| Colorectal cancer is the world’s fourth most deadly cancer(after lung,liver and stomach cancer).The intricate molecular mechanism of it has not yet been fully elucidated.The new view has found that cancer is characterized by a combination of genetic mutations and epigenetic changes.At present,epigenetic regulatory factors have attracted great attention in colorectal cancer research.Jumonji C domain-containing 1A(JMJD1A)is a histone demethylase and epigenetic regulator belonging to the lysine demethylase 3(KDM3)family of histone demethylases,also known as KDM3 A.It is an enzyme that activates target gene expression by converting dimethylated lysine 9 on histone H3 progressively into its mono-and unmethylated form.Playing a key role in determining the carcinogenic potential of colorectal cancer by controling of Wnt/β-catenin-regulated transcription.In the current study,its m RNA and protein expression was analyzed in human colorectal tumors.It was demonstrated that JMJD1 A levels were increased and correlated with a more aggressive phenotype.Downregulation of JMJD1 A in human HCT116 colorectal cancer cells caused negligible growth defects,but robustly decreased clonogenic activity.Transcriptome analysis revealed that JMJD1 A downregulation led to multiple changes in HCT116 cells,including inhibition of MYC-and MYCN-regulated pathways and stimulation of the TP53 tumor suppressor response.One gene identifed to be stimulated by JMJD1 A was α-thalassemia/mental retardation syndrome X-linked(ATRX),which encodes for a chromatin remodeler.The JMJD1 A protein,but not a catalytically inac-tive mutant,activated the ATRX gene promoter and JMJD1 A also affected levels of dimethylation on lysine 9 of histone H3.Similar to JMJD1 A,ATRX was signifcantly overexpressed in humancolorectal tumors and correlated with increased disease recurrence and lethality.Furthermore,ATRX downregulation in HCT116 cells reduced their growth and clonogenic activity.Accordingly,upregulation of ATRX may represent one mecha-nism by which JMJD1 A promotes colorectal cancer.In addition,the data presented in this study suggest that the current notion of ATRX as a tumor suppressor is incomplete and that ATRX might context dependently also function as a tumor promoter.1 Materials and methods1.1 Analysis of databases.Microarray experiments were analyzed with Oncomine and respective data downloaded from www.oncomine.org.The Human Protein Atlas served as a source for survival data(www.proteinatlas.org)with the corre-sponding RNA sequencing data originating from ‘The Cancer Genome Atlas’(TCGA).To assess coexpression of ATRX and JMJD1 A in colorectal adenocarcinomas,provisional TCGA RNA sequencing data were analyzed with cbioportal(www.bioportal.org).1.2 Sample and cell culture Colonrectal cancer tissues with corresponding normal tissues colon cancer are from OUHSC,Human colorectal carcinoma cells HCT116,SW480,DLD-1,HT-29 as well as human embryonic kidney 293 T cells(all American Type Culture Collection,Manassas,VA,USA)were grown in a humidifed,5% CO2-containing atmosphere in DMEM with 10% FBS.1.3 Analysis of protein expression The NE-PERTMnuclear and cytoplasmic extraction kit was employed on the cells.Protein expression levels were detected by Western Blot.Tissue microarray analysis(TMA)and immunohistochemical staining were used to analyze the differences.1.4 Cloning of sh RNA The retroviral vector p SIREN-Retro Q(Takara Bio USA,Inc.CA,USA)was linearized with the restriction enzymes Bam HI and Eco RI and ligated with double-stranded oligonucleotides encoding sh RNAs.The control sh RNA targets the sequence 5’-CAACAAGATGAAGAGCACCAA-3’,which displays at least four mismatches to any known human gene.The human JMJD1 A sh RNAs target the sequences 5’-GCAGGTGTCAATAGTGAT A-3’(#1 sh RNA)and 5’-GTAGACCTA GTTAATTGTA-3’(#2 sh RNA),while the human ATRX sh RNAs target the sequences5’-GGTGTTATGATCATAGGCTAT-3’(#1 sh RNA)and 5’-GGATTCAAC CTCTTGA GGATA-3’(#3 sh RNA).1.5 Proliferation analysis Employing PrestoblueTMreagent on the cells which were seeded in 96-wells for measuring growth.For clonogenic assays,cells were seeded into 6-wells.1.6 RNA sequencing and transcriptome analysis Total RNA from HCT116 cells was isolated employing TRIzol following standard procedures.RNA was further purifed with the RNeasy Mini kit and sequenced in the Targeted DNA Methylation and Mitochondrial Heteroplasmy Core at the Oklahoma