Mutations in the RNaseH2A gene are involved in Aicardi-Goutieres syndrome, an autosomal recessive neurological dysfunction; however, studies assessing RNaseH2A in relation to glioma are scarce. This study aimed to assess the role of RNaseH2A in glioma and to unveil the underlying mechanisms. RNaseH2A was silenced in glioblastoma cell lines U87 and U251. Gene expression was assessed in the cells transfected with RNaseH2A shRNA or scramble shRNA by microarrays, validated by quantitative real time PCR. Protein expression was evaluated by western blot analysis. Cell proliferation was assessed by the MTT assay; cell cycle distribution and apoptosis were analyzed by flow cytometry. Finally, the effects of RNaseH2A on colony formation and tumorigenicity were assessed in vitro and in a mouse xenograft model, respectively. RNaseH2A was successively knocked down in U87 and U251 cells. Notably, RNaseH2A silencing resulted in impaired cell proliferation, with 70.7 and 57.8% reduction in the U87 and U251 cells, respectively, with the cell cycle being blocked in the G0/G1 phase in vitro. Meanwhile, clone formation was significantly reduced by RNaseH2A knockdown, which also increased cell apoptosis by approximately 4.5-fold. In nude mice, tumor size was significantly decreased after RNaseH2A knockdown: 219.29±246.43 vs. 1160.26±222.61 mm3 for the control group; similar findings were obtained for tumor weight (0.261±0.245 and 1.127±0.232 g) in the shRNA and control groups, respectively). In the microarray data, RNaseH2A was shown to modulate several signaling pathways responsible for cell proliferation and apoptosis, such as IL-6 and FAS pathways. RNaseH2A may be involved in human gliomagenesis, likely by regulating signaling pathways responsible for cell proliferation and apoptosis.
RNaseH2Agliomacell proliferationIntroduction
Human brain glioma, also known as neuroglioma, exhibits invasive growth without an overt boundary to surrounding tissues; it has unique cell origin, pathological structures, biological features, and clinical symptoms (1–6). Malignant glioma is a tumor with extremely high malignancy and recurrence rate; low total resection rate, high postoperative recurrence and residual lesion recurrence caused by radiation, and chemotherapeutic tolerance constitute the main problems faced by neurosurgeons (7–13). Considering the bottleneck in glioma treatment, dissecting relevant molecular mechanisms and finding tumor-related gene therapy approaches in human glioma attracts increasing attention for improved diagnosis, treatment and prognosis of glioma (6,14–23).
Our laboratory and others have recently screened clinical relevant genes in glioma (24–28); notably, we found a new glioma-expressed gene that plays an important regulatory role in the proliferation, progression and apoptosis of glioma cells. The gene is called ribonuclease H2, subunit A (RNaseH2A) also known as AGS4, JUNB, RNHL, RNHIA and RNaseHI. RNaseH2A (1148 bp), located on chromosome 19 (29,30), is a component of ribonuclease H2 (RNAseH2) and is responsible for its endoribonuclease activity (31–33). RNaseH was discovered and isolated from calf thymus (34,35) and is widely distributed in mammalian cells, yeasts, prokaryotes and virus particles; it catalyzes in-nucleus degradation of RNA in DNA-RNA hybrids and is involved in reverse transcriptase of multifunctional enzymes in retroviruses, playing an important role in various stages of viral genome transcription (36,37). In eubacteria, RNaseH is required for several processes, including removal of RNA primers from Okazaki fragments, transcription of primers required for DNA polymerase I initiated DNA synthesis, and removal of R-ring to provide conditional initiating sites required for irregular DNA synthesis (38). In eukaryotes, RNaseH may play similar roles (39). Recent studies have shown that RNaseH2A mutations lead to Aicardi-Goutieres syndrome, an autosomal recessive neurological dysfunction, which mainly causes microcephaly, mental motor development retardation, cerebral calcification, increased IF-α and leukocytes in cerebrospinal fluid, fever, thrombocytopenia, and hepatitis (40–42). RNaseH2A was proposed as a putative cancer target (31). In agreement, logistic regression analysis revealed that expression levels of RNaseH2A, among other genes, were positively correlated with aggressive prostate cancer (43). However, studies assessing RNaseH2A in relation to glioma are scarce. Therefore, in the present study, we aimed to assess the role of RNaseH2A in glioma, exploring the underlying mechanisms.
Our data demonstrated that RNaseH2A silencing altered cell proliferation and clone formation and enhanced apoptosis in the glioma cells in vitro. Meanwhile, RNaseH2A-knockdown xenografted glioma cells were less tumorigenic in a nude mouse model. Moreover, microarray data confirmed that RNaseH2A regulated genes that are frequently involved in multiple important cellular regulatory processes, including focal adhesion, cancer, p53 signaling, and cell cycle. Our findings provide a strong basis for finding new gene-based therapeutic approaches against human glioma.
