Preliminary Analysis Of LOXL1’s Involvement In Hsa-miR-125a,Wnt/β-catenin Signaling Pathway In The Regulation Of Glioma Cell Growth,Apoptosis And Migration

Background and purpose: At present,the survival of malignant glioma patients has not significantly improved even after surgery,radiotherapy,chemotherapy and targeted therapy with bevacizumab.From the beginning,people had placed great hopes on targeted therapy.Chen Ricky used retrospective analysis to find that "targeted therapy has little effect on glioma" and to explore the reasons.Recent advances in the understanding of glioma molecules suggest that incorrect target molecule may be related to the failure of targeted therapy for glioma.Specifically,gliomas consists of a variety of molecular subtypes with different biological,natural history and prognosis.Therefore,WHO regards molecular subtype as one of the criteria for diagnosis and classification of glioma,and target therapy tends to be individualized.However,large data and high-throughput molecular bioinformatics technology must be introduced into clinical for individualized target molecular screening.At present,it is still in the stage of culturing cells in vitro and preclinical animal experiments for targeted therapy which has not entered clinical trials directly.The aim of this paper aims is to find out molecular data related to malignant progression of glioma from Deep Sequencing(RNA seq)database with high throughput data,which will lay a foundation for future research on target therapy.Materials and methods:The research methods are mainly divided into three parts:1.Expression and biological characterization of LOXL1 in glioma1)The correlation between LOXL1 gene expression and glioma was determined by cohort study based on data from tumor database.Tumor tissues and paraneoplastic tissues were collected from pathologically and clinically confirmed glioma patients.The expression level of LOXL1 in glioma tissues was determined by real-time fluorescence quantitative PCR and Western blot.2)The expression levels of LOXL1 were determined in NHA,U87-MG and U251 glioma cell lines.Lentiviruses containing sh RNA targeting LOXL1 gene were packaged to infect U87-MG and U251 cells and stable cell lines were established.Lentiviruses with LOXL1 c DNA were packaged and cell lines with stable over-expression of LOXL1 were established.Western blot experiments were performed to confirm the knock-down and over-expression effects of LOXL1 in the stable cell lines above.3)The effects of LOXL1 knock-down and over-expression on the growth rate,cloning formation and cell cycle of NHA,U87-MG and U251 cell lines were detected with Cell Counting Kit-8(CCK-8),plate cloning assay and flow cytometry,respectively.Determine the effects of LOXL1 gene on apoptosis and migration in NHA,U87-MG and U251 cells were detected with Annexin V-FITC/PI apoptosis detection kit and chamber transwell.2.Role of LOXL1 in regulation of malignant progression of glioma by hsa-mi R-125a1)Differential expression of micro RNAs in glioma: Deep sequencing was used to detect differentially expressed micro RNAs in glioma and normal brain tissues.2)Relationship study between LOXL1 and hsa-mi R-125a: Predict the upstream micro RNAs of LOXL1 by utilizing bioinformatics websites,such as Target Scan,mi Radna,and then confirm the targeting binding of hsa-mi R-125 a with the 3’UTR region of LOXL1 gene by double luciferase reporter gene assay.3)Regulatory effects of hsa-mi R-125 a over-expression on LOXL1: To study the effect of hsa-mi R-125 a overexpression on LOXL1 gene expression in glioma cell lines,we transfect hsa-mi R-125 a mimimics into U87-MG and U251 cells and determine the expression level of LOXL1 by Real-time PCR and Western blot after hsa-mi R-125 a mimimics transfection.4)Effects of hsa-mi R-125 a over-expression on glioma cell function: Utilizing the U87-MG and U251 cells with successfully transfected hsa-mi R-125 a mimimics,the effects of hsa-mi R-125 a on the growth rate,cloning formation and cell cycle were identified with Cell Counting Kit-8(CCK-8),plate cloning assay and flow cytometry,and the effects of hsa-mi R-125 a on apoptosis in U87-MG and U251 cells were determined with Annexin V-FITC/PI apoptosis detection kit.3.Role of LOXL1 in regulation of Wnt/β-catenin on malignant progression of glioma1)The correlation between LOXL1 and Wnt/β-catenin signal pathway was analysised by Weighted gene co-expression network analysis and GO enrichment analysis.2)Construct the Luciferase reporter plasmid of TGF/β-catenin,transfect the reporter plasmid into U87-MG and U251 cells with LOXL1 interference,and then detect the Luciferase activity.3)Lentiviruses containing sh RNA targeting LOXL1 gene and LOXL1 c DNA were constructed to infect U87-MG and U251 cells and stable LOXL1 knock-down and over-expression cell lines were established.Western blot experiments were performed to determine the distribution level of β-catenin protein in nucleus of U87-MG and U251 cells with LOXL1 RNA interference.4)Detect the effect of IWR-1-endo,which is an inhibitor of β-catenin,on proliferation and apoptosis of NHA cell lines with stable over-expression of LOXL1 by using CCK-8 and Annexin V-FITC/PI apoptosis detection kit and chamber transwell,respectively.Results and analysis: 1)The expression of LOXL1 in glioma tissue was significantly higher than that in adjacent tissues(P<0.01);the expression of LOXL1 in U87-MG and U251 was significantly higher than that in normal glioma cell line NHA(P<0.01).2)Loss of LOXL1 significantly reduced the growth rate and cloning formation ability,and led to cell cycle arrest at G0/G1 stage and increased apoptosis ratio increase in U87-MG and U251 cells.Loss of LOXL1 significantly reduced the migration potential.Overexpression of LOXL1 significantly improved the cell viability and cloning formation ability of normal glial cells,increased the S-phase distribution of glioma cells and reduced the apoptosis ratio of NHA.3)Luciferase report showed that co-transfection of hsa-mi R-125 a and LOXL1-3’UTR-wild plasmid could significantly inhibit luciferase activity,suggesting that LOXL1 was the direct target of hsa-mi R-125 a.4)hsa-mi R-125 a overexpression in U87-MG and U251 cells decreased the expression level of LOXL1 gene,significantly reduced the growth rate and cloning formation ability,and led to the cell cycle arrest at G0/G1 stage and increased apoptosis ratio.5)Weight gene co-expression network analysis and GO enrichment analysis showed that LOXL1 was associated with Wnt/β-catenin signaling pathway.6)Interference of LOXL1 gene expression in U87-MG and U251 cells significantly reduced the nuclear entry of β-catenin.The interference of LOXL1 gene significantly decreased the luciferase activity of Wnt/β-catenin reporter plasmid,and IWR-1-endo,a β-catenin inhibitor,significantly inhibited the over-expression of LOXL1 gene and promoted the proliferation of NHA cells IWR-1-endo can and significantly increased apoptosis ratio of NHA cells and reduce the migration potential.Conclusion and outlook: 1)This paper reports for the first time that LOXL1 is highly expressed in glioma tissues and cells in vitro,and this conclusion is reliable in both positive and negative ways by over-expression and interference of LOXL1.2)We first report that the LOXL1-hsa-mi R-125a-Wnt/β-catenin pathway regulating malignant progression of glioma,Our work provides a new choice for future research on target molecule therapy of glioma.