miRNA-128-3p通过下调KLHDC8A的表达抑制胶质瘤细胞的恶性生物学行为.pdf
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1、INTRODUCTIONGliomas are the deadliest brain malignancies and theirtreatment remains clinically challenging in spite of thecurrent advances in cancer therapies1,2.The molecularcomplexity3,high invasiveness,heterogeneity,andblood-brain barrier(BBB)penetrability4,as well as theunique immune environment
2、 of gliomas5all contribute toa poor prognosis of glioma patients.The recent NationalComprehensive Cancer Network guidelines(v1.2022)classified adult gliomas into 5 categories,namely adultglioma(WHO grade 1),oligodendroglioma(IDH mutantwith1p/19qco-deletion),IDH-mutantastrocytoma,glioblastoma(GBM),an
3、dintracranial/spinalependymoma.The histological features of these gliomasubtypesareanalyzedinconjunctionwiththeirmolecular diagnosis to provide prognostic confirmationfor more personalized therapies6.However,earlydiagnosis and prediction of the clinical behaviors,treatment responses,and prognosis of
4、 invasive gliomasremain challenging and require the discovery of moreeffective biomarkers.KLHDC8A,a member of the Kelch domain-containing(KLHDC)protein superfamily,is encoded bya gene located at the 1q32.1 locus7.Kelch-repeatproteins are important regulators of cellular processes(migration,morphogen
5、esis,and interaction)and somemolecular mechanisms(gene activation and proteindegradation)8and participate in tumorigenesis andneurological dysfunctions9.Previous studies showed thatKLHDC8A expression was increased in gliomas inassociation with the WHO grade10and in glioblastomatissues11,but its asso
6、ciation with the upstream regulatoryfactors hasnot beenfullyunderstood.Micro-RNAs(miRNAs)have been shown to regulatemRNA stability and translational turnover of their targetRNAsunderbothphysiologicalandpathologicalconditions12,13and participate in the regulation of a widevariety of cellular and biol
7、ogical processes14-16.AberrantmiRNA expressions have been observed in oncogeneactivation in several life-threatening cancers,includinggliomas17-20,suggesting the possibility of using miRNAsas biomarkers for cancer diagnosis and targets for cancertherapy.We previously demonstrated,by comparing mRNAan
8、d miRNA expression profiles between glioblastomaandpairedadjacenttissue,thatmiRNA-128-3pexpression was significantly reduced in glioblastoma andKLHDC8A mRNA was one of its targets in the generegulatory network21.We therefore hypothesized thatmiRNA-128-3p is capable of regulating KLHDC8AAbstract:Obje
9、ctive To determine whether miRNA-128-3p regulates malignant biological behavior of glioma cells by targetingKLHDC8A.Methods Dual-luciferase reporter assays,qRT-PCR and Western blotting were used to verify the targeting of miRNA-128-3p to KLHDC8A.Edu assay,flow cytometry,Transwell assay and would hea
10、ling assay were used to determine the effects ofchanges in miRNA-128-3p and KLHDC8Aexpression levels on malignant behavior of glioma cells.Rescue experiment was carriedout to verify that miRNA-128-3p regulated glioma cell proliferation,apoptosis,invasion and migration by targeting KLHDC8A.Results Th
11、e expression level of KLHDC8Awas significantly increased in high-grade glioma tissue and was closely related to a poorsurvival outcome of the patients.Overexpression of KLHDC8A promoted glioma cell proliferation,migration and invasion,andmiRNA-128-3p overexpression inhibited proliferative and metast
12、atic capacities of glioma cells.Mechanistically,KLHDC8Aexpression was directly modulated by miRNA-128-3p,which,by targeting KLHDC8A,inhibited malignant behavior of gliomacells.Conclusion UpregulationofmiRNA-128-3pinhibitsuncontrolledgrowthofgliomacellsbynegativelyregulatingKLHDC8Aexpression and its