Materials and methodsCell culture
The human glioblastoma (GBM) cell lines U87, U251, A373 and A172 were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in 5% CO2.
RNaseH2A knockdown in GBM cells
For RNaseH2A silencing, U85 and U251 cells were transfected with the pGCSIL-GFP plasmid containing RNaseH2A-specific shRNA, or non-effective shRNA control fragments (GeneChem, Montreal, QC, Canada).
Gene expression microarray analyses
Total RNA was extracted from the U87 cells (transfected with NC or RNaseH2A shRNA) using an RNeasy Mini-kit (Qiagen, Valencia, CA, USA), and quantified on a Nanodrop ND-1000 spectrophotometer (NanoDrop Technology). Samples with adequate RNA quality index (>7) were used for the microarray analyses.
Genome-wide transcriptome profiles were assessed by expression microarrays (GeneChip PrimeView human, Affymetrix) and validated by quantitative real-time PCR. Microarray data and fully detailed experimental procedures are published online at Gene Expression Omnibus (GEO, NCBI) (http://www.ncbi.nlm.nih.gov/geoprofiles/). Differentially expressed genes were identified using 1.5- or 2-fold change as cutoff values. Gene ontology enrichment analysis was carried out using David Functional Annotation Resources 6.7 (http://david.abcc.ncifcrf.gov/) and the KEGG pathway analysis.
Cell proliferation, apoptosis, and cell cycle analysis
GBM cells were seeded onto 96-well plates at 2×103 cells/well for culture. Cell proliferation was measured using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 96 h after transfection with NC or RNaseH2A shRNA. Briefly, 20 µl MTT solution (5 mg/ml) was added to each well for 4 h at 37°C. After the medium was aspirated, 100 µl dimethyl sulfoxide (DMSO) was added. Optical density was measured at 492 nm on a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Viability index was calculated as experimental OD value/control OD value. Three independent experiments were performed in quadruplicate.
For cell cycle analysis, the cells were stained with propidium iodide (PI) solution (50 µg/ml) and analyzed on a FACSCalibur flow cytometer 24 h after transfection.
Apoptosis was assessed using a fluorescein isothiocyanate (FITC) Annexin V staining kit (Life Technologies, Grand Island, NY, USA) followed by fluorescence-activated cell sorter (FACS) analysis according to the manufacturer's instructions.
Colony formation assay
Following treatment, adherent cells were trypsinized and counted to determine viability. Then, 800 viable cells were seeded into each well of a 6-well plate (in triplicate). Cells were allowed to adhere and grow for 10–14 days. To visualize colonies, media were removed, and cells were fixed in paraformaldehyde for 30 min and stained with Giemsa solution (ECM 550, Chemicon). Finally, colonies were counted; data are presented as mean colony number ± SEM from at least three independent experiments.
RT-PCR and quantitative real-time PCR
RNA extraction was performed with TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA); cDNA was synthesized using RT-Phusion kit (Thermo Scientific, Waltham, MA, USA). Gene-specific mRNA levels were quantified by standard RT-PCR or quantitative PCR (qPCR), using the ΔΔCt method, as previously described (44). Primer sequences are listed in Table I.
Western blot analysis
After treatment, the cells were harvested and lysed in RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS) that contained a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 4°C. Forty micrograms of protein from each lysate were fractionated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk in PBS-Tween-20 for 1 h at room temperature, the membranes were blotted with the appropriate primary antibody. Primary antibodies against RNaseH2A (1:1,000; Proteintech), IL-6 (1:2,000; Abcam), FAS (1:1,000; Abcam), IGTA2 (1:10,000; Abcam), GAPDH (1:20,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were incubated at 4°C overnight. After washing four times with TBST, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibody (Santa Cruz Biotechnology, Inc.) for 2 h. The proteins were visualized using enhanced chemiluminescence (ECL; Beyotime Institute of Biotechnology, Nantong, China).
In vivo GBM xenograft studies
All mouse care and experiments were carried out with approval of the Institutional Animal Care and Use Committee at the Beijing Tian Tan Hospital, Capital Medical University. Subcutaneous xenograft models were performed by injecting U87 cells transfected with NC or RNaseH2A shRNA as previously described (45).
Statistical analysis
Results are representative of 3 independent experiments. Data are expressed as the mean ± standard deviation (SD). Student's t-test was employed to compare groups. P<0.05 was considered statistically significant. SPSS 10.0 software (SPSS, Chicago, IL, USA) was used for statistical analyses.
