Journal: Journal of experimental & clinical cancer research : CR

Article Title: Inhibition of EZH2 via activation of SAPK/JNK and reduction of p65 and DNMT1 as a novel mechanism in inhibition of human lung cancer cells by polyphyllin I.

doi: 10.1186/s13046-016-0388-x

Figure Lengend Snippet: Fig. 3 PPI decreased protein expression of DNMT1 and EZH2 through SAPK/JNK pathway. a PC9 and A549 cells were exposed to increased concentration of PPI for 24 h. Afterwards, the expression of EZH2 and DNMT1 protein were detected by Western blot. b PC9 and A549 cells were exposed to PPI (1.6 μM) for 24 h, followed by measuring the mRNA levels by qRT-PCR. c PC9 and A549 cells were transfected with a wild type human EZH2 promoter reporter construct ligated to luciferase reporter gene and internal control for 24 h, followed by treating with PPI for an additional 24 h. Afterwards, the promoter activities were determined using the Secrete-Pair Dual Luminescence Assay Kit as described in the Materials and Methods Section. Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. d PC9 and A549 cells were treated with SP600125 (20 μM) for 2 h before exposure of the cells to PPI (1.6 μM) for an additional 24 h. Afterwards, the expression of EZH2 and DNMT1 protein were detected by Western blot using antibodies against EZH2 and DNMT1. The bar graphs represent the mean ± SD of EZH2 or DNMT1/GAPDH of three independent experiments. *Indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significant difference from PPI treated alone (P < 0.05)

Article Snippet: In the separated experiment, 2 μg of the control (pCMV6) and expression constructs containing Myc-DDK-tagged- or Myc/FLAG-tagged ORFs of human DNMT1 or EZH2 obtained from OriGene Technologies, Inc. (Rockville, MD, USA), the control (pCMV4) and p65 overexpression vector (pCMV4-p65) obtained from the Addgene (Plasmid #21966, Cambridge, MA, USA) [34] at a final concentration of 2 μg/mL, were transfected into the cells with the Lipofectamine 3000 reagent.

Techniques: Expressing, Concentration Assay, Western Blot, Quantitative RT-PCR, Transfection, Construct, Luciferase, Control, Luminescence Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Inhibition of EZH2 via activation of SAPK/JNK and reduction of p65 and DNMT1 as a novel mechanism in inhibition of human lung cancer cells by polyphyllin I.

doi: 10.1186/s13046-016-0388-x

Figure Lengend Snippet: Fig. 4 Activation of SAPK/JNK pathway-mediated inhibition of p65 expression contributed to the PPI-decreased protein expression of DNMT1. a PC9 and A549 cells were exposed to increased concentration of PPI for 24 h, followed by measuring the protein expression of p65 and p50 by Western blot. The bar graphs represent the mean ± SD of p65/GAPDH of three independent experiments. b PC9 and A549 cells were treated with SP600125 (20 μM) for 2 h before exposure of the cells to PPI (1.6 μM) for an additional 24 h. Afterwards, the expression of phosphorylation of SAPK/JNK and p65 protein were detected by Western blot. c PC9 and A549 cells were transfected with control and p65 overexpression vector for 24 h before exposing the cells to PPI for an additional 24 h. Afterwards, p65 and DNMT1 protein expressions were determined using Western blot. Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. *Indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significant difference from PPI treated alone (P < 0.05)

Article Snippet: In the separated experiment, 2 μg of the control (pCMV6) and expression constructs containing Myc-DDK-tagged- or Myc/FLAG-tagged ORFs of human DNMT1 or EZH2 obtained from OriGene Technologies, Inc. (Rockville, MD, USA), the control (pCMV4) and p65 overexpression vector (pCMV4-p65) obtained from the Addgene (Plasmid #21966, Cambridge, MA, USA) [34] at a final concentration of 2 μg/mL, were transfected into the cells with the Lipofectamine 3000 reagent.

