MiR-506 Inhibits The Proliferation And Invasion By Targeting IGF2BP1 In Glioblastoma And The Mechanism Of Its Effect

Background and Aims Glioblastoma multiforrne is the most common malignant tumor in central nervous system. The World Health Organization(WHO) classified the glioblastoma in 2007. According to the WHO’s classification to the glioblastoma, the glioblastoma is designated as the Grade IV brain tumor, which is the highest malignant form of glioblastoma, and constituting about 50% of all the tumor in central nervous system. There are two forms of the primary and secondary of glioblastoma, according to the reasons leading to the glioblastoma. As we all know, most of the glioblastoma are the primary, and that are more common in adults older than 50 years old. The age of secondary glioblastoma population is relatively young, common in adults less than 45 years old. The current study suggests that: the primary glioblastoma with the higher malignant characteristics and mortality than the secondary. In recent years, along with the more in-depth research, people divided glioblastoma into 4 types according to the difference phenotypes of glioblastoma: Classical, Mesenchymal, Proneural and Neural. Because of different reactions to the radiotherapy or chemotherapy treatment, and different biological characteristics of the glioblastoma, the scientists give the different Category of glioblastoma phenotypes. Studies shown that four different genes phenotypes of glioblastoma corresponding to different stages of neural development, the most important thing is that the different genes phenotypes of prognosises are not the same to the clinical outcomes. Micro RNAs(mi RNAs) is the important member in the non-coding RNA family. The structure of mi RNAs composed of about 22 nucleotides in length, highly consistent of single stranded RNA in evolution. The mi RNAs regulated the expression of targete m RNA mainly by leading to the target m RNA degradation or translational inhibition. The abnormal expressions of mi RNAs have a close relationship to the human tumors development. In recent years, many studies found that mi RNAs are closely associated with cell proliferation, invasion, migration, and other related acts in the cell formation process. Currently, there are many reports about micro RNA-506(mi R-506) in other kinds of tumors, such as stomach cancer, liver tumors, breast cancer, osteosarcoma cancer, bladder cancer and so on. But the current study suggests that: mi RNA-506 may both play the role of leading cancer gene and a suppressor gene in the tumor development. Until now, the role of mi R-506 in glioblastoma generate remains unknown. To explore the biological characteristics of mi R-506 in glioblastoma process, we introduce mi R-506 mimic to promote its expression in glioblastoma cells, then we use bioinformatics and molecular biology identify its target gene preliminary. This paper further analyzed the mi R-506 and the target gene IGF2BP1 on glioblastoma differentiation and proliferation regulation characteristics.First part: The expression, clinical relevance and bio-functions of mi R-506 in tumor tissue from glioblastoma patientsObjective: To determine the expression of mi R-506 in tumor tissue from glioblastoma patients, and evaluate the clinical relationship between mi R-506 expression and clinical characteristics including prognosis of patients, and then further study the biological function of mi R-506 in the progress of glioblastoma proliferation and invasion.1. The relative expression of mi R-506 in glioblastoma cell lines(U87, U251, LN229 and A172) and the normal human astrocytes cell line(NHAs) were detected by q RT-PCR technology. 2.The effect of mi R-506 overexpression on cell migration and invasion of glioblastoma cell was determined by MTT after cells transfected with mi R-506 mimics and the influence on cell cycle was detected by Flow Cyto Metry. 3. The effect of mi R-506 overexpression on cell migration and invasion of glioblastoma was determined by scratch repair and matrix hit transwell experiments. the expression of invasion related protein was detected by Westernblot. 4.The effect of mi R-506 on glioblastoma cell proliferation was determined by clone forming experiment. Methods:Results: 1. Compared with the NHAs, the expression of mi R-506 in glioblastoma cell lines(U87, U251, LN229 and A172) was significant decreased. There were statistically differences(P<0.05). 2. Mi R-506 significantly declined the U87 cells proliferation after mi R-506 mimic transfection, there were statistical significance(P<0.05). CCK8 assay showed that mi R-506-transduced U87 cells exhibited significantly lower growth rates than scramble groups. Cell cycle assays also showed that U87 transfected with mi R-506 mimics had an obvious cell cycle arrest at the G0/G1 phase. 