ResultsThe RNaseH2A gene is expressed in GBM cells
Four GBM cell lines, including U87, U251, A373 and A172 were assessed for RNaseH2A expression by RT-PCR. RNaseH2A was detected in all of the cell lines (Fig. 1A).
RNaseH2A silencing suppresses glioma cell proliferation in vitro and in vivo
Two cell lines (U87 and U251) were selected for RNA interference experiments, to assess RNaseH2A function in glioma cells. As shown in Fig. 1B and C, RNaseH2A was successfully silenced in both cell lines. RNaseH2A mRNA expression was reduced to a greater extent in the U251 cell line.
Cell proliferation analysis was performed in the two glioma cell lines (U87 and U251) transfected with RNaseH2A-shRNA. Both glioma cell lines showed significantly reduced viability after RNaseH2A silencing (Fig. 2A and B). Five days after shRNA transfection, cell viability was reduced by 70.7 and 57.8% in the U87 and U251 cells, respectively. In accordance, colony formation was also significantly decreased (P<0.05) in the U87 cells after RNaseH2A knockdown, compared with the control values (Fig. 2C). Furthermore, xenografted tumor growth in the RNaseH2A-shRNA group was markedly reduced compared with that of the normal control group in vivo (Fig. 2D–F). Indeed, 34 days after U87 cell injection, tumor volumes were 219.29±246.43 and 1160.26±222.61 mm3 in the RNaseH2A-shRNA and normal control groups, respectively; tumor weights of 1.127±0.232 and 0.261±0.245 g were obtained from the control and RNaseH2A-silenced cells, respectively (P<0.01). These findings demonstrated that RNaseH2A was required for glioma cell proliferation, both in vitro and in vivo.
RNaseH2A silencing causes cell cycle arrest in the G0/G1 phase and apoptosis in glioma cells
To explore the mechanism underlying the observed impaired growth of glioma cells after RNaseH2A silencing, cell cycle distribution was assessed by flow cytometry. RNaseH2A silencing resulted in an increased percentage of U87 cells in the G0/G1 phase, with 75.14±1.004 and 61.51±0.43% obtained respectively, in the RNaseH2A knockdown and control groups, respectively (P<0.01, Fig. 3A).
The effect of RNaseH2A on apoptosis in glioma cells (Fig. 3B) was also investigated. As shown in Fig. 3B, RNaseH2A knockdown induced cell apoptosis by 4.5-fold compared to the normal control cells.
RNaseH2A silencing induces differential expression of genes involved in multiple cell signaling pathways
To further explore the biological significance of RNaseH2A in glioma cells, gene expression profiles of U87 cells transfected with or without RNaseH2A shRNA were assessed. As shown in the heat map (Fig. 4A), gene expression profiles in the NC and RNaseH2A shRNA groups were remarkably different. A total of 821 upregulated and 941 downregulated genes were found in the RNaseH2A shRNA-transfected U87 cells. GO and KEGG pathway analysis revealed that the differentially expressed genes are frequently involved in multiple important cellular regulatory processes, including focal adhesion, cancer, p53 signaling, and cell cycle (Fig. 4B). Therefore, our data suggest that RNaseH2A shRNA affects various genes involved in important biological processes.
To validate the microarray data, qRT-PCR and western blot analysis were carried out. Protein and gene expression levels were consistent with the microarray data. For instance, IL-6 mRNA levels were decreased significantly in the RNaseH2A shRNA group compared to the control group, while FAS mRNA amounts were increased significantly in the RNaseH2A shRNA group compared to the control group (P<0.001); western blotting yielded similar results. These findings indicated that IL-6 and FAS might be suitable RNaseH2A target genes (Fig. 4C and D).
Discussion
RNaseH2A was expressed in all glioma cells assessed. Notably, RNaseH2A knockdown by shRNA silencing resulted in reduced proliferation and viability of the tumor cells, which were blocked in the G0/G1 phase. In addition, RNaseH2A-knockdown cells showed increased apoptosis in vitro. In agreement, clone formation (in vitro) and tumor growth (in vivo) were significantly decreased after RNaseH2A silencing. Gene-chip based transcriptome assay indicated that RNaseH2A plays an important role in several signaling pathways involved in cell proliferation, including the IL-6 and FAS pathways.