Techniques: Activation Assay, Inhibition, Expressing, Concentration Assay, Western Blot, Phospho-proteomics, Transfection, Control, Over Expression, Plasmid Preparation

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Inhibition of EZH2 via activation of SAPK/JNK and reduction of p65 and DNMT1 as a novel mechanism in inhibition of human lung cancer cells by polyphyllin I.

doi: 10.1186/s13046-016-0388-x

Figure Lengend Snippet: Fig. 5 Exogenously expressed EZH2 not only restored cell growth, but also feedback antagonized PPI increased SAPK/JNK signaling. a PC9 and A549 cells were transfected with the control or expression constructs of DNMT1 for 24 h before exposing the cells to PPI for an additional 24 h. Afterwards, EZH2 and DNMT1 protein expression were determined using Western blot. b–c PC9 and A549 cells were transfected with the control or expression constructs of EZH2 for 24 h before exposing the cells to PPI for an additional 24 h. Afterwards, EZH2 and DNMT1 protein expression (b) and cell viability (c) were determined using Western blot and MTT assays, respectively. d PC9 and A549 cells were transfected with the control or expression constructs of EZH2 for 24 h before exposing the cells to PPI for an additional 24 h. Afterwards, EZH2, phosphor-SAPK/JNK were determined using Western blot. Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. *Indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significant difference from PPI treated alone (P < 0.05)

Article Snippet: In the separated experiment, 2 μg of the control (pCMV6) and expression constructs containing Myc-DDK-tagged- or Myc/FLAG-tagged ORFs of human DNMT1 or EZH2 obtained from OriGene Technologies, Inc. (Rockville, MD, USA), the control (pCMV4) and p65 overexpression vector (pCMV4-p65) obtained from the Addgene (Plasmid #21966, Cambridge, MA, USA) [34] at a final concentration of 2 μg/mL, were transfected into the cells with the Lipofectamine 3000 reagent.

Techniques: Transfection, Control, Expressing, Construct, Western Blot

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Inhibition of EZH2 via activation of SAPK/JNK and reduction of p65 and DNMT1 as a novel mechanism in inhibition of human lung cancer cells by polyphyllin I.

doi: 10.1186/s13046-016-0388-x

Figure Lengend Snippet: Fig. 6 Effects of PPI in subcutaneous xenograft model. Mice (n = 12/group) were divided to 3 groups [Con (saline), Low (L, 1 mg/kg) and High (H, 3 mg/kg)], and PPI was given by intraperitoneal injection for up to 27 days. a The xenografts were assessed by in vivo bioluminescence imaging at the sixth and end of the experiments (on day 6 and 27). The tumor growth was monitored by injecting luciferin in the mice followed by measuring bioluminescence and analyzed as described in the Materials and Methods section. Representative images are shown. b and c The xenografts were harvested on day 27, and the size and weight of tumors were determined. The bar graphs represented the tumor weight and size of mice results of as mean ± SD. d–e At the end of the experiments, xenografted tumors were isolated from individual animals and the corresponding lysates were detected p-SAPK/JNK, DNMT1, p65 and EZH2 proteins by Western blot and Immunohistochemistry as described in the Materials and Methods sections. Scale bar 50 μM. GAPDH was used as loading control for Western blot. Values in bar graphs were given as the mean ± SD from three independent Western blot experiments. *Indicates the significant difference from untreated control (p < 0.05). f The diagram shows that PPI inhibits growth of NSCLC cells through SAPK/JNK-mediated inhibition of p65 and DNMT1, subsequently; this results in the reduction of EZH2 gene expression. The interactions among p65, DNMT1 and EZH2, and feedback regulation of SAPK/JNK by EZH2 converge on the overall responses of PPI

Article Snippet: In the separated experiment, 2 μg of the control (pCMV6) and expression constructs containing Myc-DDK-tagged- or Myc/FLAG-tagged ORFs of human DNMT1 or EZH2 obtained from OriGene Technologies, Inc. (Rockville, MD, USA), the control (pCMV4) and p65 overexpression vector (pCMV4-p65) obtained from the Addgene (Plasmid #21966, Cambridge, MA, USA) [34] at a final concentration of 2 μg/mL, were transfected into the cells with the Lipofectamine 3000 reagent.

