The Expression And Mechanism Of The Long Non-coding RNA CRNDE In Gliomas

BackgroundGlioma,a devastating invasive cerebral tumor,is the leading cause of central nervous system tumor-related death in adults,and its early diagnosis and treatment remain difficult medical problems.Malignant gliomas infiltrate the brain parenchyma as they grow and commonly recur after surgery,radiotherapy,and/or chemotherapy.Research has shown that some lncRNAs;which represent a prominent subtype of regulatory ncRNA,are closely associated with glioma pathogenesis and progression.Genes that encode proteins and pass on genetic information only account for about 1.5%of the whole genome sequences while a large number of noncoding regulatory sequences are transcribed into non coding RNA,which include small RNA(sRNA),represented by miRNA and siRNA,and lncRNA.In recent years,attentions have been switched from studies of miRNA and siRNA to lncRNA holding larger quantity and variety.LncRNA,with noncoding transcript length of more than 200nt,has the specific and comprehensive intramolecular secondary structures which possess multiple binding sites for proteins,or interact with DNA or RNA via the complementary base pairing principle,and thus form the lncRNA-involved gene expression regulatory network.The colorectal neoplasia differentially expressed(CRNDE)gene,initially identified in colorectal cancer,is located on chromosome 16 and encodes a lncRNA that was proposed to serve as a serum-based diagnostic and prognostic tumor biomarker.The overexpression pattern of CRNDE in glioma was also confirmed by high throughput microarray data and revealed a potential role in the malignant properties of glioma.CRNDE-mediated tumor pathogenesis and progression have recently been revealed in gliomas.Zhang et al analyzed the microarray data of glioma samples retrieved from the GEO database and discovered that 129 IncRNAs are differentially expressed in gliomas compared to in normal brain tissues,including the upregulation of CRNDE,with an average increase of 14-32-fold.The overexpression pattern of CRNDE in gliomas was also confirmed using high throughput microarray and revealed its potential role in the malignant properties of gliomas.Although previous research suggest a significant role for CRNDE in glioma growth and progression,in-depth analyses of the underlying mechanisms have yet to be undertaken.Increasing evidence has shown that lncRNAs may act as competing endogenous RNAs(ceRNAs)that sequester miRNAs and hinder them from binding to their mRNA targets,thus altering the expression of the corresponding proteins.Our study started with CRNDE expression in clinical glioma specimens and cell lines,and we systemically explored the relationship between the CRNDE-miR-136-5p-Bcl-2/Wnt2 regulatory pathway and gliomas in vitro,which deepened our understanding of the mechanisms of CRNDE in gliomas.ObjectivesPart Ⅰ Study on the expression and function of CRNDE in gliomas1.To investigate the expression levels of CRNDE in glioma specimens and different human glioma cell lines.2.To investigate the effect of CRNDE on proliferation,migration,invasion,and apoptosis of glioma cellsPart Ⅱ Study on the mechanism of CRNDE in gliomas1.To investigate whether CRNDE,as a ceRNA,provides a network regulatory model for ceRNA-miRNA-mRNA interactions,underlining a new point of view for the elucidation of glioma pathogenesis,which suggests that targeting the regulatory circuitry may be an ideal diagnostic marker to support diagnosis even when in the early clinical stages of gliomas and may provide a novel strategy for therapy of gliomas associated with the regulatory circuitry.MethodsPartⅠ Study on the expression and function of CRNDE in gliomas 1.To investigate the relevance of CRNDE in glioma development,we first used qRT-PCR to determine CRNDE expression levels on specimens from 47 glioma patients.CRNDE expression was further measured in high-grade and low-grade glioma specimens2.CRNDE expression was further measured in four human glioma cell lines(U87,U251,A172,and T98G)and were selected for further studies assessing the functional role of CRNDE.3.To investigate the effect of CRNDE on biological activity of glioma cells,U87 cells were transfected with shRNAs(shRNA1 and shRNA3)targeting CRNDE(sh-CRNDE)to knockdown this lncRNA and U251 cells were transfected with pEX-2-CRNDE to upregulate CRNDE expression.The CCK8 assay,wound-healing assay,Matrigel invasion assay and flow cytometry analysis were next applied to quantify the effect of CRNDE on proliferation,migration,invasion,and apoptosis of glioma cells separately.Part Ⅱ Study on the mechanism of CRNDE in gliomas1.To test the hypothesis that CRNDE acts as a ceRNA,we first accessed the bioinformatics database starBase v2.0 to search for potential CRNDE/miRNA interactions.To verify this prediction,we generated wild type(Wt)CRNDE luciferase plasmids containing potential miR-136-5p binding sites,as well as mutant variants