Gene dysregulation is a hallmark of tumor genesis and progression (46–51), with post-transcriptional regulation of messenger RNA constituting an important step. Ribonucleases (RNases) catalyze RNA breakdown, thus influencing mRNA turnover and gene expression; their dysfunction is linked to various types of tumors. For instance, failure to recruit PARN, a poly A ribonuclease, has been observed in malignant glioma (52,53). In addition, reduction and/or depletion of XRN1, a 5′–3′ exonuclease which initiates mRNA decay, is implicated in primary osteogenic sarcoma and its derived cell lines (54). Furthermore, the gene encoding truncated RNase L is positively correlated with hereditary prostate cancer (55), while moderated reduction in enzyme activity is related to a higher risk of prostate and colorectal cancers (56) as well as pancreatic carcinoma (57–59). IRE1, a transmembrane endoribonuclease found in the endoplasmic reticulum (60), can act as a tumor suppressor, deciding the fate of cancer cells (61). RNases from the miRNA pathway, including Drosha, Dicer, and Ago2, are also implicated in tumor biology. For instance, elevated expression of Drosha was found in esophageal cancers (62), with its suppression leading to decreased cancer cell proliferation; in addition, elevated mRNA levels and genomic copy numbers of Drosha were found in clinical cervical squamous cell carcinoma samples and derived cell lines (63). Several studies have reported overexpression of Dicer in multiple cancers, including salivary gland, lung, prostate, and ovary carcinomas, as well as Burkitt's lymphoma; Ago2-overexpression was also found in these cancers (64–68).
RNaseH2A is a component of the heterotrimeric type II ribonuclease H enzyme (RNAseH2), which provides the main ribonuclease H activity (31–33). Consistent with our findings, a previous study indicated that RNaseH2A is a putative anticancer drug target in transformed stem cells (31). However, how RNaseH2A is involved in tumor genesis and progression remains unclear. In this study, gene silencing and transcriptome profiling were combined to assess downstream signaling pathways mediating the effect of RNaseH2A. Multiple genes involved in important cellular processes, including focal adhesion, cancer, p53 signaling, and cell cycle, were differentially expressed after RNaseH2A silencing. Further analysis demonstrated that IL-6 was downregulated after RNaseH2A knockdown. GBM cells produced IL-6 in vitro and in vivo (69,70), and Goswami et al reported that IL-6 mediated autocrine growth promotion in the human GBM multiforme cell line U87MG (71); in addition, targeting IL-6Rα or IL-6 expression in GSCs impaired cell growth and increased the survival of mice bearing intracranial human glioma xenografts (71), in agreement with our findings of decreased xenograft growth after RNaseH2A silencing. We also found that FAS expression was upregulated after RNaseH2A silencing, which is consistent with the enhanced apoptosis described above after RNaseH2A silencing. FAS receptor is a death receptor on the surface of cells that leads to programmed cell death (72). Activation of FAS was shown to initiate apoptosis in different glioma cell lines (73). Fas-ligand, which is expressed in GBM cell lines and primary astrocytic brain tumors (74), can lead to >90% inhibition of clonal tumor cell growth in high grade gliomas ex vivo (73). Thus, silencing of RNaseH2A may inhibit proliferation and promote apoptosis in glioma cells, via downregulation of IL-6 and upregulation of FAS simultaneously.
In summary, we unveiled a possible mechanism by which RNaseH2A contributes to glioma cell proliferation. Indeed, RNaseH2A silencing resulted in growth arrest and apoptosis in glioma cells, possibly through IL-6 and FAS regulation. These findings indicate that RNaseH2A upregulation may contribute to gliomagenesis and progression, via modulation of factors involved in cell growth and apoptosis. Therefore, RNaseH2A should be considered as a potential molecular target for glioma diagnosis and treatment.
