CircPTN Sponges MiR-145-5p And MiR-330-5p To Promote Proliferation And Stemness In Glioma

Background:Previous studies have demonstrated that the profile of circRNAs between tumor and normal tissues varied greatly.However,the role of circRNAs in glioma is unknown.We analyzed the data of circRNA between 27 glioma tissues and 19 normal brain tissues sequenced by Song et al,we found circPTN highly expressed in glioma tissues,but the role it plays and how it regulates in glioma is unknown.Objective:To determine the role circPTN plays in glioma,study how it influence in glioma,and explore its potential as diagnosis biomarker in glioma.Methods:qRT-PCR was used to determine the expression of circPTN in glioma cell lines/astrocyte and glioma tissues/normal brain tissues.χ2 test was utilized to analyze the association between the expression of circPTN and clinicopathological data.Treatment of RNase R and actinomycin D were performed for comparing the stability of circPTN with linear RNA.Fluorescence in situ hybridization(FISH)was used to ensure the sublocation of circPTN in glioma cell.Stably transfected with circPTN/sh-circPTN by lentivirus in U87/U251 cells were utilized to explored circPTN’s influence in glioma biological behavior.Assays of Dual luciferase reporter system and RNA pull down were performed to study the interaction between circPTN and miR-145-5p/miR-330-5p.The expression of targeted proteins of miR-145-5p/miR-330-5p(SOX9,ITGA5)and stemness markers(SOX9,SOX2,CD133,Nestin),were measured by western blot.Tumor sphere formation assays were performed to determine the self-renewal ability.Intracranial xneograft mice model was established to study circPTN’s influence in glioma in vivo.Results:The expression of circPTN was significantly higher in glioma cell lines(P<0.05)and glioma tissues(P<0.05),compared with astrocyte and normal brain tissues.The expression was not associated with age(P=0.961),gender(P=0.091)and IDH mutation(P=0,185),but associated with glioma grade(P=0.012).CircPTN was resistance to RNase R treatment and more stable,than linear RNA(P<0.05).We determined that circPTN was mainly localized in cytoplasm.By performing assays of CCK-8,EdU and flow cytometry,we found circPTN significantly promote glioma cell proliferation(P<0.05)and transition of G1→S.By conducting assays of dual-luciferase reporter system and RNA pull down,we confirmed that circPTN could sponge miR-145-5p and miR-330-5p.Moreover,circPTN could rescue the down-regulation of SOX9 and ITGA5,by miR-145-5p and miR-330-5p,respectively.In a word,circPTN rescues the inhibition of PI3K/AKT pathway by miR-145-5p/miR-330-5p to promote proliferation of glioma cells.CircPTN significantly promoted self-renewal of glioma stem cells by sponging miR-145-5p(P<0.05),while the regulation disappeared when binding sites of miR-145-5p were mutated.In intracranial xneograft model,circPTN elevated tumor growth and shorten the survival time of mice.Conclusions:circPTN rescues the inhibition of PI3K/AKT pathway by miR-145-5p/miR-330-5p to promote proliferation of glioma cells,and facilitates glioma stem cells self-renewal by sponging miR-145-5p.circPTN is a promising biomarker in diagnosis of glioma.