Research On The Mechanism Of Pseudogene UBDP1 Competitive Binding MicroRNA And Regulating Malignant Proliferation And Invasion Of Gliobastoma

ObjectiveGlioblastoma is one of the most common and malignant brain tumors.At present,the standardized treatment of glioblastoma still can not get a satisfactory prognosis.Pseudogene is a widely expressed non-coding gene,which is derived from mutation of coding gene.The coding gene before mutation is called true gene.Many studies have confirmed that pseudogenes play an important role in the progress of human cancer.This study aims to find the pseudogenes that affect the malignant progression of glioblastoma,and to explore the key molecular mechanism of their influence on the malignant progression of glioblastoma,so as to provide new possibilities for the targeted therapy of glioblastoma.MethodsFirst,we analyzed the gene expression profile of glioblastoma and normal brain tissue that we had completed in our team’s early work(GEO dataset: GSE51146),and a pseudogene UBDP1 was screened out.UBD is the true gene of UBDP1 and was confirmed to be related to the prognosis of gliomas.RT-q PCR experiment of 30 glioblastoma samples and 15 normal brain tissues samples collected in clinical practice was performed to verified the abnormal expression of UBDP1 and its true gene UBD in glioblastoma,and the Kaplan-Meier survival analysis and Cox survival analysis of the relative expression of q PCR and the clinical information of 30 glioblastoma samples were also performed.Next,we explored the distribution of UBDP1 and UBD in glioblastoma cells by FISH and immunofluorescence experiments of the glioblastoma cell lines U87 and U251.The UBDP1,UBD overexpression / interference stable U87 and U251 Cell lines were constructed and CCK-8 cell proliferation and Transwell cell migration and invasion experiments were carried out to explore the effect of UBDP1 and UBD on the function of glioblastoma cells.The regulatory effect of UBDP1 and UBD on the malignant progression of glioblastoma in vivo was studied by constructing Intracerebral tumor model in nude mice and carrying out survival analysis of nude mice and immunohistochemistry experiment of tumor samples in nude mice.Finally,we searched for micro RNA with the same binding sites as UBDP1 and UBD by bioinformatics,and miR-6072 was screened out by western blot of micro RNA overexpression / interference U87 and U251 Cell lines,and the binding of miR-6072 and UBDP1/UBD in cells was verified by luciferase reporter gene experiment.The results of rescue experiment of UBDP1,miR-6072,UBD and phenotypic reversion experiment of UBDP1,miR-6072 were carried out to explore the mechanism of UBDP1,miR-6072,UBD.ResultsRT-q PCR result of 30 cases of glioblastoma and 15 cases of normal brain tissue showed that the expression of UBDP1 and UBD in glioblastoma was significantly higher than that in normal brain.The results of Cox survival analysis and Kaplan-Meier survival analysis showed that the expressions of UBDP1 and UBD in glioblastoma were independent prognostic factors of patients with glioblastoma;FISH and protein immunofluorescence test results of U87 and U251 showed that UBDP1 and UBD were mainly distributed in the cytoplasm of glioblastoma cells;CCK-8 cell proliferation experiments and Transwell cell migration and invasion experiments of UBDP1,UBD overexpression / interference U87/U251 cell lines showed that UBDP1 and UBD promoted the proliferation,invasion and migration of glioblastoma cells;survival analysis of brain tumor bearing nude mice showed that the survival time of brain tumor bearing nude mice overexpressed with UBDP1 and UBD was significantly lower than that of control group;the survival time of brain tumor bearing nude mice overexpressed with UBDP1 and UBD was significantly shorter than that of control group;Immunohistochemical results showed that the proportion of Ki-67 in tumor samples with overexpression of UBDP1 and UBD was significantly higher than that in the control group.The results of Western blot of miR-6072 overexpression / interference transients in U87 and U251 cell lines showed that miR-6072 inhibited the expression of UBD;luciferase reporter gene assay showed that both UBDP1 and UBD could bind to mir-6072 in cells;the rescue assay of UBDP1,mir-6072 and UBD showed that the expression of UBD was significantly increased in UBDP1 overexpression stable transfected U87 and U251 cells,and the expression of UBD was significantly increased in UBDP1 overexpression stable transfected U87 and U251 cells.The results of phenotype reversion assay of UBDP1 and miR-6072 showed that after overexpression of miR-6072 in UBDP1 overexpression stably transfected U87 and U251 cells,the proliferation,migration and invasion ability of cells that had been significantly increased decreased.ConclusionPseudogene UBDP1 promotes the proliferation,migration and invasion of glioblastoma by competitively binding mir-6072 to promote the expression of UBD.