Acknowledgments
This study was supported by the National Key Technologies R&D Program of the Ministry of Science and Technology of China (no. 2013BAI09B03)
ReferencesAdamczykLAWilliamsHFrankowAEllisHPHaynesHRPerksCHollyJMKurianKMCurrent understanding of circulating tumor cells–potential value in malignancies of the central nervous systemFront Neurol6174201510.3389/fneur.2015.00174Aparicio-BlancoJTorres-SuárezAIGlioblastoma multiforme and lipid nanocapsules: a reviewJ Biomed Nanotechnol1112831311201510.1166/jbn.2015.208426295134CuddapahVARobelSWatkinsSSontheimerHA neurocentric perspective on glioma invasionNat Rev Neurosci15455465201410.1038/nrn376524946761ErricoACNS cancer: new options for glioblastomaNat Rev Clin Oncol11124201424535164ParsonsDWJonesSZhangXLinJCLearyRJAngenendtPMankooPCarterHSiuIMGalliaGLAn integrated genomic analysis of human glioblastoma multiformeScience32118071812200810.1126/science.1164382187723962820389WestphalMLamszusKCirculating biomarkers for gliomasNat Rev Neurol11556566201510.1038/nrneurol.2015.17126369507ZhangZZShieldsLBSunDAZhangYPHuntMAShieldsCBThe art of intraoperative glioma identificationFront Oncol5175201510.3389/fonc.2015.00175262841964520021SamdaniAFTorre-HealyAKhalessiAMcGirtMJalloGICarsonBIntraventricular ganglioglioma: a short illustrated reviewActa Neurochir (Wien)151635640200910.1007/s00701-009-0246-0GautschiOPvan LeyenKCadoschDHildebrandtGFournierJYFluorescence guided resection of malignant brain tumors-breakthrough in the surgery of brain tumorsPraxis Bern (1994)986436472009In German10.1024/1661-8157.98.12.643SignorelliFGuyotatJElisevichKBarbagalloGMReview of current microsurgical management of insular gliomasActa Neurochir (Wien)1521926201010.1007/s00701-009-0450-yBelloLFavaECarrabbaGPapagnoCGainiSMPresent day's standards in microsurgery of low-grade gliomasAdv Tech Stand Neurosurg35113157201020102113FurnariFBFentonTBachooRMMukasaAStommelJMSteghAHahnWCLigonKLLouisDNBrennanCMalignant astrocytic glioma: genetics, biology, and paths to treatmentGenes Dev2126832710200710.1101/gad.159670717974913LacroixMAbi-SaidDFourneyDRGokaslanZLShiWDeMonteFLangFFMcCutcheonIEHassenbuschSJHollandEA multivariate analysis of 416 patients with glioblastoma multiforme: prognosis, extent of resection, and survivalJ Neurosurg95190198200110.3171/jns.2001.95.2.0190OhgakiHKleihuesPGenetic alterations and signaling pathways in the evolution of gliomasCancer Sci10022352241200910.1111/j.1349-7006.2009.01308.x19737147MarumotoTSayaHMolecular biology of gliomaAdv Exp Med Biol746211201210.1007/978-1-4614-3146-6_122639155PatelMVogelbaumMABarnettGHJalaliRAhluwaliaMSMolecular targeted therapy in recurrent glioblastoma: current challenges and future directionsExpert Opin Investig Drugs2112471266201210.1517/13543784.2012.70317722731981SpasicMChowFTuCNagasawaDTYangIMolecular characteristics and pathways of Avastin for the treatment of glioblastoma multiformeNeurosurg Clin N Am23417427201210.1016/j.nec.2012.05.00222748654ZhuJJWongETPersonalized medicine for glioblastoma: current challenges and future opportunitiesCurr Mol Med13358367201323331008GoodenbergerMLJenkinsRBGenetics of adult gliomaCancer Genet205613621201210.1016/j.cancergen.2012.10.00923238284AssiHCandolfiMBakerGMineharuYLowensteinPRCastroMGGene therapy for brain tumors: basic developments and clinical implementationNeurosci Lett5277177201210.1016/j.neulet.2012.08.003229069213462660CarénHPollardSMBeckSThe good, the bad and the ugly: epigenetic mechanisms in glioblastomaMol Aspects Med34849862201310.1016/j.mam.2012.06.0073714597RizzoDRuggieroAMartiniMRizzoVMauriziPRiccardiRMolecular biology in pediatric high-grade glioma: impact on prognosis and treatmentBioMed Res Int2015215135201510.1155/2015/215135264489304584033MischelPSCloughesyTFTargeted molecular therapy of GBMBrain Pathol135261200310.1111/j.1750-3639.2003.tb00006.x12580545ZhangLChenLHWanHYangRWangZFengJYangSJonesSWangSZhouWExome sequencing identifies somatic gain-of-function PPM1D mutations in brainstem gliomasNat Genet46726730201410.1038/ng.2995248803414073211WanWXuXJiaGLiWWangJRenTWuZZhangJZhangLLuYDifferential expression of p42.3 in low- and high-grade gliomasWorld J