,10). Out of these miRNA molecules, miRNA 218 (miR-218) has been revealed to be downregulated in human glioblastoma multiforme (GBM) specimens compared with the adjacent brain tissue that is devoid of tumor cells (11)(12)(13)(14). Accumulated data have demonstrated that the upregulati...