| BackgroundAs one of the most common forms of brain cancer in adults,high-grade gliomas are notorious for its diffuse infiltrative growth as well as for its notorious resistance to different treatment strategies.Although improvements have been made by combining neurosurgery,chemotherapy,and radiotherapy,the prognosis and survival rate for glioma patients is still poor,and the 5-year survival rate is less than 5%.It is therefore important to elucidate the molecular mechanisms involved in glioma progression and to develop new strategies for glioma treatment.Over the past decade,microRNA(miRNA)expression has been a hot topic in cancer biology.MiRNAs are a class of endogenous non-coding RNAs that are 19-25 nucleotides in length.MiRNAs interact with mRNAs through complementary base-pairing with their 3'-untranslated region and thereby influence the translation or stability of the target mRNA molecule.MiRNAs contribute to diverse biological processes and their dysregulation is linked to the progression and metastasis of human cancers.These insights have made miRNAs targets for novel therapeutic approaches and recently several miRNA-targeted therapeutics have reached clinical development.In glioma's abnormal miRNA expression can affect cell proliferation,invasion,and chemo-and radio-resistance.The Wnt3a gene is clustered on human chromosome 1q42 and contributes to the accumulation of P-catenin,thereby activating the canonical Wnt signaling pathway.The Wnt3a protein is often highly expressed in tumor tissue.Interestingly,Wnt3a expression is associated with the clinical grade and aggressiveness of gliomas.In the present study,we explored the role of miR-497 in glioma cells and investigated the underlying mechanism.We revealed that miR-497 expression was significantly down-regulated in glioma tissues and cell lines.The Wnt3a protein was identified as a down-stream target of miR-497 and mediated miR-497's function in cell growth and invasion.In addition,Wnt3a promoted c-jun expression and thereby negatively modulated miR-497 expression.Results1.miR-497 expression was down-regulated in glioma tissues and cell linesWe measured the miR-497 expression in glioma tissues and normal brain tissues by the use of RT-PCR assay.It was revealed that the expression level of miR-497 was significantly down-regulated in glioma tissues when compared with that in normal brain tissues.In parallel,the expression level of miR-497 was significantly down-regulated in glioma cell lines when compared with that in normal brain cell line(NBC).Interestingly,glioma patients with high miR-497 expression had better disease-free survival and over-all survival rate than those with low miR-497 expression.2.Wnt3a was a direct target of miR-497 in gliomaBy using bioinformatic analysis(TargetScan and miRanda)it was predicted that Wnt3a was one of miR-497 targets.To examine whether miR-497 directly targeted the Wnt3a 3'-TR,the Wnt3a 3'-UTR was subcloned into the luciferase reporter plasmids,including the predicted miR-497 recognition site(WT)or a mutated sequence(MUT).There was a significant decrease in luciferase activity in U251 cells transfected with the wild-type miR-497 vector,when compared with cells transfected with the mutant vector.Subsequently,by RT-PCR and western blot assay we found that miR-497 inhibited Wnt3a mRNA and protein levels,respectively.More important,we observed a negative association between miR-497 and Wnt3a expression in glioma tissues.3.Wnt3a mediated miR-497's effect in glioma cellsWe established U251 cells that stably expressed miR-497 by using lentiviral vector-mediated overexpression(LV-miR-497).Those cells transduced with a control lentiviral vector(LV-ctrl)were used as negative control.We measured the transduction efficiency by using RT-PCR assay.As expected,restoration of miR-497 inhibited glioma cell growth,as determined by the MTT assay.Similarly,U251 cells treated with LV-miR-497 formed smaller and fewer colonies than cells treated with LV-ctrl.We next examined whether miR-497 controlled cell cycle progression.It was observed that miR-497 decreased the proportion of S phase and increased the proportion of G1 phase.We also examined the Gl/S phase checkpoint proteins(ie.cyclin D1,CDK4,and E2F1)in LV-miR-497 cells.It was observed that miR-497 decreased these protein expression.Taken together,these data revealed that miR-497 decreased glioma cell growth through regulating cell cycle progression.Subsequently,we examined whether miR-497 affected glioma cell migration.The transwell assay revealed that miR-497 inhibited glioma cell migration.The epithelial-mesenchymal transition(EMT)phenotype is involved in promoting cancer cell migration.We then examined the impact of miR-497 on EMT.The E-cadherin expression was elevated,while N-cadherin and Vimentin expressions were downregulated in LV-miR-497 cells.These data implicated that restoration of miR-497 may reverse EMT phenotype and decrease cell migration.Rescue experiment was performed to determine whether miR-497 exerted its function mainly through Wnt3a.miR-497 suppressive effect of Wnt3a was abolished when Wnt3a was over-expressed.In addition,it was revealed that overexpression of Wnt3a rescued miR-497's effect on cell growth,cell cycle distribution,invasion and EMT phenotype.In all,the above data suggested that miR-497 inhibited glioma cell growth and migration through its down-stream target Wnt3a.4.Wnt3a negatively regulated miR-497 expression through c-junWe further explored the underlying mechanism for miR-497 down-regulation in glioma.The UCSC and PROMO bioinformatics software were to analyze a 1-kb region upstream of the