MiR-185 Targets the DNA Methyltransferases 1 and Regulates Global DNA Methylation in human glioma

<p>Abstract</p> <p>Background</p> <p>Perturbation of DNA methylation is frequent in cancers and has emerged as an important mechanism involved in tumorigenesis. To determine how DNA methylation is modified in the genome of primary glioma, we used Methyl-DNA immunoprecipitation (MeDIP) and Nimblegen CpG promoter microarrays to identify differentially DNA methylation sequences between primary glioma and normal brain tissue samples.</p> <p>Methods</p> <p>MeDIP-chip technology was used to investigate the whole-genome differential methylation patterns in glioma and normal brain tissues. Subsequently, the promoter methylation status of eight candidate genes was validated in 40 glioma samples and 4 cell lines by Sequenom's MassARRAY system. Then, the epigenetically regulated expression of these genes and the potential mechanisms were examined by chromatin immunoprecipitation and quantitative real-time PCR.</p> <p>Results</p> <p>A total of 524 hypermethylated and 104 hypomethylated regions were identified in glioma. Among them, 216 hypermethylated and 60 hypomethylated regions were mapped to the promoters of known genes related to a variety of important cellular processes. Eight promoter-hypermethylated genes (ANKDD1A, GAD1, HIST1H3E, PCDHA8, PCDHA13, PHOX2B, SIX3, and SST) were confirmed in primary glioma and cell lines. Aberrant promoter methylation and changed histone modifications were associated with their reduced expression in glioma. In addition, we found loss of heterozygosity (LOH) at the miR-185 locus located in the 22q11.2 in glioma and induction of miR-185 over-expression reduced global DNA methylation and induced the expression of the promoter-hypermethylated genes in glioma cells by directly targeting the DNA methyltransferases 1.</p> <p>Conclusion</p> <p>These comprehensive data may provide new insights into the epigenetic pathogenesis of human gliomas.</p

Influences of image resolution on herbaceous root morphological parameters

Root images of four herbaceous species (including Plantago virginica,Solidago canadensis,Conyza canadensis and Erigeron philadelphicus) were obtained by using EPSON V7000 scanner with different resolutions.Root morphological parameters including root length,diameter,volume and area were determined by using a WinRhizo root analyzing software.The results show a distinct influence of image resolution on root morphological parameter.For different herbaceous species,the optimal resolutions of root images,which would produce an acceptable precision with relative short time,vary with different species.For example,a resolution of 200 dpi was recommended for the root images of Plantago virginica and S.Canadensis, while 400 dpi for Conyza canadensis and Erigeron philadelphicus

Circulating MicroRNA-21 Is a Potential Diagnostic Biomarker in Gastric Cancer

MicroRNA-21 was upexpressed in gastric cancer (GC) indicating that it is a potential diagnostic biomarker for GC. In this study, 50 GC patients and 50 healthy controls were recruited. miR-21 levels in serum and peripheral blood mononuclear cells (PBMCs) were quantified using quantitative real-time PCR. CA199, and CEA were measured using electrochemiluminescence assay. The sensitivity and specificity of circulating miR-21, CA199 and CEA in GC diagnosis, the correlation of circulating miR-21 to clinicopathological features, and the diagnostic value of miR-21 in different GC stages were determined. The levels of miR-21 in both serum and PBMCs increased significantly in GC patients comparing to healthy controls; however, no correlation was observed between circulating miR-21 level and clinicopathological features. The sensitivity and specificity of miR-21 in serum and PBMCs, and CA199 and CEA in GC diagnosis were 88.4%, 79.6%, 81.3%, 73.4%, 60.5%, 55.9%, and 68.6%, 59.3%, respectively. The positive prediction rates of circulating miR-21 in GC stages I to IV were all around 90%, while those of CA199 and CEA were around or less than 50%. Our data suggest circulating miR-21 (both in serum and in PBMCs) can serve as a good biomarker for GC and could be used in diagnosis of early (stage I) and late GC (stage IV)

Surface Grain Refinement of 304L Stainless Steel by Combined Severe Shot Peening and Reversion Annealing Treatment