Surg Oncol12185201410.1186/1477-7819-12-185249277514073174MeltonCReuterJASpacekDVSnyderMRecurrent somatic mutations in regulatory regions of human cancer genomesNat Genet47710716201510.1038/ng.3332260534944485503PetersITezvalHKramerMWWoltersMGrünwaldVKuczykMASerthJImplications of TCGA network data on 2nd generation immunotherapy concepts based on PD-L1 and PD-1 target structuresAktuelle Urol464814852015In German26560846ChenXShiKWangYSongMZhouWTuHLinZClinical value of integrated-signature miRNAs in colorectal cancer: miRNA expression profiling analysis and experimental validationOncotarget637544375562015264620344741947MoellingKBroeckerFThe reverse transcriptase-RNase H: from viruses to antiviral defenseAnn NY Acad Sci1341126135201510.1111/nyas.1266825703292NatiqAElalaouiSCMieschSBonnetCJonveauxPAmzaziSSefianiAA new case of de novo 19p13.2p13.12 deletion in a girl with overgrowth and severe developmental delayMol Cytogenet740201410.1186/1755-8166-7-40249633504068972FlanaganJMFunesJMHendersonSWildLCareyNBoshoffCGenomics screen in transformed stem cells reveals RNASEH2A, PPAP2C, and ADARB1 as putative anticancer drug targetsMol Cancer Ther8249260200910.1158/1535-7163.MCT-08-063619139135FengSCaoZIs the role of human RNase H2 restricted to its enzyme activity?Prog Biophys Mol BiolNov192015Epub ahead of print26603688ReijnsMABubeckDGibsonLCGrahamSCBaillieGSJonesEYJacksonAPThe structure of the human RNase H2 complex defines key interaction interfaces relevant to enzyme function and human diseaseJ Biol Chem2861053010539201110.1074/jbc.M110.1773943060506HausenPSteinHRibonuclease H. An enzyme degrading the RNA moiety of DNA-RNA hybridsEur J Biochem14278283197010.1111/j.1432-1033.1970.tb00287.x5506170SteinHHausenPEnzyme from calf thymus degrading the RNA moiety of DNA-RNA hybrids: effect on DNA-dependent RNA polymeraseScience166393395196910.1126/science.166.3903.3935812039CotéMLRothMJMurine leukemia virus reverse transcriptase: structural comparison with HIV-1 reverse transcriptaseVirus Res134186202200810.1016/j.virusres.2008.01.001182947202443788MizunoMYasukawaKInouyeKInsight into the mechanism of the stabilization of moloney murine leukaemia virus reverse transcriptase by eliminating RNase H activityBiosci Biotechnol Biochem74440442201010.1271/bbb.9077720139597SchultzSJChampouxJJRNase H activity: structure, specificity, and function in reverse transcriptionVirus Res13486103200810.1016/j.virusres.2007.12.007182618202464458RiceGIForteGMSzynkiewiczMChaseDSAebyAAbdel-HamidMSAckroydSAllcockRBaileyKMBalottinUAssessment of interferon-related biomarkers in Aicardi-Goutières syndrome associated with mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, and ADAR: a case-control studyLancet Neurol1211591169201310.1016/S1474-4422(13)70258-8241833094349523OrcesiSLa PianaRFazziEAicardi-Goutieres syndromeBr Med Bull89183201200910.1093/bmb/ldn04919129251CrowYJAicardi-Goutières syndromeHandb Clin Neurol11316291635201310.1016/B978-0-444-59565-2.00031-9CrowYJManelNAicardi-Goutières syndrome and the type I interferonopathiesNat Rev Immunol15429440201510.1038/nri385026052098WilliamsKALeeMHuYAndreasJPatelSJZhangSChinesPElkahlounAChandrasekharappaSGutkindJSA systems genetics approach identifies CXCL14, ITGAX, and LPCAT2 as novel aggressive prostate cancer susceptibility genesPLoS Genet10e1004809201410.1371/journal.pgen.1004809254119674238980LivakKJSchmittgenTDAnalysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT MethodMethods25402408200110.1006/meth.2001.1262DaiBWanWZhangPZhangYPanCMengGXiaoXWuZJiaWZhangJSET and MYND domain-containing protein 3 is overexpressed in human glioma and contributes to tumorigenicityOncol Rep3427222730201526328527YunKFidlerAEEcclesMRReeveAEInsulin-like growth factor II and WT1 transcript localization in human fetal kidney and Wilms' tumorCancer Res535166517119938221652Pritchard-JonesRODunnDBQiuYVareyAHOrlandoARigbyHHarperSJBatesDOExpression of VEGFxxxb, the inhibitory isoforms of VEGF, in malignant melanomaBr J Cancer97223230200710.1038/sj.bjc.6603839175956662360298IsmailPMLuTSawadogoMLoss of USF transcriptional activity in breast cancer cell