3.Transwell invasion assays were utilized to examine the effect of mi R-506 on cell invasion. The invasive potential of the U87 cells was decreased after transfection with mi R-506 mimics when compared with cells transfection with scramble mimics, there were statistically differences(P<0.05). The nvasion-associated protein test results: the expression levels of MMP1, MMP2,and MMP9 were significantly declined after transfecting mi RNA-506 mimic(P<0.05), and the MMP11 protein level had no change.Conclusion: The expression of mi R-506 was closely related to clinical phenotypes and clinical prognosis in glioblastoma patients and mi R-506 overexpression can significantly inhibit glioblastoma proliferation and invasion. Mi R-506 may affect the gliblastoma cell invasion by adjusting MMP1, MMP2, MMP9 protein expression. These results suggested that mi R-506 could be considered as a tumor suppressor gene in the development of glioblastoma.Second part: The molecular mechanism study of mi R-506 in the glioblastoma developmentObjective: Bioinformatics techniques were used to screening and identified the mi RNA gene targeting mi R-506, and the molecular mechanisms involved in glioblastoma progression were investigated.Methods: 1. Use The micro RNA online database(targetscan mi Rbase mi Rwalk) to predict the target gene of mi R-506. 2. Westernblot and Luciferase technique were used to detect the pecific target of mi R-506 for predicting target genes IGF2BP1. 3. The effect of mi R-506 overexpression and target genes overexpression were detected by MTT via building overexpression plasmid of IGF2BP1. 4. By using gene chip and immunochemistry technique to analysis glioblastoma gene expression profile and the relationship of IGF2BP1 expression level with clinical features and prognosis in glioblastoma tissue.Results: 1.We found 3’UTR of IGF2BP1 containing the conserved putative mi R-506 binding sites using the commonly cited programs(Target Scan). Overexpression of mi R-506 repressed the activity of p GL3-WT-IGF2BP1-3’UTR plasmid in U87 cells, without changes in luciferase activity of p GL3-MUT-IGF2BP1-3’ UTR plasmid. Moreover, mi R-506 inhibited the m RNA expression of IGF2BP1 in the U87 cells. mi R-506 suppressed the protein level of IGF2BP1 in the U87 cells. 2. Knockdown of IGF2BP1 inhibited U87 cells proliferation and enhanced cell cycle arrest at the G0/G1 phase. 3. Over-expression of IGF2BP1 enhanced U87 cells proliferation and inhibited cell cycle arrest at the G0/G1 phase. 4. The IGF2BP1 expression results and glioblastoma clinical pathology parameters showed: the glioblastoma prognosis was related to IGF2BP1 expression level(P = 0.001); Kaplan-Meier analysis showed that the survival time of glioblastoma patients with low IGF2BP1 expression was longer than who with high IGF2BP1 expression, suggesting that the IGF2BP1 expression level was closely related clinical outcomes in glioblastoma patients.Conclusion: 1. Mi R-506 targeted regulation of the expression of IGF2BP1 in glioblastoma; After silence IGF2BP1 expression in glioblastoma that can decreased proliferation significantly, cell cycle obviously stagnation; overexpressed after IGF2BP1 all this is revere. 2. The survival time of high expression of IGF2BP1 was shorter than low expression. 3. Mi R-506 inhibited glioblastoma by negatively regulate the target gene expression IGF2BP1; mi R-506 may become a new therapeutic target for glioblastomatreatment in the future.The full text conclusions: 1. The expression of mi R-506 in glioblastoma tissues and cells were significantly decreased. 2. Mi R-506 overexpression significantly inhibited glioblastoma proliferation, migration and invasion in vivo and vitro, cell cycle arrest, which indicated that mi R-506 may play a tumor suppressor genes role in the development and progression of glioblastoma. 3. Mi R-506 could target regulation the expression of IGF2BP1, resulting in the inhibition of IGF2BP1 expression, glioblastoma proliferation was significantly decreased and cell cycle obviously stagnation.The glioblastoma cells proliferation was partially recovered and cell cycle arrest significantly reduced after IGF2BP1 overexpression. 4. The survival time of glioblastoma patients with IGF2BP1 high expression significantly shorter than patients with lower expression of IGF2BP1, and the expression of IGF2BP1 is closely related to the patient’s metastasis and recurrence. 5. Mi R-506 could negatively regulated the expression of IGF2BP1, resulting in the inhibition of glioblastoma proliferation. The mi R-506/IGF2BP1 played an important role in the development of glioblastoma and may become a new therapeutic target for glioblastoma treatment in the future.