Nathan Shock Center of Excellence in the Biology of Aging(Oklahoma City,OK,USA).Data was made into Venn diagram and Ingenuity Pathway Analysis(Qiagen)was performed with both canonical pathway and upstream regulator analysis.1.7 Identifcation of potential target gene Human 293 T or HCT116 cells were transfected with an ATRX luciferase reporter construct and JMJD1A(wild-type or H1120A/D1122 G catalytic mutant),Resultant relative luciferase activity is measured.Chromatin immunoprecipitation assay with293 T cells transfected with indicated Flag-tagged JMJD1 A expression constructs and the ATRX luciferase reporter gene.1.8 Statistics Statistical significance was assessed with one or two-way analysis of variance(ANOVA)with post hoc Dunnett’s or Tukey’s multiple comparisons test,an unpaired Student’s t-test,or a log-rank test.P<0.05 was deemed to show a statistically signifcant difference.2 Results2.1 JMJD1 A expression in colorectal cancer According to publicly available databases,JMJD1 A was significantly overexpressed in colorectal cancer(P<0.05)and correlated with a more aggressive tumor phenotype.High JMJD1 A m RNA levels were significantly associated with decreased survival(P=0.0132).Western Blot showed that JMJD1 A was expressed in human colorectal cancer cell lines HCT-116,SW480,DLD-1 and HT-29.With biochemically fractionating HCT116 and DLD-1,JMJD1 A was present in both the cytoplasm and nucleus.Tissue microarray staining showed a significant overexpression of JMJD1 A in the tumors other than normal tissues.2.2 Impact of JMJD1 A on HCT116 cells JMJD1 A downregulation has only negligible effects on HCT116 cell growth.Clonogenic activity of HCT116 colorectal cancer cells was robustly reduced upon JMJD1 A downregulation with either of the two sh RNAs.2.3 Transcriptome analysis Compared to control sh RNA,281 genes were >1.5-fold upregulated with both JMJD1 A sh RNAs and 192 genes were >1.5-fold downregulated.JMJD1 A downregulation led to stimulation of TP53-and TGF-β1-regulated and inhibition of MYC-and MYCN-driven pathways.the PPARA upstream regulator pathway was the most signifcantly stimulated pathway upon JMJD1 A sh RNA expression.2.4 Identifcation of ATRX as a potential JMJD1 A target gene JMJD1 A downregulation by sh RNA#1 or sh RNA#2 led to a 2.6-fold or 3.5-fold decrease in ATRX m RNA levels.Accordingly,ATRX protein levels were reduced in the presence of JMJD1 A sh RNAs.JMJD1 A and ATRX m RNA levels were positively correlated.293 T or HCT116 cells were transfected with an ATRX luciferase reporter construct and JMJD1A(wild-type or H1120A/D1122 G catalytic mutant),relative luciferase activity is higher in the wild-type,lower in the mutant.JMJD1 A overexpression expectedly reduced dimethylation of histone H3 on lysine 9,but not dimethylation on lysine 36.On the other hand,the H1120A/D1122 G mutant predictably elevated levels of dimethylation on lysine 9 of histone H3.2.5 ATRX in colorectal cancer According to publicly available databases,ATRX m RNA was significantly overexpressed in colorectal tumors(P < 0.05).High levels of ATRX expression were more likely in patients with recurrence(P = 0.01)and death(P = 0.017).Cell proliferation assays showed that down-regulation of ATRX expression led to a signifcant reduction of HCT116 cell growth(P < 0.0001)and caused a reduction in clonogenic activity.3 Conclusion3.1 JMJD1 A m RNA is overexpressed in colorectal tumors and associated with a more aggressive phenotype.Implicating that JMJD1 A overexpression may contribute to the development of colorectal cancer.3.2 Clonogenic activity of HCT116 colorectal cancer cells is robustly reduced upon JMJD1 A downregulation.Indicate that JMJD1 A can infuence the physiology of HCT116 cells in a manner that is predicted to be tumor promoting.3.3 JMJD1 A pleiotropically affects the gene expression program of HCT116 colorectal cancer cells and may thereby be a determinant of their oncogenic potential.3.4 JMJD1 A can stimulate ATRX gene transcription.It can interact with the ATRX promoter and induces it by a mechanism that involves reduction of H3K9me2 levels.3.5 ATRX is overexpressed in colorectal cancer and its expression level is positively correlated with worse outcomes of the disease.ATRX downregulation leds to a signifcant reduction of HCT116 cell growth and clonogenic activity.ATRX is a promoter of colorectal cancer. |
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