MiR-186 And Apollon Affect The Proliferation And Invasion Of Human Glioma Cells

Glioma is the most common malignant brain tumor,and its efficacy is still unsatisfactory due to its unique invasive growth pattern.In recent years,the molecular targeted therapy of tumor has been developing rapidly.It is important to explore the molecular biological mechanism of glioma and find a new effective target for the treatment of glioma.The study of targets involved in RNA regulation or protein expression remains hot in the field of glioma.This study is divided into two parts,which are related to the study of MicroRNA and inhibitor of apoptosis proteins.The first part:MicroRNA-186 targets IGF-1R and inhibits cell proliferation and invasion in gliomaMicroRNA(miRNA)belongs to a class of small,non-coding RNA s that can promote the degradation of target genes or inhibit their translation at post-transcriptional level.Studies have shown that aberrant expression of miRNA play an important role in various tumors including glioma.MiRNAs can act as tumor suppressor genes or oncogenes to participate in the development of tumors.Recent miRNA expression profiling studies have shown that miR-186 is downregulated in a variety of tumors,but its potential functions in tumorigenesis and its underlying mechanisms have not been elucidated.The present study was aimed to investigate the expression level and effects of microRNA-186(miR-186)and IGF-1R on glioma and the underlying molecular mechanism.IGF-1R,a tyrosine kinase receptor,has previously been demonstrated that could abnormally express in many malignant tumors and participate in the development of tumors by promoting cell proliferation,inhibiting apoptosis and so on.Considering that both miR-186 and IGF-IR are associated with invasive ability of tumor,we hypothesized that miR-186 and IGF-IR may have a targeting relationship using biological prediction software.The present study was aimed to investigate the expression level and effects of microRNA-186(miR-186)on glioma and its underlying molecular mechanism.Here,RT-qPCR was performed to detect miR-186 expression in glioma tissues and cell lines.Cell proliferation and invasion were assessed by using MTT assay and cell invasion assay,respectively.Bioinformatic analysis and luciferase reporter assay were adopted to identify IGF-1R as a novel target gene of miR-186.The expression level of IGF-1R mRNA was also measured using RT-qPCR.The relationship betweenmiR-186 and IGF-1R expression was evaluated with spearman' s correlation analysis.Furthermore,the regulatory effect of miR-186 on IGF-1R mRNA and protein expression was determined using RT-qPCR and western blot.Finally,we used the small interfering RNA technique to silence the IGF-1R gene,RT-qPCR and Western blot methods to detect the expresion of IGF-1R mRNA and protein.We also detected the proliferation and invasion of the cells by MTT and Transwell methods.Our results showed that miR-186 was significantly downregulated in glioma tissues and cell lines.Resumption of miR-186 expression suppressed cell proliferation and invasion of glioma.IGF-1R was validated as a direct target gene of miR-186.In addition,IGF-1R mRNA was upregulated and inversely correlated with miR-186 expression in glioma tissues.Moreover,the effects of IGF-1R knockdown on glioma cells proliferation and invasion were similar with those induced by miR-186 overexpression.These findings demonstrated that miR-186 acted as a tumor suppressor by targeting IGF-1R in glioma,suggesting miR-186 as a potential therapeutic target for the treatments of this disease.The second part:Apollon overexpression promotes cell proliferation of glioma,and predicts patients' poor prognosis.Recent studies have shown that the occurrence and development of malignant tumors are related to the imbalance of apoptosis.As the largest member of the family of inhibitors of apoptosis proteins,Apollon has been reported to play a carcinogenic role in a variety of human tumors.In this part,we aimed to investigate the clinical impact of apollon dysregulation in patients with gliomas,and its effect in malignant phenotypes of glioma cells.Study methods:Apollon expression in human gliomas tissues was examined by immunohistochemistry.Associations between apollon expression and various clinicopathological factors as well as patients' prognosis were then evaluated.Followed by the transfection of an apollon-specific small interfering RNA in glioma cells,Apollon protein expression was detected by Western Blot and cell proliferation and invasion were measured by cck-8 and transwell assays in vitro.Results showed that immunostainings of apollon protein were mainly shown in tumor cell cytoplasm of glioma tissues,but weakly or negatively observed in nonneoplastic brain tissues.Statistically,the immunoreactive score(IRS)of apollon protein in glioma tissues was significantly higher than that in the corresponding nonneoplastic brain tissues.Additionally,patients with higher IRS often had advanced WHO grade and shorter overall survival time.Further multivariate analysis identified apollon overexpression as an independent prognostic factor of glioma patients.Functionally,in vitro experiments revealed that loss of apollon could efficiently suppress proliferation and invasion of glioma cells.In conclusion,these findings imply that the overexpression of apollon may be implicated into tumorigenesis and various pathological changes of human gliomas.Importantly,this protein might be a potential prognostic marker and novel therapeutic target for patients with this tumor.