transcription start site of miR-497.We identified two c-jun-binding motifs inside the putative promoter region upstream of the miR-497 transcriptional start site(TSS)at-978 to-972 and-952 to-946.These transcription factor-binding sites(TFBSs)were named A and B.The Chromatin immunoprecipitation(ChIP)assay revealed that c-jun protein was recruited to the both two binding sites in the putative miR-497 promoter.It was found that c-jun down-regulation elevated miR-497 promoter luciferase activity and increased miR-497 expression.Previous studies demonstrated that Wnt3a increased c-jun expression.We supposed that Wnt3a regulated miR-497 expression through c-jun in glioma cells.We used si-RNA to knock down Wnt3a expression.It was revealed that Wnt3a down-regulation decreased c-jun,but increased miR-497 expression.However,when c-jun was overexpressed,miR-497 expression was elevated in si-Wnt3a cells.U251 cells were then transfected with plasmid pcDNA-3.1-Wnt3a.It was revealed that Wnt3a over-expression increased c-jun but decreased miR-497 expression.However,when c-jun was inhibited,miR-497 expression was down-regulated in pcDNA-3.1-Wnt3a cells.In all,these data suggest that Wnt3a negatively regulated miR-497 expression through c-jun.5.miR-497 inhibited cell growth in vivoTo confirm whether miR-497 inhibited tumor growth in vivo,LV-miR-497 or LV-ctrl cells were injected into the backs of nude mice.LV-miR-497 cell-derived xenograft tumors grew more slowly when compared with LV-ctrl cell-derived xenograft tumors.In addition,the mean weight of LV-miR-497 cell-derived xenograft tumors was significantly lower than that of LV-ctrl cell-derived xenograft tumors.Tumor sections were stained for proliferation marker Ki-67 and results suggested that miR-497 inhibited the tumor proliferation rate of glioma cells in vivo.Conclusions and DiscussionMiRNAs contribute to the development and metastasis of glioma and have been suggested as novel therapeutic targets to treat glioma.The role of miR-497 in the development of cancer has been documented.In non-small cell lung cancer,miR-497 expression is down-regulated and able to inhibit cell growth and invasion by targeting VEGF-A and YAP1.Similar results were found in a serious of studies,which revealed that miR-497 functioned as a tumor suppressor.Previous studies demonstrated that reduced expression of microRNA-497 was associated with poor prognosis in human gliomas.However,the biological functions and underlying mechanisms of miR-497 in glioma are still poorly understood.In consistence with previous study,we found that miR-497 was down-regulated in glioma tissues and cell lines.Wnt3a was identified as a downstream target of miR-497.Wnt3a overexpression has been reported in a variety of human cancers.In glioma cells miR-497 negatively regulated the expression of Wnt3a,as determined by luciferase reporter and western blot assays.A previous study suggested that Wnt3a expression was a significant prognostic factor of glioma and might play an important role in cell invasion.We hypothesized that miR-497 exerted its function through targeting Wnt3a.To examine this possibility,we re-introduced Wnt3 a into the LV-miR-497 cells and observed that Wnt3 a overexpression reversed the tumor-suppressing effects of miR-497 on glioma cells.Interestingly,we also found that there was a negative correlation between miR-497 and Wnt3a expression in glioma tissues.Subsequently,we performed functional experiment and explored the role of miR-497 in glioma cells.As determined by the MTT and colony formation assays,miR-497 inhibited cell growth ability.We then examined whether miR-497 affected cell cycle distribution,which was a predominant factor promoting tumor cell growth.It was observed that miR-497 delayed cell cycle from G1 phase to S phase.This data suggested that miR-497 may decrease glioma cell growth ability through affecting cell cycle distribution.In consistence with the in vitro study,the in vivo study revealed that miR-497 inhibited cell growth in the nude mice.Tumor invasion was a key step during cancer development.The Boyden assay revealed that miR-497 decreased glioma cell invasion ability.The EMT phenotype was tightly associated with cell invasion ability and we thus asked whether miR-497 inhibited EMT phenotype.As determined by the western blot assay,miR-497 increased E-cadherin expression while decreased N-cadherin and Vimentin expression.These data implated that miR-497 may inhibit EMT phenotype and ultimately decreased glioma cell invasion.Finally,we asked the underlying mechanism that led to the dys-regulation of miR-497 in glioma.Recruitment of specific transcription factors often led to abnormal miRNAs expression at genetic or epigenetic levels.Among these transcription factors,c-jun played a key role in cell growth and metastasis during cancer development.Two putative biding sites of c-jun in the region upstream of miR-497 locus were found.The ChIP-PCR and luciferase assays demonstrated that c-jun directly bound at miR-497 promoter.The PCR assay revealed that c-jun regulated miR-497 expression.We also demonstrated that Wnt3a regulated c-jun expression and thereby controlled miR-497 expression.In all,our findings revealed that Wnt3a regulated c-jun-miR-497 axis.In summary,our results provided the first evidence that the Wnt3a//c-jun/miR-497 feedback axis controlled glioma cell growth and invasion.Because down-regulation of miR-497 was associated with a poor prognosis and the restoration of miR-497 decreased cell growth and invasion,targeting miR-497 might be a potential treatment for glioma. |
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