Oncogenic ZEB2/miR-637/HMGA1 signaling axis targeting vimentin promotes the malignant phenotype of glioma

Glioma is the most common primary tumor of central nervous system. We previously confirmed that zinc finger E-box binding homeobox (ZEB) 2 promotes malignant progression glioma, while microRNA-637 (miR-637) associated with favorable prognosis in glioma. This study aimed to investigate potential interaction between ZEB2 and miR-637 its downstream signaling pathway The results revealed could directly bind elements promoter promote cell proliferation, migration, invasion via downregulation. Subsequent screening HMGA1 was a direct target miR-637, drive phenotype glioma by suppressing both vitro vivo. Furthermore, cytoplasmic vimentin observed, inhibition abolish increased migration induced overexpression. Both were an unfavorable Additionally, upregulated found isocitrate dehydrogenase (IDH) wild-type 1p/19q non-codeletion diffusely infiltrating In conclusion, we identified oncogenic ZEB2/miR-637/HMGA1 axis targeting brain adults. Glioblastoma (GBM) type characterized high level infiltration, therapeutic resistance, poor prognosis, modest median survival 14.2 months, even under radiation plus temozolomide therapy.1Johnson D.R. O’Neill B.P. United States before during era.J. Neurooncol. 2012; 107: 359-364Crossref PubMed Scopus (476) Google Scholar Genetic changes, including 1/2 mutation, codeletion, O6-methylguanine-DNA methyltransferase (MGMT) methylation have recently been accepted as crucial molecular characteristics for prediction subgroup stratification.2Brito C. Azevedo A. Esteves S. Marques A.R. Martins Costa I. Mafra M. Bravo J.M. Roque L. Pojo Clinical insights gained refining 2016 WHO classification diffuse gliomas with: EGFR amplification, TERT mutations, PTEN deletion MGMT methylation.BMC Cancer. 2019; 19: 968Crossref (16) Scholar, 3Bund Guergova-Kuras Cicek A.E. Moussallieh F.M. Dali-Youcef N. Piotto Schneider P. Heller R. Entz-Werle Lhermitte B. et al.An integrated genomic metabolomic approach defining time adult oligodendrogliomas patients.Metabolomics. 15: 69Crossref (2) 4Harat Blok Harat Soszyńska K. impact adjuvant radiotherapy on prognostic markers gliomas.OncoTargets Ther. 12: 2215-2224Crossref (10) Besides these genetic modifications, overexpression protein-coding genes non-coding RNAs (ncRNAs)5Reon B.J. Anaya J. Zhang Y. Mandell Purow Abounader Dutta Expression lncRNAs Low-Grade Gliomas Multiforme: An Silico Analysis.PLoS Med. 2016; 13: e1002192Crossref (51) dysregulated post-translational modification6Yin Park G. Lee J.E. Choi E.Y. J.Y. Kim T.H. Jin X. Jung Shin D. al.DEAD-box RNA helicase DDX23 modulates malignancy elevating miR-21 biogenesis.Brain. 2015; 138: 2553-2570Crossref (42) can also modulate Thus, into their value are crucial. Emerging evidence has indicated ncRNAs, constituting vast majority human transcriptome considered non-functional,7Kent O.A. Steenbergen Das Vivo Nanovector Delivery Heart-specific MicroRNA-sponge.J. Vis. Exp. 2018; 136: 57845Google regulate gene expression involved variety cancers.8Sharma Pavlasova Seda V. Cerna Vojackova E. Filip Ondrisova Sandova Kostalova Zeni P.F. al.miR-29 Modulates CD40 Signaling Chronic Lymphocytic Leukemia Targeting TRAF4: Axis Affected BCR inhibitors.2020https://doi.org/10.1182/blood.2020005627Crossref (5) 9Tristán-Ramos Rubio-Roldan Peris Sánchez Amador-Cubero Viollet Cristofari Heras S.R. suppressor microRNA let-7 inhibits LINE-1 retrotransposition.Nat. Commun. 2020; 11: 5712Crossref (3) 10Wei Lu Zhou Zhao Lyu Yin Shi Z. You Y.P. EIF4A3-induced circular ASAP1(circASAP1) tumorigenesis resistance glioblastoma NRAS/MEK1/ERK1/2 signaling.Neuro. Oncol. (Published online September 14, 2020)https://doi.org/10.1093/neuonc/noaa214Crossref (6) As important component oncogenesis either blocking mRNA translation or promoting degradation.11Ding Lan Xiong Ao H. Feng Gu Yu Cui Q. Dual Role MicroRNAs Colorectal Cancer Progression.Int. Mol. Sci. 2791Crossref (49) Scholar,12Huang novel regulatory role lncRNA-miRNA-mRNA cardiovascular diseases.J. Cell. 22: 5768-5775Crossref (120) marker targets 3ʹ-untranslated region (UTR) Akt1.13Que T. Song Liu Zheng Long Li Wang al.Decreased miRNA-637 growth, Akt1.Oncogene. 