The Mechanism Of MiR-101 Inhibited Proliferation And Invasion Of Glioblastoma Cells In Vitro And In Vivo

Background and objective:Glioma is the most common type of primary malignant central nervous system(CNS) tumor, the incidence of the tumor is about 46%. The 2007 World Health Organization(WHO) classifiation of CNS tumors separates glioma into grades I–IV, whereby grade I and II are defied as low grade and grade III and IV as high grade(also known as malignant glioma) in clinical practice. Treatment of surgical resection for malignant glioma, which is characteristic of high aggressive, is mostly unrealizable. The 95%median survival of patients with newly diagnosed glioblastoma(GBM) is only 3 months if they receive none therapeutic strategies, and even though treatment of surgical resection followed by adjuvant radiation and chemotherapy, the median survival of patients is not significant extension.MicroRNA(miRNA)is an endogenous non-coding small molecules RNA and widespread in animal and plant. It is consider as the common target for multiple genes, and meanwhile effect on many target moleculars. The regulation mechanism of miRNA is mainly inhibit translation of target genes through specific recognition sites in post-transcription, or direct reduce the downstream gene expression level. miRNA plays a very important role in the process of occurrence and development of a wide variety of tumors. There is no exception that the expression profile of miRNA in GBM is abnormal. Many researches showed that miRNA subtype is involved in main signal paths in glioma, it imply the abnormal expression of miRNA is related to the proliferation and invasion of glioma. In addition, transfect ectogenic miRNA could rescue the phenotype resulting from down expression of miRNA subtype and has potential clinical significance.It has been reported that low expression of miR-101 was accompanied by poorer survival rates in patients with liver cancer and present a positive correlation with the occurrence of glioma. In addition, the results showed that miR-101 can directly inhibit EZH2 and c-Myc at the expression lever, thereby inhibiting the proliferation and and invasion of glioma cells. However, the activity and function of downstream gene of miR-101 in malignant glioma remain unknown. Therefore, the study of mechanisms between mi R-101 and glioma becomes very important and has potential clinical significance.According to the analyzed by bioinformatics software and related literature reviewed, the study was focus on the relationship between miR-101 and the proliferation and invasion of glioma cells. At first, qRT-PCR was used to assess expression lever of miR-101 in glioma primary tumor specimens and normal brain tissues. Then, Lentiviral transfection was used to generate U251-MG and U87-MG cell lines that knock-down of miR-101, and assays that assess cell proliferation, migration and invasion were used to characterize the in vitro properties of the glioma cells through a series of experiments, including MTT experiment, scratch test, Transwell experiment and nude mouse tumorigenicity assay. In order to clarify the molecular mechanism, we analyze the 3’UTR region of the target gene via target gene prediction software, and then connect it to the reporter vector pGL3-Control by molecular cloning techniques. Subsequently, we confirmed that in glioma SOX9 was the direct target of miR-101 using luciferase reporter gene assay, Western blot and qRT-PCR and other methods. At last, we analyzed the influence in glioma cell lines treated with SOX9 silence.Using qRT-PCR, we show that the protein of miR-101 expression was significant decreased in glioma tissues compared to vector controls, overexpression of miR-101 increases the growth, migration of glioma cells, including the nude mouse tumorigenicity assay. we confirmed that in glioma SOX9 was the direct target of miR-101 using luciferase reporter gene assay, Western blot and qRT-PCR and other methods. Knock-down SOX9 can inhibited the ability of the proliferation and invasion of glioma cells by the technology of Lentiviral transfection.In conclusion, we firstly confrimed that miR-101 can act directly on SOX9 and then inhibit the ability of proliferation and migration in brain glioma.