The present study proposes a novel method, i.e., combined severe shot peening (SP) and reversion annealing treatment, to grain-refine the surface layers of 304L austenitic stainless steel. Steel specimens were shot-peened at 0.7 MPa for 30 min, introducing 40% vol. &alpha;&prime; martensite, and then were annealed at 700 or 800 &deg;C for different durations (30 s). As annealing reversed &alpha;&prime; martensite to austenite, the obtained surface layers consist of fully austenitic ultrafine grains. The smallest grain size obtained is about 500 nm at the top surface. SP elevates the microhardness to more than 500 HV. Although the grain-refined surface layers produced by the combined method are not as hard as that treated by only SP, they are harder (e.g., the specimen annealed at 700 &deg;C for 30 s using a heating rate of 50 &deg;C/s exhibited a peak microhardness of 400 HV) than the untreated surface layer (225 HV) due to grain refinement. Moreover, due to the absence of &alpha;&prime; martensite, they have higher corrosion resistance in H2SO4 solution than that treated by only SP

LNA-anti-miR-182 and -381 suppressed the promoter activity of <i>BRD7</i> by down-regulating AP2, SP1, and E2F6, and up-regulating c-Myc.

<p>(A) LNA-mediated miR-182 and -381 silencing down-regulated expression of K-Ras, p-c-Raf, pERK, PI-3K, and pAKT, but the silencing had no effect on N-Ras, total ERK and AKT expression, as shown by Western blot (left) and gray image scanning (right). (B) LNA-mediated miR-182 and -381 silencing down-regulated the expression of AP2, SP1, and E2F6, and up-regulated the expression of c-Myc, as shown by Western blot (top) and gray image scanning (bottom). (C) PD98059 or LY294002 reversed the miR-182 and miR-381 mimics-induced expression of AP2, SP1, E2F6, and c-Myc. AP2, SP1, E2F6, and c-Myc expression were assessed by Western blot (left) and gray image scanning (right). (D) EMSA confirmed that LNA-mediated miR-182 and -381 silencing or ectopic <i>LRRC4</i> expression promoted the <i>BRD7</i> promoter association of c-Myc and disrupted that of AP2, SP1, and E2F6. Mutant, nuclear protein +200×mutant probe + wild biotin-probe; Competitor, nuclear protein +200×competitor cold probe + wild biotin-probe; No extracts, no nuclear protein + wild biotin-probe; Scrambled, nuclear protein of transfected miRNA negative control + wild biotin-probe; LNA-182, nuclear protein of transfected LNA-miR-182 inhibitors + wild biotin-probe; LNA-381, nuclear protein of transfected LNA-miR-381 inhibitors + wild biotin-probe; LRRC4, nuclear protein of transfected LRRC4 gene + wild biotin-probe. (E) Luciferase assays confirmed the inhibition of LNA-mediated miR-182 and -381 silencing, or siRNA of AP2, SP1, and E2F6, and c-Myc overexpression on the promoter activity of <i>BRD7</i> gene. * <i>p</i><0.05 compared with the control (siRNA including SP1, AP2, and E2F6 <i>vs.</i> siRNA scrambled or pCMV-HA-c-Myc <i>vs.</i> pCMV-HA). (F) EMSA indicated that PD98059 and LY294002 reversed the association of AP2, SP1, and E2F6 or c-Myc with the <i>BRD7</i> promoter that was induced by miR-182 and miR-381. Mutant, nuclear protein+200×mutant probe + wild biotin-probe; Competitor, nuclear protein +200×competitor wild probe + wild biotin-probe; No extracts, no nuclear protein + wild biotin-probe; Mock, nuclear protein+ wild biotin-probe; PD98059, nuclear protein with PD98059 + wild biotin-probe; LY294002, nuclear protein with PD98059 + wild biotin-probe; Scrambled, nuclear protein of transfected miRNA negative control + wild biotin-probe; LNA-182, nuclear protein of transfected LNA-miR-182 inhibitors + wild biotin-probe; LNA-381, nuclear protein of transfected LNA-miR-381 inhibitors + wild biotin-probe; 182M, nuclear protein of transfected miR-182 mimics + wild biotin-probe; 381M, nuclear protein of transfected miR-381 mimics + wild biotin-probe. (G) siRNA-AP2, siRNA-SP1, siRNA-E2F6, and c-Myc overexpression affected endogenous expression of <i>BRD7</i> at the protein (left) and mRNA (right) levels. * <i>p</i><0.05 compared with control (LNA-182 and LNA-381 <i>vs.</i> scrambled; si-SP1, si-AP2 and si-E2F6 <i>vs</i>. si-SC; pCMV-HA-c-Myc <i>vs</i>. pCMV-HA).</p