linesOncogene1855825591199910.1038/sj.onc.120293210523835MacéKAguilarFWangJSVautraversPGómez-LechónMGonzalezFJGroopmanJHarrisCCPfeiferAMAflatoxin B1-induced DNA adduct formation and p53 mutations in CYP450-expressing human liver cell linesCarcinogenesis1812911297199710.1093/carcin/18.7.1291CoxCBignellGGreenmanCStabenauAWarrenWStephensPDaviesHWattSTeagueJEdkinsSA survey of homozygous deletions in human cancer genomesProc Natl Acad Sci USA10245424547200510.1073/pnas.040859310215761058555487DoyleGABourdeau-HellerJMCoulthardSMeisnerLFRossJAmplification in human breast cancer of a gene encoding a c-myc mRNA-binding proteinCancer Res6027562759200010850408SuswamELiYZhangXGillespieGYLiXShackaJJLuLZhengLKingPHTristetraprolin down-regulates interleukin-8 and vascular endothelial growth factor in malignant glioma cellsCancer Res68674682200810.1158/0008-5472.CAN-07-275118245466LaiWSKenningtonEABlackshearPJTristetraprolin and its family members can promote the cell-free deadenylation of AU-rich element-containing mRNAs by poly(A) ribonucleaseMol Cell Biol2337983812200310.1128/MCB.23.11.3798-3812.200312748283155217ZhangKDionNFuchsBDamronTGitelisSIrwinRO'ConnorMSchwartzHScullySPRockMGThe human homolog of yeast SEP1 is a novel candidate tumor suppressor gene in osteogenic sarcomaGene298121127200210.1016/S0378-1119(02)00929-012426100RökmanAIkonenTSeppäläEHNupponenNAutioVMononenNBailey-WilsonJTrentJCarptenJMatikainenMPGermline alterations of the RNASEL gene, a candidate HPC1 gene at 1q25, in patients and families with prostate cancerAm J Hum Genet7012991304200210.1086/34045011941539447604KrügerSSilberASEngelCGörgensHMangoldEPagenstecherCHolinski-FederEvon Knebel DoeberitzMMoesleinGDietmaierWGerman Hereditary Non-Polyposis Colorectal Cancer ConsortiumArg462Gln sequence variation in the prostate-cancer-susceptibility gene RNASEL and age of onset of hereditary non-polyposis colorectal cancer: a case-control studyLancet Oncol6566572200510.1016/S1470-2045(05)70253-916054567BartschDKFendrichVSlaterEPSina-FreyMRiederHGreenhalfWChaloupkaBHahnSANeoptolemosJPKressRRNASEL germline variants are associated with pancreatic cancerInt J Cancer117718722200510.1002/ijc.2125415981205ShookSJBeutenJTorkkoKCJohnson-PaisTLTroyerDAThompsonIMLeachRJAssociation of RNASEL variants with prostate cancer risk in Hispanic Caucasians and African AmericansClin Cancer Res1359595964200710.1158/1078-0432.CCR-07-070217908993RennertHZeigler-JohnsonCMAddyaKFinleyMJWalkerAHSpanglerELeonardDGWeinAMalkowiczSBRebbeckTRAssociation of susceptibility alleles in ELAC2/HPC2, RNASEL/HPC1, and MSR1 with prostate cancer severity in European American and African American menCancer Epidemiol Biomarkers Prev14949957200510.1158/1055-9965.EPI-04-063715824169SidrauskiCWalterPThe transmembrane kinase Ire1p is a site-specific endonuclease that initiates mRNA splicing in the unfolded protein responseCell9010311039199710.1016/S0092-8674(00)80369-49323131DaviesMPBarracloughDLStewartCJoyceKAEcclesRMBarracloughRRudlandPSSibsonDRExpression and splicing of the unfolded protein response gene XBP-1 are significantly associated with clinical outcome of endocrine-treated breast cancerInt J Cancer1238588200810.1002/ijc.2347918386815SugitoNIshiguroHKuwabaraYKimuraMMitsuiAKureharaHAndoTMoriRTakashimaNOgawaRRNASEN regulates cell proliferation and affects survival in esophageal cancer patientsClin Cancer Res1273227328200610.1158/1078-0432.CCR-06-051517121874MuralidharBGoldsteinLDNgGWinderDMPalmerRDGoodingELBarbosa-MoraisNLMukherjeeGThorneNPRobertsIGlobal microRNA profiles in cervical squamous cell carcinoma depend on Drosha expression levelsJ Pathol212368377200710.1002/path.217917471471KaulDSikandKDefective RNA-mediated c-myc gene silencing pathway in Burkitt's lymphomaBiochem Biophys Res Commun313552554200410.1016/j.bbrc.2003.12.002FlavinRJSmythPCFinnSPLaiosAO'TooleSABarrettCRingMDenningKMLiJAherneSTAltered eIF6 and Dicer expression is associated with clinicopathological features in ovarian serous carcinoma patientsMod Pathol21676684200810.1038/modpathol.2008.3318327211ChioseaSJelezcovaEChandranUAcquafondataMMcHaleTSobolRWDhirRUp-regulation of dicer, a component of the MicroRNA machinery, in prostate adenocarcinomaAm J