34: 4952-4963Crossref (83) Further such signal transducer activator transcription 3 (STAT3) SOX10,14Huang Yuan Cheng Xu Xia Circular circHIPK3 Promotes Cell Metastasis through miR-637/STAT3 Osteosarcoma.BioMed Res. Int. 2020: 2727060Crossref Scholar,15Jin Z.P. Wu Liao X.B. Y.X. J.P. CircRNA EPHB4 stem properties proliferation sponging up-regulating SOX10.Mol. October 21, 2020)https://doi.org/10.1002/1878-0261.12830Crossref validated so far, but there remains lack knowledge regarding upstream regulation. family comprises two factors, ZEB1 (also known SMAD-interacting protein-1 SIP1), which element CACCT(G) clusters critical regulators cellular plasticity.16Hill Browne Tulchinsky ZEB/miR-200 feedback loop: at crossroads transduction cancer.Int. 2013; 132: 745-754Crossref (170) 17Drápela Bouchal Jolly M.K. Culig Souček ZEB1: A Critical Regulator Plasticity, DNA Damage Response, Therapy Resistance.Front. Biosci. 7: 36Crossref (22) 18Nam E.H. Y.K. J.W. upregulates integrin α5 cooperation Sp1 induce epithelial-mesenchymal transition cancer cells.Carcinogenesis. 33: 563-571Crossref (77) our previous study, pivotal biomarker inducing epithelial–mesenchymal (EMT) process cycle progression.19Qi Peng Fang Luo W. al.ZEB2 mediates multiple pathways regulating invasion, apoptosis glioma.PLoS ONE. e38842Crossref (137) recent showed inhibited several miRNAs, miR-153,20Zhou Xie Gong Zi Wen MicroRNA-153 functions SET7 ovarian cells.Oncol. Rep. 111-120Crossref (29) miR-155,21Brown C.Y. Dayan Wong S.W. Kaczmarek Hope C.M. Pederson S.M. Arnet Goodall G.J. Russell Sadlon T.J. Barry S.C. FOXP3 miR-155 cooperate control invasive breast cells down independently ZEB1.Oncotarget. 9: 27708-27727Crossref (14) miR-30a,22di Gennaro Damiano Brisotto Armellin Perin Zucchetto Guardascione Spaink H.P. Doglioni Snaar-Jagalska B.E. al.A p53/miR-30a/ZEB2 controls triple negative aggressiveness.Cell Death Differ. 25: 2165-2180Crossref (47) interactions significantly suppressed EMT process. On other hand, miRNA repressive factor. For example, reciprocal miR-203; miR-203 3ʹ-UTR region, within miR-203.23Duan Fu Gao Deng Direct suppresses reduces lung adenocarcinoma chemoresistance.Acta Biochim. Biophys. Sin. (Shanghai). 48: 1042-1049Crossref (25) Though regulator process,24Zhang Yao LINC00473 impairing miR-637/CDK6 axis.Artif. Cells Nanomed. Biotechnol. 47: 3896-3903Crossref (9) association unraveled. Herein, this regulation extend current Two putative sites (CACCT) (Figure 1A). To validate result, performed chromatin immunoprecipitation (ChIP) assays along electrophoresis analysis using primers specifically amplified regions. protein recruit predicted (Figures 1B 1C). further support results, luciferase then constructs containing site 1 constitutively active reporter gene. accordance ChIP assay transfection ZEB2-expressing plasmid decreased when it coupled construct, knockdown siZEB2 led activity 1D). electrophoretic mobility shift (EMSA) no band detected nuclear extract incubated (WT) probes, observed mutant (Mut) ZEB probes added S1). Having ZEB2, examined levels correlation clinical samples. Consistent higher high-grade (grade III IV) compared low-grade I II), 1E). evident 1F). Collectively, suggested targeted might exert inhibitory effect expression, although needed. factor process, glioma.19Qi contrast, glioma.13Que Building assumption facilitate miR-637. prove this, transfected U251 U87 short hairpin (shRNA) 2A). investigated ZEB2/miR-637 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) 2B) 5-ethynyl-2ʹ-deoxyuridine (EdU) incorporation 2C 2D) ectopic lentiviral particles mimics viability mediated cells. Similar case invasion. Transwell 2E 2F) Boyden 2G 2H), not only changed shape small round promoted migratory cells, concomitant attenuated alone 2E–2H). transcriptional searched TargetScan, miRDB, PITA databases elucidate mechanism determined microarray-based signatures (NC), absolute fold-change >1.5 p <0.05 differentially expressed (DEGs). Since be indirect intersection downregulated examined. 