The Mechanism Of TIAR/LOXL1-AS1/miR-374b-5P Axis Modulating Vasculogenic Mimicry In Glioma

Objective: Glioma is common acknowledged as one of the most common primary malignant tumors in the nervous system,characterized by low survival rates,high mortality,and high recurrence rates.The median survival time of glioma is only 12?15months.Although surgery,radiotherapy,chemotherapy and other therapeutic methods have made great progress in the glioma treatment,the prognosis of glioma patients is still not optimistic due to some special reasons.Thus,the vasculogenic mimicry(VM)in tumors comes into view.VM is a novel vascular mechanism in which blood vessels are not surrounded by tumor cells and stromal membranes instead of endothelial cells.In addition,VM is found in multiple tumors,such as glioma,gastric cancer,and hepatocellular cancer,and its appearance is associated with a worse prognosis in patients.Therefore,it is urgent to find efficient molecular targets for treatment of glioma and VM.RNA binding proteins(RBPs)play an indispensable role in splicing,transcription,translation,and stability during the development and progression of tumors.TIAR(TIA-1 related protein),a member of the TIA family,has not been reported in glioma,especially in VM.Long non-coding RNAs(lnc RNAs)are non-coding RNAs with more than 200 nucleotides in length.Currently,more and more evidences indicate that lnc RNAs are involved in the process of glioma.Recent studies have shown that LOXL1-AS1(LOXL1 antisense RNA1)plays an oncogenic role in various tumors,such as breast cancer,ovarian cancer,and colorectal cancer.However,the role and mechanism of LOXL1-AS1 in vasculogenic mimicry in glioma remain unclear.MiRNA regulates the biological behaviors of tumor cells by targeting the 3'UTR of downstream proteins.Downregulation of miR-374b-5P enhances chemotherapy resistance in pancreatic cancer.However,the role and mechanism of miR-374b-5P in glioma are still unknown.This study aims to explore the mechanism of TIAR,LOXL1-AS1,miR-374b-5P,MMP14 and VEGFA regulating VM formation in glioma,and to find novel potential targets for glioma therapy.Methods:1.The expressions of TIAR,lnc RNA LOXL1-AS1,and miR-374b-5P in glioma tissues and cell lines were detected by quantitative real-time PCR(q RT-PCR)and western blot.2.Glioma cells U87 and U251 were transfected,and those with stable expression of TIAR,LOXL1-AS1,miR-374b-5P,MMP14 and VEGFA plasmids were screened.The transfection efficiency of the transfected cells was verified by western blot and q RTPCR.3.The role of TIAR,LOXL1-AS1,miR-374b-5P,MMP14,and VEGFA in malignant biological behaviors and VM formation of glioma cells was evaluated by proliferation,migration,invasion,and tube formation.4.We evaluated whether TIAR,LOXL1-AS1,and miR-374b-5P modulated VM formation in vivo by the establishment of xenograft model in nude mice as well as CD34 and PAS dual staining.Results:1.TIAR was expressed at low levels in glioma cells and tissues,and TIAR overexpression inhibited the biological behaviors and VM of glioma cells.2.LOXL1-AS1 was expressed at high levels in glioma cells and tissues,and LOXL1-AS1 knockdown inhibited the biological behaviors and VM of glioma cells.3.Overexpressed TIAR negatively regulates the expression of LOXL1-AS1 by reducing the stability of LOXL1-AS1,and thereby attenuated the malignant biological behaviors and VM ability of glioma cells.In addition,overexpression of LOXL1-AS1 reversed the tumor-inhibiting effect of TIAR overexpression on glioma cells.4.miR-374b-5P was expressed at low levels,and miR-374b-5P overexpression inhibited the biological behaviors and VM of glioma cells.5.Downregulation of LOXL1-AS1 played sponge-like effects on miR-374b-5P,and significantly inhibited cell proliferation,migration,invasion and VM formation.Silencing miR-374b-5P reversed the inhibitory effects of LOXL1-AS1 knockdown in glioma.6.Overexpression of miR-374b-5P inhibited the malignant biological behaviors and VM of glioma by modifying MMP14 and VEGFA.Expressions of MMP14 and VEGFA reversed the inhibition of malignant biological behaviors and VM of overexpressed miR-374b-5P glioma cells.7.In vivo experiments proved that TIAR overexpression,LOXL1-AS1 knockdown and miR-374b-5P overexpression all inhibited the growth and VM of the nude mouse transplantation tumor,and prolonged the survival of nude mice.Conclusion:1.TIAR and miR-374b-5P were expressed at low levels in glioma tissues and cells,while LOXL1-AS1 was highly expressed in glioma tissues and cells.The overexpression of TIAR and miR-374b-5P and the silence of LOXL1-AS1 significantly inhibited VM formation and malignant biological behaviors of glioma cells,such as proliferation,migration,and invasion.2.TIAR negatively regulates LOXL1-AS1 expression by decreasing its stability.LOXL1-AS1 binds to miR-374b-5P and miR-374b-5P binds to MMP14 and the 3'UTR region of VEGFA mRNA.3.Overexpressed TIAR increased the negative regulation of miR-374b-5P on MMP14 and VEGFA by reducing the stability of LOXL1-AS1 and the binding of LOXL1-AS1 to miR-374b-5P,and inhibited the proliferation,migration,invasion and other malignant biological behaviors as well as VM formation of glioma cells.4.TIAR overexpression,LOXL1-AS1 knockdown and miR-374b-5P overexpression alone or in combination significantly inhibited the growth and VM formation of transplanted tumor in nude mice,and prolonged the survival period of nude mice.5.TIAR/LOXL1-AS1/miR-374b-5P axis plays an important role in regulating biological behaviors and VM of glioma cells.