Pathol16918121820200610.2353/ajpath.2006.060480170716021780192ChioseaSJelezcovaEChandranULuoJManthaGSobolRWDacicSOverexpression of Dicer in precursor lesions of lung adenocarcinomaCancer Res6723452350200710.1158/0008-5472.CAN-06-353317332367ChioseaSIBarnesELLaiSYEgloffAMSargentRLHuntJLSeethalaRRMucoepidermoid carcinoma of upper aerodigestive tract: clinicopathologic study of 78 cases with immunohistochemical analysis of Dicer expressionVirchows Arch452629635200810.1007/s00428-007-0574-518239938WangHLathiaJDWuQWangJLiZHeddlestonJMEylerCEElderbroomJGallagherJSchuschuJTargeting interleukin 6 signaling suppresses glioma stem cell survival and tumor growthStem Cells2723932404200910.1002/stem.188196581882825688ChangCYLiMCLiaoSLHuangYLShenCCPanHCPrognostic and clinical implication of IL-6 expression in glioblastoma multiformeJ Clin Neurosci12930933200510.1016/j.jocn.2004.11.01716326273GoswamiSGuptaASharmaSKInterleukin-6-mediated autocrine growth promotion in human glioblastoma multiforme cell line U87MGJ Neurochem7118371845199810.1046/j.1471-4159.1998.71051837.x9798907GreenDRLlambiFCell Death SignalingCold Spring Harb Perspect Biol72015pii: a00608010.1101/cshperspect.a00608026626938FreiKAmbarBAdachiNYonekawaYFontanaAEx vivo malignant glioma cells are sensitive to Fas (CD95/APO-1) ligand-mediated apoptosisJ Neuroimmunol87105113199810.1016/S0165-5728(98)00065-49670851GratasCTohmaYVan MeirEGKleinMTenanMIshiiNTachibanaOKleihuesPOhgakiHFas ligand expression in glioblastoma cell lines and primary astrocytic brain tumorsBrain Pathol7863869199710.1111/j.1750-3639.1997.tb00889.x9217971
Gene expression levels of RNaseH2A in human glioblastoma cells silenced or not for RNaseH2A. (A) RNaseH2A mRNA in different glioma cell lines, assessed by qRT-PCR. (B) Silencing efficacy of RNaseH2A shRNA in the U87 cells, as assessed by qRT-PCR. (C) Silencing efficacy of RNaseH2A shRNA in U251 cells, as assessed by qRT-PCR. Data are represented as the mean ± SD. Each data point was determined from at least three replicates; *P<0.05 or **P<0.01, significant difference between the RNaseH2A shRNA and the shRNA control groups.
Effects of RNaseH2A silencing on glioma cell proliferation in vitro and in vivo. RNaseH2A silencing inhibits the growth of (A) U87 and (B) U251 glioma cells. (C) RNaseH2A silencing reduces colony formation of U87 cells. (D-F) RNaseH2A silencing inhibits tumor growth in vivo. shRNA control- and RNaseH2A shRNA-transfected U87 cells were injected subcutaneously into nude mice. Tumor volumes were calculated daily. Mice were then sacrificed and tumors dissected, photographed and weighed. Quantitative data are represented as the mean ± SD. Each data point was determined from at least three replicates; *P<0.05 or **P<0.01, significant difference between the RNaseH2A shRNA and the shRNA control groups (n=10).
RNaseH2A knockdown alters glioma cell cycle progression and apoptosis. (A) Effects of RNaseH2A knockdown on (A) cell cycle progression and (B) apoptosis. Quantitative data are expressed as mean ± SD. Three representative flow-cytograms for the control and RNaseH2A groups, respectively, are presented for cell cycle distribution and apoptosis assays. Each data point was determined from at least three replicates; *P<0.05 or **P<0.01, significant difference between the RNaseH2A shRNA and the shRNA control groups.
RNaseH2A knockdown alters mRNA and protein levels of several genes involved in multiple cellular signaling pathways. (A) Heat map showing differentially expressed genes after RNaseH2A silencing. Rows and columns represent genes and samples, respectively. The intensity of each color denotes the standardized ratio between each value and the average methylation/expression of the gene across all samples. Red and green indicate increased and decreased transcript levels, respectively. (B) KEGG pathway analysis of differentially expressed genes (cutoff of 1.5- or 2-fold). (C) Validation of microarray results by qPCR. (D) Validation of microarray results by western blot analysis are represented as the mean ± SD; *P<0.05, **P<0.01 or ***P<0.001, significant difference between the RNaseH2A shRNA and the shRNA control groups.