RIZ1 Negatively Regulates UbcH10 Via Targeting C-Myc In Malignant Meningioma

Meningioma is one of the most common primary tumors of central nervous system(CNS),making up about 30% of intracranial primary tumors. In the light of 2016 World Health Organization (WHO) classification criteria, meningioma can be classified as benign,atypical or anaplastic (malignant). Compared with the benign ones, high-grade meningioma has worse prognosis and higher risk of recurrence. Despite advanced therapies for high-grade meningioma, including surgery and radiation, the prognosis of these patients have not been improved significantly. Thus, other treatments, such as gene targeting therapy, have been rapidly developed, and finding new molecular targets for innovative approaches to reduce recurrence and improve survival is urgently needed.Retinoblastoma protein-interacting zinc finger proteinl (RIZ1),which is considered to be an important tumor suppressor gene, is located on 1p36. Recent studies have indicated that the expression of RIZ1 is closely related to the occurrence and development of glioma,breast cancer, hepatocellular carcinoma, colon cancer and other tumors.In our previous study, we found that the expression of RIZ1 protein was negatively correlated with the pathological grade of meningioma, and over-expression of RIZ1 in malignant meningioma cell lines can inhibit cell proliferation and activate G2/M checkpoint mechanism to promote its apoptosis. Further study showed that forced expression of RIZ1 could decrease the expression of c-Myc.UbcH10, also named UBE2C, is an important member of ubiquitin-conjugating enzymes family and exerts oncogenic function. Our previous study found that the expression of UbcH10 protein in meningioma tissues was positively correlated with the pathological grade of meningiomas, even more important, patients with higher UbcH10 expression had poor prognosis. It is also found that over-expression of c-Myc could promote the expression of UbcH10. Therefore, we hypothesized that RIZ1 may regulate the function of c-Myc and UbcH10 in meningiomas, and maintain the malignant proliferation of meningioma cells.At the beginning of this study, we cultured and identified 2 primary anaplastic meningioma cells from fresh surgical specimens. The relationship between RIZ1 and UbcH10 was studied by immunohistochemistry and overexpression experiments. The biological function of UbcH10 in malignant meningioma primary cells was observed through the in vitro experiments. Mechanistically, we demonstrated that RIZ1 regulates UbcH10 expression in a c-Myc-dependent manner by ChIP assay and Luciferase reporter assay. This study can be divided into four parts. The first part: primary malignant meningioma cells culture; the second part: study on the relationship between RIZ1 and UbcH10; the third part: study on the biological effects of UbcH10 konckdown in primary malignant meningioma cells; the fourth part: study on the mechanism of RIZ1 regulates UbcH10 expression in a c-Myc-dependent manner.Part Ⅰ Primary malignant meningioma cells cultureObjective: To culture the primary malignant meningioma cells which provided the carrier for following in vitro experiments.Methods: The fresh specimens of human meningioma were obtained through surgery,and the primary cells were cultured by chemical digestion method. Specific molecular markers of the cells were detected for identification by immunofluorescence assay. Finally,cells were implanted in nude mice and the markers of the xenograft tumors were analyzed by immunohistochemical staining.Results: We cultured and identified 2 primary anaplastic meningioma (WHO Ⅲ) cells,cells were bipolar or multipolar morphology, and proliferated quickly. The cells went through more than 50 serial passages. Epithelial membrane antigen (EMA) and vimentin(VIM) were positive in the cells which were detected by immunofluorescence assay, the molecular markers were also positive in xenograft tumors of the nude mice, the percent of Ki-67-positive cells were 40% and 33% respectively. In addition, the same methods were used to culture and identify 2 primary cells of endothelial cell meningioma (WHO Ⅰ) for control experiments. WHO I meningioma cells grew slowly after about 10 circles of passage.Conclusion: After primary cell culture and identification, we obtained 2 WHO Ⅲ and 2 WHO I primary meningioma cells for following experiments.Part Ⅱ Research of the relationship between RIZ1 and UbcH10Objective: To confirm that UbcH10 is a downstream molecular of RIZ1.Methods: The expression of RIZ1 and UbcH10 in four primary meningioma cells was detected by western blotting. The over expression plasmid of RIZ1 was constructed and transfected into two primary WHO III meningioma cells, and the expression of UbcH10 was observed after transfection. Finally, the expression of RIZ1 and UbcH10 in 63 patients with different pathological grades of meningiomas were analyzed by immunohistochemistry, and the correlation between RIZ1 and UbcH10 was statistically analyzed.Results: The expression of RIZ1 decreased with the increase of pathological grade,on the contrary, the UbcH10 expression was positively correlated with the malignancy grade. Next, we transfected the overexpression plasmid of RIZ1 into WHO Ⅲ meningioma cells, and then decreased expression of UbcH10 was detected. Immunohistochemistry revealed that RIZ1 expression had an obvious negative correlation with UbcH10 expression by means of Spearman rank correlation (P<0.0001).Conclusion: UbcH10 is a downstream molecular of RIZ1Part Ⅲ Function study of UbcH10 knockdown in primary malignant meningioma cellsObjective: To identify the functional role of UbcH 10 in primary malignant meningioma cells.Methods: Primary malignant meningioma cells were transiently transfected with siRNA targeting UbcH10. Interference efficiency was detected by real-time PCR and western blotting. A series of cell functional experiments were carried out in the primary cells 24h after transfection, and the effects of UbcH10 knockdown in the primary cells were examined.Results: UbcH10 expression level was down-regulated in siRNA group compared with NC group. We assessed the effect of siRNA on the proliferation by using WST-1 assay.Compared with NC group, cell proliferation was significantly inhibited in cells with UbcH10 silenced. We analyzed the cell cycle distribution by flow cytometry. It was revealed that the population of G2/M phase in primary cells transfected with UbcH10 siRNA was significantly increased. Next, we studied the effect of RNA interference on cell motility through transwell assays. The results showed that suppressing of UbcH10 expression brought out a strong inhibitory motility of the cells. We explored the influence of UbcH10 knockdown on apoptosis. The number of apoptotic cells were significantly increased by interference with UbcH10 siRNA.Conclusion: UbcH10 is an important molecule that affects the occurrence and development of meningiomas.Prat Ⅳ Mechanism of RIZ1 regulates UbcH10 expression in a c-Myc-dependent mannerObjective: Study on the molecular mechanism of RIZ1 regulates UbcH10 via c-Myc in transcriptional regulation.Methods: ChIP assay was performed to validate whether c-Myc was able to bind to the UbcH10 promoter region. Luciferase reporter gene assay was used verify whether RIZ1 regulated UbcH10 in a c-Myc dependent manner. c-Myc stably knocked-down primary malignant meningioma cells were established by the transfection of lentivirus vector. The expression of UbcH10 was detected after transfection of RIZ1 overexpressing plasmid in primary cells with different c-Myc backgrounds.Results: c-Myc was able to bind to the UbcH10 promoter region by performing ChIP assay. Luciferase reporter gene assay revealed that RIZ1 regulates UbcH10 promoter activity via targeting c-Myc. Transfection of pIRES2-RIZ1 into primary cells induced a significant decrease in UbcH10 expression, however, almost no reduction of UbcH10 expression was observed after transfection of pCMV-RIZ1 into the c-Myc shRNA-transduced cells,both at the mRNA level and protein level.Conclusion: RIZ1 inhibits tumor progression in meningioma via the c-Myc/BbcH10 axis.