Disturbing miR-182 and -381 Inhibits BRD7 Transcription and Glioma Growth by Directly Targeting <i>LRRC4</i>

<div><p>Inactivated <i>LRRC4</i> has been clinically detected in gliomas, and promoter hypermethylation has been implicated as the mechanism of inactivation in some of those tumors. Our previous researches indicated that <i>LRRC4</i> is a target gene of miR-381, the interaction of miR-381 and <i>LRRC4</i> is involved in glioma growth. In this study, we demonstrate that <i>LRRC4</i> is a target gene of the other microRNA, miR-182. We found that the high expression of miR-182 and miR-381 in gliomas are involved in pathological malignant progression. The silencing of miR-182 and miR-381 inhibited the proliferation <i>in vitro</i> and growth of glioma cell with <i>in vivo</i> magnetic resonance imaging by intracranial transplanted tumor model in rats. We also demonstrated that BRD7, a transcriptional cofactor for p53, is highly expressed and negatively correlated with <i>LRRC4</i> expression in gliomas. Disturbing miR-182 and miR-381 affected transcriptional regulation of the <i>BRD7</i> gene. This finding was verified by ectopic overexpression of <i>LRRC4</i> or restoration of endogenous <i>LRRC4</i> expression by treatment with the DNA demethylating agent 5-Aza-dC. Taken together, miR-182 and miR-381 may be a useful therapeutic target for treatment of glioma.</p></div

LNA-anti-miR-182 and -381 had anticancer effects on intracranial transplanted tumors by surpassing the blood-brain barrier.

<p>(A) Intraperitoneal injection of LNA-anti-miR-182 and/or -381 oligonucleotides inhibited the growth of intracranial transplanted tumors in Sprague-Dawley rats (top and middle, MRI; bottom, HE staining of coronal section). (B) LNA-anti-miR-182 and/or -381 oligonucleotides reduced expression of miR-182 and -381 (ISH), increased expression of LRRC4, and reduced expression of BRD7 and Ki-67 (IHC).</p

miRNA-182 and miR-381 or <i>BRD7</i> expression is inversely related to <i>LRRC4</i> expression in gliomas.

<p>(A) miRNA-182 and miR-381 or <i>BRD7</i> expression is inversely related to <i>LRRC4</i> expression in glioma tissues. miR-182 and miR-381 expression levels were assessed by ISH. IHC was used to detect the protein expression of <i>LRRC4</i> and <i>BRD7</i>. (B) miRNA-182 and miR-381 or <i>BRD7</i> expression is inversely related to <i>LRRC4</i> expression in normal brain and WHO grade I, II, III astrocytomas, and grade IV glioblastoma. (C) The total gray value of (B). Image analysis and total gray value were estimated by the GSM-2000P pathology image analysis system.</p

<i>LRRC4</i> is a target gene of miR-182.

<p>(A) Schema of the interaction sites between miR-182 and the 3′-UTRs of <i>LRRC4</i> (B) (B) Luciferase assay of U251 glioma cells co-transfected with pMIR-REPORT–WT/mutant 3′-UTR <i>LRRC4</i> and miR-182 or scrambled control as indicated. * <i>p</i><0.05. (C) Western blot showing the protein expression of <i>LRRC4</i> after miR-182 was transfected into U251/L cells for 48 h. miR-182 mimics inhibited the protein expression of <i>LRRC4</i>. GAPDH was used as a loading control. (D) qRT-PCR showing the mRNA level of <i>LRRC4</i> after miR-182 mimic was transfected into U251/L cells for 48 h. miR-182 mimic down-regulated the mRNA level of <i>LRRC4</i>. * <i>p</i><0.05.</p