Journal: EBioMedicine

Article Title: LncRNA HOTAIR epigenetically suppresses miR-122 expression in hepatocellular carcinoma via DNA methylation

doi: 10.1016/j.ebiom.2018.08.055

Figure Lengend Snippet: HOTAIR epigenetically suppressed miR-122 through DNMTs in HCC cells. (a–g) DNMT1, DNMT3A, and DNMT3B expression were suppressed by siHOTAIR-1 and promoted by pHOTAIR at both mRNA (a–c) and protein (d–g) levels in HCC cells. (h) either siDNMT1, or siDNMT3A, or siDNMT3B increased miR-122 expression in HCC cells. (i) either siDNMT1, or siDNMT3A, or siDNMT3B rescued the HOTAIR-induced miR-122 suppression in HCC cells. (j) knockdown of HOTAIR caused a significant reduction of DNMTs binding to the miR-122 promoter region in HCC cells analyzed by ChIP assay. Data are representative of three independent experiments and are presented as mean ± SD. *P < 0.05, **P < 0.01 (Student's t-test).

Article Snippet: Human DNMT1-siRNA, DNMT3A-siRNA and DNMT3B-siRNA were purchased from Origene.

Techniques: Expressing, Binding Assay

Journal: EBioMedicine

Article Title: LncRNA HOTAIR epigenetically suppresses miR-122 expression in hepatocellular carcinoma via DNA methylation

doi: 10.1016/j.ebiom.2018.08.055

Figure Lengend Snippet: EZH2 mediated HOTAIR-induced DNMTs expression in HCC cells. (a–e) knockdown of EZH2 by siEZH2-1 downregulated the expression levels of DNMT1, DNMT3A, and DNMT3B mRNA (a–c) and protein (d–e) in HCC cells. (f–j) siEZH2-1 reversed the upregulation of DNMTs mRNA (f–h) and protein (i–j) expression induced by HOTAIR in HCC cells. (k) siEZH2-1 abrogated the suppression of miR-122 induced by HOTAIR in HCC cells. Data are representative of three independent experiments and are presented as mean ± SD. *P < 0.05, **P < 0.01 (Student's t-test).

Article Snippet: Human DNMT1-siRNA, DNMT3A-siRNA and DNMT3B-siRNA were purchased from Origene.

Techniques: Expressing

Journal: EBioMedicine

Article Title: LncRNA HOTAIR epigenetically suppresses miR-122 expression in hepatocellular carcinoma via DNA methylation

doi: 10.1016/j.ebiom.2018.08.055

Figure Lengend Snippet: HOTAIR expression negatively correlated with miR-122 and positively correlated with CCNG1 in HCC. (a–c) the statistically significant association between HOTAIR, miR-122 and CCNG1 expression in HCC specimens. (d–f) the expression of HOTAIR, CCNG1, DNMT1, DNMT3A, DNMT3B, and EZH2 in xenograft tumors were analyzed by qRT-PCR and WB. (g) the schematic overview of HOTAIR/miR-122 mediated tumorigenesis in HCC. Data are representative of three independent experiments and are presented as mean ± SD. *P < 0.05, **P < 0.01 (Student's t-test).

Article Snippet: Human DNMT1-siRNA, DNMT3A-siRNA and DNMT3B-siRNA were purchased from Origene.

Techniques: Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Male-Specific Long Noncoding RNA TTTY15 Inhibits Non-Small Cell Lung Cancer Proliferation and Metastasis via TBX4

doi: 10.3390/ijms20143473

Figure Lengend Snippet: TTTY15 interacts with DNA methyltransferases (DNMT) 3A and decreases the binding of DNMT3A to the TBX4 promoter. ( A ) Cell fractionation assays were performed to determine the distribution of TTTY15 expression between the cell cytoplasm and nucleus. ( B ) TTTY15 RNA coimmunoprecipitated with DNMT1, DNMT3A, or IgG was quantified by qRT-PCR. PCR products were then loaded onto a 3% agarose gel for confirmation. ( C ) DNMT3A turned out to be upregulated in human NSCLC samples compared to the adjacent normal tissue samples (n = 37 for each group). ( D ) The knockdown of DNMT3A increased the expression levels of TBX4 in A549 and H441 cells. ( E ) The knockdown of TTTY15 did not affect the protein expression levels of DNMT3A. ( F ) Chromatin immunoprecipitation (ChIP)-qPCR was performed to quantify the binding of DNMT3A to the TBX4 promoter (P1: −1734 to −1626 bp; P2: −740 to −672 bp). ( G ) Methylation-specific PCR was performed to analyze the methylation status on the CpG islands of the TBX4 promoter in A549/scramble and A549/sh TTTY15 (Me: methylated; Un: unmethylated; H 2 O was used as a negative control). Data are presented as the mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical analysis was conducted by Student’s t -test ( n = 3).

Article Snippet: The siRNAs for targeting TBX4 and DNMT3A mRNA were constructed by MDBio Inc. (Taipei, Taiwan).