of each site.These plasmids were co-transfected with miR-136-5p mimics or miR-NC into HEK293T cells,and then luciferase assays were performed.To investigate the correlation analysis of the expression of CRNDE and miR-136-5p in gliomas,we then used qRT-PCR to determine CRNDE expression levels in the 47 clinical glioma specimens and in our four glioma cell lines,2.To investigate the effect of miR-136-5p on biological activity of glioma cells,U87 cells were transfected with miR-136-5p mimics to upregulate miR-136-5p expression.The CCK8 assay,wound-healing assay,Matrigel invasion assay and flow cytometry analysis were next applied to quantify the effect of miR-136-5p on proliferation,migration,invasion,and apoptosis of glioma cells separately.3.We performed a bioinformatics analysis on TargetScan,miRanda,and DAVID and identified several genes as candidate targets of miR-136-5p.To determine whether CRNDE acted as a ceRNA by sequestering miR-136-5p and hindering it from binding to its target mRNAs,the expression of candidate miR-136-5p targets was examined by qRT-PCR in U251 cells transfected with pEX-2-CRNDE and in U87 cells transfected with miR-136-5p mimics or sh-CRNDE.To evaluate whether these changes were accompanied by corresponding variations in protein expression,Bcl-2 and Wnt2 levels were determined by western blot.ResultsPart Ⅰ Study on the expression and function of CRNDE in gliomas1.CRNDE transcripts were dramatically upregulated in tumor samples,compared with normal brain tissues.CRNDE expression was significantly higher in patients with high-grade(WHO grades Ⅲ/Ⅳ),compared with both low-grade(WHO grades Ⅰ/Ⅱ)glioma and control samples.2.CRNDE was also upregulated in all four glioma cell lines.Among these,the U87 cell line expressed relatively high CRNDE levels,whereas relatively low CRNDE expression was detected in U251 cells.Therefore,the U87 and U251 cell lines were selected for further studies assessing the functional role of CRNDE.3.U87 cells were transfected with shRNAs(shRNA1 and shRNA3)targeting CRNDE(sh-CRNDE)to knockdown this lncRNA.By inducing and silencing CRNDE expression,we detected a positive correlation between CRNDE levels and the proliferative,migratory,and invasive capacities of glioma cells,which were concomitant with a decreased apoptosis rate.Part Ⅱ Study on the mechanism of CRNDE in gliomas1.We searched for potential binding sites of CRNDE to miRNAs on bioinformatics databases and found a putative,specific binding site for miR-136-5p.Such interaction was confirmed by dual luciferase activity assays.The results predicted that miR-136-5p can bind the lncRNA product of the CRNDE gene.To verify this prediction,we generated wild type(Wt)CRNDE luciferase plasmids containing potential miR-136-5p binding sites,as well as mutant variants of each site.These plasmids were co-transfected with miR-136-5p mimics or miR-NC into HEK293T cells,and then luciferase assays were performed.Luciferase activity in the pEX-2-CRNDE Wt+miR-136-5p group was lower than in the pEX-2-CRNDE Wt group,the pEX-2-CRNDE-Mut+miR-13 6-5p group,and the pEX-2-CRNDE-Wt+miR-NC group(P<0.01).These results indicated that miR-136-5p could suppress the activity of a luciferase reporter harboring CRNDE-Wt,and does not affect CRNDE-Mut activity.2.Meanwhile,experiments in human glioma cells showed that CRNDE not only binds miR-136-5p but also reduces its expression,which is consistent with the inverse correlation between CRNDE and miR-136-5p expression observed in clinical glioma specimens.This relationship was examined experimentally in glioma cells transfected with either pEX-2-CRNDE or sh-CRNDE.Results of qRT-PCR showed that miR-136-5p expression was upregulated in U87 cells transfected with sh-CRNDE and downregulated in U251 cells transfected with pEX-2-CRNDE.Taken together,these data suggest that CRNDE binds to miR-136-5p and negatively regulates its expression in glioma cells.3.MiR-136-5p overexpression inhibited tumor cell proliferation,migration,and invasion,and enhanced apoptosis,implying that miR-136-5p exerts a tumor suppressive function in glioma cells.4.We run bioinformatics prediction analyses on TargetScan,miRanda,and DAVID,which identified Bcl-2 and Wnt2 as downstream target genes of miR-136-5p.The mRNA and protein expression of Bcl-2 and Wnt2 was significantly decreased in U87 cells transfected with miR-136-5p mimics or sh-CRNDE.Conversely,the expression of both genes was increased in U251 cells transfected with pEX-2-CRNDE.ConclusionsPart Ⅰ Study on the expression and function of CRNDE in gliomas1.We confirmed high CRNDE expression in gliomas.2.These findings indicate an oncogene-like role for CRNDE in the pathogenesis and development of glioma.Part Ⅱ Study on the mechanism of CRNDE in gliomasWe therefore propose that CRNDE functions as a competing endogenous RNA(ceRNA)that binds to and negatively regulates miR-136-5p,thereby protecting Bcl-2 and Wnt2 from miR-136-5p-mediated inhibition in glioma.