The Function And Mechanism Of CircUCHL3 In Glioblastoma

Aims: Among adult brain tumors,glioblastoma(GBM)is the tumor with the highest recurrence rate and malignancies(WHO Grade IV).It is also one of the intracranial tumors with the worst survival prognosis.Less than 10% of the patients can achieve a 5-year survival rate.The median survival is just one year.Even after aggressive treatment,almost all GBM patients eventually relapse and die quickly within a year.GBM is a highly invasive and heterogeneous complex disease,and its pathogenesis has not been completely clarified.Circular RNA(circRNA),as a member of non-coding RNA that has attracted much attention,may be involved in the occurrence and development of a variety of cancers by regulating the transcription of cancer genes,and is a promising new marker for cancer diagnosis.This project intends to identify circRNAs related to the occurrence of GBM through transcriptome sequencing of GBM tissues,and explore their effects on the biological functions of GBM cells and the mechanism of action,so as to provide biomarkers for the prognosis of GBM and potential intervention targets for the treatment of GBM.Methods: 1)Target circRNA screening and annular structure identification: find and screen the circRNA with significant differential expression in 3 GBM simples and 3 adjacent simples from Air Force Military Medical University Tangdu Hospital,performed RNA-seq.Bioinformatics analysis of differentially expressed circRNAs was performed using R language,circUCHL3 was the most obvious up-regulated expression were screened out,so we choose to explore the function of circUCHL3 in GBM cells.Designed the convergent primer and divergent primer,and performed reverse transcription PCR with specific reverse primers,q PCR products were electrophoretized in agarose gel.Next,the products of agarose gel were sequenced and compared with circ BASE database.The stability of circUCHL3 was tested by RNase R tolerance experiments.Real-time quantitative PCR was used to detect the expression of circUCHL3 in GBM and adjacent tissues to determine whether circUCHL3 is dysregulated in GBM.2)Explore the biological function of circUCHL3: Overexpress and knock down circUCHL3 in U251 and U87 GBM cell line,and detect cell proliferation,colonation,migration,invasion,apoptosis and cell cycles by Transwell,CCK8 and Flow cytometry,respectively.3)Explore the mechanism of circUCHL3 in GBM.According to the results of bioinformatics co-expression analysis,we find the target protein ASS1 that can be regulated by circUCHL3 and we verify the regulated ability of circUCHL3.Change the expression level of circUCHL3 in U251 and U87 cells,and check whether the expression level of ASS1 changes by Western blot.From the function enrichment and the study,we found the AMPK/mTOR signal pathway related ASS1.Results:1)Target circRNA screening and annular structure identification: the differential circRNAs detected in RNA-seq data were screened,and the differential expression of circUCHL3 was significantly up-regulated(Fold change =2.9);In GBM tissues and normal tissues,convergent primers across splicing sites were designed and q PCR experiment was performed to verify the expression level of circUCHL3.It was further indicated that circUCHL3 was up-regulated in GBM tissues.The amplification products were sequenced by Sanger.The results were compared with the sequence in circ BASE database,and the circular structure of circUCHL3 was identified.These results indicate that circUCHL3 is formed by the back splice of exons 5-8 of UCHL3 gene on chromosome 13.RNase R resistance experiment results showed that circUCHL3 was more resistant to Rnase R than linear m RNA UCHL3,which further verified the existence of its circular structure.2)Explore the biological function of circUCHL3: Using U87/U251 as in vitro model,the expression level of circUCHL3 was knocked down or overexpressed.The experimental results showed that after overexpression of circUCHL3,the proliferation ability,migration and invasion ability of cells were significantly increased,cell apoptosis was slowed down,and the ratio of S/G2 phase of cells was increased.Contrary results were obtained after knockdown,indicating that circUCHL3 could positively regulate GBM cell proliferation and progression.3)Explored the mechanism of circUCHL3 regulating GBM: the co-expression network based on RNA-seq data showed that the expression level of ASS1 was significantly correlated with circUCHL3.In humans,the key rate-limiting enzyme of arginine synthesis is ASS1,which is involved in the progression of various tumors.At the same time,functional enrichment analysis based on the GO functional annotation and KEGG database showed that ASS1 and AMP-activated protein kinase,AMPK is closely related to the activity of mTOR signaling pathway.The results showed that the overexpression of circUCHL3 upregulated the expression of ASS1,further activated the activity of AMPK/mTOR signaling pathway,and increases the phosphorylated expression level,but does not change the unphosphorylated protein level.Conclusions: 1)This study explored the bio-function and mechanism of circUCHL3 in the development of GBM by using human GBM samples,and GBM cell lines.The experimental results show that: circUCHL3 is a highly stable circular RNA.Its expression in GBM tissues and cells is significantly up-regulated,which suggesting that circUCHL3 may play an important role in the development of GBM.2)The results indicated that circUCHL3 knock down could effectively suppress the cell proliferation,migration and invasion,block the cell cycle and promote the cell apoptosis of glioma cells U251 and U87 in vitro,whereas the upregulation of circUCHL3 exerted opposite effects.3)CircUCHL3 can inhibit the biological function of AMPK/mTOR signal path by regulating ASS1 which ultimately hinders the progress of GBM.This study provides new ideas and theoretical basis for the treatment of GBM by exploring the function and potential mechanism of circRNA in GBM.