25 overlapping microarray bioinformatic 3A). had already well apoptosis.19Qi literature search us postulate PI3K/Akt candidate due pleiotropic effects glioma25Yu Xue Linc00152 miR-103a-3p/FEZF1/CDC25A pathway.Mol. 2017; 16: 110Crossref (79) 26Koul Shen Y.W. Kondo Bankson Ronen Kirkpatrick D.L. Powis Yung W.K. Cellular vivo PI3K inhibitor, PX-866, against glioblastoma.Neuro-oncol. 2010; 559-569Crossref (86) 27Kaushik N.K. Kaushik Yoo K.C. Uddin J.S. S.J. Low doses PEG-coated gold nanoparticles sensitize solid tumors cold plasma PI3K/AKT-driven suppress transformation inhibiting growth EMT.Biomaterials. 87: 118-130Crossref because Akt1 miR-637.13Que Therefore, expanded findings, focusing protein, pathway.28Wei F. Wei J.C. Yang Chen W.L. H.C. Hu Cao PD-L1 colorectal expansion activating HMGA1-dependent pathways.Cancer Lett. 450: 1-13Crossref quantitative real-time PCR 3B) western blot 3C) level. HMGA1, vector constructed sequence (HMGA1 WT 3ʹ-UTR) Mut 3D). co-transfection inhibitor 3E, columns 2). mutation successfully abrogate 4). Consistently, 3F), 3G). expression. Although remained unelucidated. MTT first evaluate viability, revealing rescue 4A). EdU demonstrated rate rescued group treatment 4B 4C). Regarding resulting from 4D–4G). Briefly, downregulating vitro. whether act mediating biological established subcutaneous xenograft model examine relationship HGMA1 more biologically relevant setting. U87/NC, U87/HMGA1, U87/miR-637, U87/miR-637/HMGA1 subcutaneously implanted nude mice, respectively. mice sacrificed 28 days after implantation, weighed. U87/HMGA1 formed larger than U87/NC 5A, panels 2), exhibited U87/miR-637 Greater weight group, difference statistically significant sample size 5B). evaluated Ki-67 tumors. proliferative confirmed, whereas 5C 5D). GBM stemness modifying architecture SOX2, one major stemness.29Lopez-Bertoni Lal Michelson Guerrero-Cázares Quiñones-Hinojosa Laterra Epigenetic modulation miR-296-5p:HMGA1 regulates Sox2 cells.Oncogene. 35: 4903-4913Crossref (38) It reported cancer.30Pegoraro Ros Piazza Sommaggio Ciani Rosato Sgarra Del Sal Manfioletti metastatic processes basal-like stemness.Oncotarget. 4: 1293-1308Crossref (93) N-cadherin, vimentin, β-catenin following E-cadherin 6A). Interestingly, anti-HGMA1 coimmunoprecipitation (coIP) intermediate filament immature astrocytes, 6B, panel 1). Reciprocal anti-vimentin antibody similarly tissue samples, 6C), positive 6D). based CGGA TCGA cohorts supported result S2). Vimentin member filaments contraction migration.31Li Sun Ban function vimentin: predicting melanoma hematogenous metastasis.J. Clin. 29: 109Crossref (56) focused examining While vitro, 6E–6H). interact ZEB2/miR-637/HMGA1/vimentin cohort 69 prospectively collected tissues department. astrocytic progenitors, astrocytoma There patient’s age gender (Table Notably, HGMA1, lower cohort, markedly IDH 7A–7D), according World Health Organization (WHO) system (CNS) classification.Table 1Correlation clinicopathological factors axisCharacteristicsnZEB2 expressionmiR-637 expressionHMGA1 expressionVimentin expressionHighLowpHighLowpHighLowpHighLowpGenderMale4017220.28614210.1220200.4519210.35Female291414131213161613Age>5018990.336590.1631080.241170.11<50512227222423282427Histological typeAT4621230.27918210.21322240.1024220.03OT156958510411Other844446271WHO gradeI + II34429<0.001248<0.001826<0.001826<0.001III IV352773252510278Abbreviations: AT, tumor; OT, oligodendroglial WHO, Organization. Open table new tab Abbreviations: glioma,19Qi correlated Kaplan-Meier overall patients poorer 7E 7H), differences low significant. scale databases, similar 7F 7I 7G 7J, respectively). Taken together, aggressive pattern date, engagement ncRNAs remain inadequately characterized, among ncRNAs. cancer,32Polytarchou Iliopoulos Struhl circuit reinforces state.Proc. Natl. Acad. USA. 109: 14470-14475Crossref (107) transforming factor-β-induced miR-192 overexpression, leading p53 upregulation.33Deshpande S.D. Putta Lai Bitzer Nelson R.G. Lanting L.L. Kato Natarajan Transforming cross talk pathogenesis diabetic nephropathy.Diabetes. 62: 3151-3162Crossref (126) Our findings built upon repress upregulation plays cancers. NUPR1 shown lead cells.34Wang Jiang LncRNA FAL1 carcinogenesis miR-637/NUPR1 Biochem. Biol. 106: 46-56Crossref Moreover, supporting hamper thyroid carcinoma pancreatic ductal adenocarcinoma.35Xu R.L. He Tang J