MiRNA-204-5p Targeting RAB22A Regulates The Mechanism Of Proliferation, Migration And Invasion Of Gliomas

High-grade gliomas are the most common form of adult malignant brain tumors, and are characterized by rapid progression, high resistance to radiotherapy or chemotherapy, as well as exhibit extremely poor clinical prognosis. Similar to other tumors, glioma mainly results from genetic risk factors and environmental carcinogenic factors. Some genetic diseases, environmental carcinogenic factors may also be associated with the occurrence of glioma. Consequently, a better understanding of the molecular mechanisms involving glioma formation and development will be helpful to highlight novel therapeutic targets and develop strategies for the treatment of gliomas. miRNA is a class of non coding single stranded small molecule RNA with 5’end band with phosphoric acid group of 20-24 nucleotides, and 3’end band with hydroxyl. miRNAs have emerged as key factors involved in several biological processes, including development, differentiation, cell proliferation, and tumorigenesis. The dysregulation of miRNAs in cancer has been repeatedly described, miRNAs is also involved in the pathological process of many malignant tumors, and the abnormal expression of miRNA is also related to the glioma. miR-204 is one of the most common abnormal changes of miRNA, miR-204 in the mature process can be divided the miR-204-5p and miR-204-5p. miR-204-5p has been reported to function as a tumor suppressor in a variety of human cancers through different mechanisms, suggesting its extensive function in tumor stage, lymph node metastasis, poor prognosis. However, the role of miR-204-5p in glioma was not well known. RAB22A is usually activated by binding GTP in the transport vesicles and then hydrolysed to generate GDP-bound RABs after membrane fusion. Although RAB22A has recently been reported to be upregulated in some cancers, little is known about its role in human tumorigenesis, especially in human glioma. but its role is limited to the human cancer. miR-204-5p has been reported to function as a tumor suppressor in a variety of human cancers through different mechanisms, suggesting its extensive function in tumorigenesis. We demonstrated that the expression of miR-204-5p is significantly correlated to the glioma by the clinical and in vitro and in vivo ways, Further mechanistic investigations revealed that miR-204-5p inhibits gliomas cell growth, migration and invasion by directly targeting RAB22A, which appears to be a new treatment strategy in gliomas.In the first part of this study, we detected the expression of miR-204-5p cluster in the freshly removed glioma samples by In-Situ Hybrization and qRT-PCR respectively. The results showed that all of miR-204-5p down-regulated in glioma tissue comparing to normal brain tissue. Moreover, the expression level was positively correlated to the glioma WHO grade. These results implied that miR-204-5p might has a role in glioma development and progression.In the second part of this study, we used lentiviral expression vector(LEV) to construct miR-204-5p overexpression system. At the same time, we selected miR-204-5p of antisense oligo nuclear nucleotide as the inhibitor, miR-204-5p homologs as the mimics. We studied the biological role of miR-204-5p in glioma cell growth in LN229 and U87 cell line. By MTT assay, colony formation, edu experiments, we found that compared to the LEV group, the proliferation of glioma cells were greatly decreased in the Lev-miR-204-5p group; By Transwell(Boyden) and chamber experiments, we found that compared to the LEV group, the cell migration and invasion abilitys were greatly decreased in the Lev-miR-204-5p group. We also analyzed predicted targets of each miRNA by bioinformatics approaches analysis. According to the bioinformatic prediction, the majority of targets of these miRNAs concentrate on the individual development and differentiation. We demonstrated that miR-204-5p directly targets RAB22A to inhibit proliferation, migration and invasion in glioma cells by the dual-luciferase activity test, qRT-PCR and Western blot. RAB22A overexpression could significantly increase cell growth, migration and invasion. These data suggested that miR-204-5p may be considered to be a tumor suppressor through directly targeting RAB22A.In the third part of this study, we established orthotopic transplantation model system in nude mice. The results showed that miR-204-5p transfected LN229 cells displayed a marked reduction of the tumor, it also can reduce the expression of Ki-67. Kaplan-Meier survival analysis demonstrated that miR-204-5p significantly prolonged survival of glioma in mice.Taken together, miR-204-5p may play an important functional role in decreasing the ability of neoplastic cells to grow and invade tissue in glioma in the clinical, in vitro and in vivo, Overexpression of miR-204-5p is able to suppress glioma cell growth and metastasis through directly targeting RAB22A. So the miR-204-5p has significant value as an unfavorable progression indicator for glioma patients, and may serve as a therapeutic target in the future.