Techniques: Binding Assay, Cell Fractionation, Expressing, Quantitative RT-PCR, Agarose Gel Electrophoresis, Chromatin Immunoprecipitation, Methylation, Negative Control

Journal: Nature Communications

Article Title: Obesity-induced DNA hypermethylation of the adiponectin gene mediates insulin resistance

doi: 10.1038/ncomms8585

Figure Lengend Snippet: ( a ) Dnmt1 mRNA levels in adipocytes from NCD- ( n =4) or HFD-fed ( n =3) mice and in adipocytes from WT ( n =4) or db/db mice ( n =4). ( b ) Correlation between body mass index and DNMT1 mRNA levels in human adipocytes. mRNA levels were measured by qPCR. r 2 and P values are indicated on the graph. ( c – e ) DNMT1 was suppressed by small interfering RNA in 3T3-L1 cells ( n =3). ( c ) Dnmt1 and adiponectin mRNA levels. mRNA levels were measured by qPCR. ( d ) Adiponectin protein levels were determined by western blot analysis. ( e ) Bisulfite sequencing data at the R2. ( f – h ) DNMT1 overexpression in differentiated 3T3-L1 cells ( n =3). ( f , g ) Dnmt1 and adiponectin mRNA and protein levels were measured by qPCR and western blot analysis. ( h ) Degree of R2 DNA methylation was examined by bisulfite sequencing. ( i ) AluI restriction sites in R2. Red and grey arrows indicate the AluI restriction sites and CpG locations in the R2, respectively. Double headed arrow points to PCR amplified region. ( j ) Restriction enzyme accessibility assay in adipocytes from HFD-fed ( n =5) or NCD-fed ( n =5) mice. EcoRI and BamHI, which do not digest R2, were used as negative controls. Results are expressed as the mean±s.e.m. Similar results were obtained at least more than three independent experiments. * P <0.05; ** P <0.01; *** P <0.001 in a two-tailed Student's t- test. siNC; negative control siRNA. See for full-length images of blots.

Article Snippet: Small interfering RNA duplexes were designed and purchased from Bioneer (DNMT1, 1350629; DNMT3a, 1350650). pcDNA3-DNMT1 vector was kindly donated by Dr Francois Fuks (Free University of Brussels, Belgium).

Techniques: Small Interfering RNA, Western Blot, Methylation Sequencing, Over Expression, DNA Methylation Assay, Amplification, Two Tailed Test, Negative Control

Journal: Nature Communications

Article Title: Obesity-induced DNA hypermethylation of the adiponectin gene mediates insulin resistance

doi: 10.1038/ncomms8585

Figure Lengend Snippet: ( a – d ) 3T3-L1 adipocytes were pretreated with DMSO (white bars) or RG108 (blue bars; 100 μM) for 24 h before TNFα treatment (hatched bars; 10 ng ml −1 ) for 24 h ( n =3). ( a ) Relative DNMT enzymatic activity. ( b ) mRNA levels of Dnmt1 , adiponectin and Mcp-1 . mRNA levels were measured by qPCR. ( c , d ) Bisulfite sequencing results of the adiponectin promoter R2 ( c ) and R1 ( d ) in 3T3-L1 cells treated with TNFα. Quantification of the 5-mC levels in the adiponectin promoter R2 and R1. ( e , f ) In 3T3-L1 adipocytes, DNMT1 was suppressed by small interfering RNA. The cells were then incubated with or without TNFα (10 ng ml −1 ) for 24 h ( n =3). ( e ) mRNA levels of adiponectin, Dnmt1 and Mcp-1 in negative control (NC) or DNMT1 suppressed 3T3-L1 adipocytes. mRNA levels were measured by qPCR. ( f ) R2 DNA methylation levels were measured by bisulfite sequencing. ( g , h ) 3T3-L1 adipocytes were pretreated with DMSO (white bars) or RG108 (blue bars; 100 μM) for 24 h before TNFα treatment (hatched bars; 10 ng ml −1 ) for 24 h ( n =3). ( g ) R2 ChIP analysis. Quantification of DNMT1 and MeCP2 relative recruitment and H3K9Ac levels using qPCR. ( h ) Restriction enzyme accessibility assay. After restriction with endonucleases, purified gDNA was amplified and quantified using qPCR. All results are expressed as mean±s.e.m. * P <0.05; ** P <0.01; *** P <0.001 in a two-tailed Student's t -test. Cntl, control.

Article Snippet: Small interfering RNA duplexes were designed and purchased from Bioneer (DNMT1, 1350629; DNMT3a, 1350650). pcDNA3-DNMT1 vector was kindly donated by Dr Francois Fuks (Free University of Brussels, Belgium).

Techniques: Activity Assay, Methylation Sequencing, Small Interfering RNA, Incubation, Negative Control, DNA Methylation Assay, Purification, Amplification, Two Tailed Test, Control