The Mechanism Of MiR-199a-5p Participated In The Regulation Of Proliferation,Migration And Invasion Of Glioma Cells Via CELSR1

ObjectiveGlioma,accounting for about 40% of all brain tumors,is characterized by high morbidity and poor survival.High-grade gliomas are obviously characterized by high invasiveness,high proliferation rate and poor differentiation.Previous research achievements have greatly increased life expectancy and quality of life.However,the molecular mechanisms underlying its pathology remain unclear.CELSR1 is a member of the flamingo subfamily,which is a part of the cadherin superfamily.CELSR1 is likewise a member of planar cell polarity signaling pathway.Mutations of CELSR1 is linked to the severe neural tube defect craniorachischisis.Besides,aberrant CELSR1 expression is reported in a variety of tumors(including lung cancer,breast cancer,hepatocellular carcinoma,ductal carcinoma is situ,oral squamous cell carcinoma,lymphoid leukemia,and glioma).However,until now,the biological function of CELSR1 has not been identified in cancers including glioma.MicroRNAs(miRNAs),a type of non-coding RNA,are single-stranded RNAs of 18?24 nucleotides in length.miRNAs regulate gene expression at a post-transcriptional level by degrading or repressing target mRNAs,resulting in translational repression or mRNA degradation.Our previous study reported that miR-199a-5p suppresses glioma cell proliferation,migration,and invasion by inhibiting MAGT1.However,the correlation between CELSR1 and miR-199a-5p has not been reported.The purpose of our study was to investigate the biological function of CELSR1 and the upstream regulatory mechanism in glioma.Given that the glioma biomarkers are scarce for clinical,our study on CELSR1 has significant implications in the diagnosis and treatment of glioma.Methodswe evaluated the expression of CELSR1 in glioma by TCGA?GEPIA tool,RT-qPCR,and Western blot assays.CCK-8,wound healing and transwell invasion assays were respectively performed to detect the effect of CELSR1 on cell proliferation,migration,and invasion.The upstream regulatory miRNAs of CELSR1 were predicted by Target Scan and validated by luciferase activity reporter assay.Results(1)CELSR1 is overexpressed in gliomaUsing Cancer Hallmarks Analytics Tool(CHAT),we found that CELSR1 is associated with genome instability and mutation,evading growth suppressors,inducing angiogenesis,invasion and metastasis,and sustaining proliferative signaling.To determine the expression levels of CELSR1 in glioma,we analyzed the TCGA datasets by GEPIA tools and detected the protein and mRNA expression levels of CELSR1 in glioma cells.TCGA datasets shows that CELSR1 is significantly overexpressed in glioma patients(P<0.05).Besides,Western blot and RT-qPCR assays respectively revealed that the protein and mRNA expression levels of CELSR1 are increased in glioma cells(P<0.001).(2)CELSR1 promoted glioma cell proliferation,migration and invasionTo investigate the biological function of CELSR1 in glioma,we significantly knocked down and overexpressed CELSR1 expression in U251 and U118 cells(P<0.01).CCK-8 assays revealed that CELSR1 significantly increased cell viability,and blockade of CELSR1 significantly inhibited cell viability in U251 and U118 cells(P<0.01).Besides,wound healing assay found that CELSR1 significantly promoted cell migration(P<0.01).Transwell invasion assay revealed that CELSR1 significantly promoted cell invasion(P<0.01).(3)CELSR1 was a direct target of miR-199a-5pTo determinate the upstream regulation mechanism of CELSR1,we predicted the correlated miRNA using targetscan database.We found that CELSR1 probably is a direct target of miR-199a-5p.To further investigate the correlation between miR199a-5p and CELSR1,we transfected miR-199a-5p mimics and inhibitors in U251 and U118 cells(P<0.01)and detected the CELSR1 expression levels in different groups.RT-qPCR assays found that miR-199a-5p mimics significantly inhibited CELSR1 mRNA expression(P<0.01)and miR-199a-5p inhibitors significantly promoted CELSR1 mRNA expression(P<0.01).Western blot assays revealed that miR-199a-5p mimics significantly inhibited CELSR1 protein expression and miR-199a-5p inhibitors significantly promoted CELSR1 protein expression(P<0.05).Hence,to further verified the binding sites between miR-199a-5p and CELSR1,we detect the relative luciferase activity after co-transfection assays.The dual-luciferase report system revealed that miR-199a-5p mimics significantly inhibits the luciferase activity of CELSR1-wt 3'UTR,and miR-199a-5p inhibitors significantly increases the luciferase activity of CELSR1-wt 3'UTR among U251 and U118 cells,whereas miR-199a-5p mimics or inhibitors do not regulate the luciferase activity of CELSR1-mut 3'UTR among U251 and U118 cells(P<0.05).(4)CELSR1 was an effector for the role of miR-199a-5pTo further illuminate the regulatory process of miR-199a-5p/CELSR1 axis on cell biological function,we co-transfected CELSR1 and miR-199a-5p mimics(P<0.05)and detected cell proliferation,migration and invasion in U251 and U118 cells.CCK-8assays revealed that miR-199a-5p mimics significantly reversed the effect of CELSR1 promoting cell proliferation(P<0.01).Wound healing assay found that miR-199a-5p mimics significantly reversed the effect of CELSR1 promoting cell migration(P<0.01).Transwell invasion assay found that miR-199a-5p mimics significantly reversed the effect of CELSR1 promoting cell invasion(P<0.01).Besides,we found that CELSR1 significantly reversed the effect of miR-199a-5p inhibiting the mRNA expression of ROCK1,Galectin1,and Snail.Conclusion(1)In glioma,CELSR1 regulated cell proliferation,migration,and invasion.miR-199a-5p inhibits CELSR1 expression at a post-transcriptional level by degrading CELSR1 mRNA.(2)CELSR1 promotes cell migration and invasion regulated by miR-199a-5p in glioma(3)CELSR1 acts as an oncogene promoting glioma cell proliferation,migration,and invasion,which is regulated by miR-199a-5p.