13、downstream effectors,suggesting that the miRNA-128-3p-KLHDC8A axis may serve as a potential prognosticindicatorandatherapeutictargetfordevelopingnewstrategiesforgliomatreatment.Keywords:glioma;miRNA-128-3p;KLHDC8A;biomarker;migration;proliferationmiRNA-128-3p inhibits malignant behavior of glioma ce
14、lls bydownregulating KLHDC8AexpressionYU Zhengtao1,LI Jiameng1,JIANG Junwen1,LI You1,LIN Long1,XIAYing1*,WANG Lei2*1Department of Neurosurgery,Affiliated Haikou Hospital of Xiangya School of Central South University,Haikou 570208,China;2Department ofNeurosurgery,Hunan Cancer Hospital and Affiliated
15、Cancer Hospital of Xiangya School of Medicine,Central South University,Changsha410006,ChinaOriginalArticleReceived:2023-05-23Accepted:2023-09-18Supported by Hainan Provincial Natural Science Foundation ofChina(No.820MS163),Scientific Research Project of HunanProvincial Health Commission(No.20200709)
16、,and HainanCerebrovascular Diseases Clinical Medical Research Center(LCYX202206).*Correspondingauthors:XIAYing,E-mail:;WANGLei,E-mail:.J South Med Univ,2023,43(9):1447-1459doi 10.12122/j.issn.1673-4254.2023.09.011447expression in glioblastoma.In this study,we investigatedthe cross-talk between miRNA
17、-128-3p and KLHDC8Ain glioma cells,and the results suggest that the oncogenicexpressionofKLHDC8Aisinstrumentalforthemalignant phenotype of glioblastoma and associated withpoortreatment responsesof thepatients.METHODSBioinformaticanalysisBioinformatic analysis was performed as describedpreviously21.B
18、riefly,miRNA expression profile dataset(GSE90603)andtranscriptomeprofiledataset(GSE90598)weredownloadedfromcombinedmiRNA-mRNA microarray chips in the Gene ExpressionOmnibus(GEO)database.The expressions of miRNAand mRNA were detected using the platform GPL21582and GPL17692.The differentially expresse
19、d miRNAs(DEMs)and differentially expressed genes(DEGs)wereidentified using the R limma software package.FunRichwas used to perform functional enrichment analysis;themiRNAs and their target gene regulatory network wasconstructed usingCytoscape 3.6.1 software.Survival analysis and gliomagrading analys
20、isThe Chinese Glioma Genome Atlas(CGGA)database(http:/ and survival analyses.This database of over 2000glioma tissue samples contains miRNA microarrays(198samples)and matched clinical data that can be used toassess the impact of certain miRNAs on glioma patientsurvival.The miRNA expression levels be
21、tween grade IIand grade IV gliomas were analyzed using CGGAdatabase.The median expression value of each DEM inglioma specimens was calculated to define the highexpression(above the median)group and low expression(below the median)group.Combined with the prognosticresults obtained from the CGGA datab
22、ase(includingoverall survival data),Kaplan-Meier survival curves ofthe two groups of patients were drawn,and log-rank testwas used to evaluate the relationship between the geneexpression level and the patients survival.A P value lessthan 0.05wasconsideredstatisticallysignificant.Cell cultureHuman gl
23、ioma lines U87-MG(#BNCC100646;BNCC,China)and U251(#BNCC337874;BNCC,China)andnormal human astrocytes(NHAs)(#QCB849;QCHENG,China)were cultured routinely in DMEM medium(#R8758,Sigma,USA)supplemented with 10%fetalbovine serum(FBS;Gibco,USA)and 1%Pen-Strep(#SV30010,Beyotime,China)at 37 in 5%CO2in ahumidi
24、fied chamber.Cell transfectionOE-KLHDC8A,OE-NC,KLHDC8A siRNA,and siRNANC were purchased from RiboBio.miRNA-128-3pmimic,mimicNC,miRNA-128-3pinhibitor,andinhibitor NC were purchased from HonorGene.For celltransfection,the cells were seeded in a 6-well plate,andwhen the cell confluence reached 60%,the
25、culturemedium was removed.95 L of serum-free culturemedium was added to each sterile centrifuge tube,followed by addition of 5 L of each of the 8 plasmids(ata concentration of 20 mol/L and 5 L of Lipofectamine2000 reagent(Invitrogen,USA).After gentle mixing,themixtures were incubated at room tempera
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