Techniques: Saline, Injection, In Vivo, Imaging, Isolation, Western Blot, Immunohistochemistry, Control, Inhibition, Gene Expression

Journal: Cell reports

Article Title: Decreased KAT5 Expression Impairs DNA Repair and Induces Altered DNA Methylation in Kidney Podocytes.

doi: 10.1016/j.celrep.2019.01.005

Figure Lengend Snippet: Figure 7. KAT5 Increases Nephrin Expression through Reductions in DNA DSBs and DNA Methylation at the Promoter Region in Cultured Human Podocytes (A) (Left) Real-time RT-PCR analysis and (right) western blots of nephrin expression in cultured human podocytes with normal (6 mM) or high (30 mM) glucose treatment. (B) Methylation-specific PCR (MSP) analysis of the nephrin promoter region in cultured human podocytes with normal (6 mM) or high (30 mM) glucose treatment. (C) (Left) Real-time RT-PCR analysis and (right) western blots of nephrin expression in KAT5-overexpressed cultured human podocytes. (D) Methylation-specific PCR (MSP) analysis of the nephrin promoter region in KAT5 overexpressed cultured human podocytes. (E and F) Chromatin immunoprecipitation (ChIP) assay for DNMT1 (E) and DNMT3B (F) in the promoter region of nephrin in KAT5-overexpressed cultured human podocytes. (G) gH2AX foci was decreased in the nephrin promoter region by ChIP assay. Control, podocytes transfected with control vector. KAT5, KAT5-overexpressing podocytes. Experiments were performed in podocytes cultured with 30 mM of D-glucose (C–G). n = 6 in each group. Data represent mean ± SEM. *p < 0.05 and **p < 0.01 versus controls by two-tailed Student’s t test.

Article Snippet: pCMV-SPORT6-KAT5 and pCMV-SPORT6 were purchased from the RIKEN Center for Life Science Technologies (Hyogo, Japan), DNMT1 cDNA clone (SC325419) was purchased from Origene and transient transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

Techniques: Expressing, DNA Methylation Assay, Cell Culture, Quantitative RT-PCR, Western Blot, Methylation, Chromatin Immunoprecipitation, Control, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Molecular cancer

Article Title: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

doi: 10.1186/1476-4598-12-99

Figure Lengend Snippet: Figure 1 Mahanine restores RASSF1A expression by demethylating its promoter and all three DNMTs control RASSF1A expression. A. PC3 cells were treated with DMSO control or 10 μM mahanine for 1 and 3 days. Methylation-specific PCR was performed to detect the methylated (M) and un-methylated (UM) status of RASSF1A promoter. B. PC3 cells were treated with DMSO control or 10 μM mahanine for 1 and 3 days, following which RASSF1A expression was assessed by RT-PCR. GAPDH was used as an internal control. C. PC3 cells were transfected with shRNA for DNMT1, DNMT3A, DNMT3B or scrambled shRNA. Forty-eight hours after transfection, cells were harvested for RT-PCR analyses to assess RASSF1A expression. GAPDH was used as an internal control. For DNMT3A, two shRNAs were used to confirm the result. D. BPH1 cells were transfected with expression vectors of DNMT1, DNMT3A, DNMT3B or empty vector control. Forty-eight hours after transfection cells were collected for RT-PCR analyses to determine RASSF1A, DNMT1, DNMT3A and DNMT3B expression levels. GAPDH was used as an internal control.

Article Snippet: PC3 cells were transfected with DNMT shRNAs (OriGene Technologies Inc., Rockville, MD) and RASS F1A expression vector [34].

Techniques: Expressing, Control, Methylation, Reverse Transcription Polymerase Chain Reaction, Transfection, shRNA, Plasmid Preparation

Journal: Molecular cancer

Article Title: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

doi: 10.1186/1476-4598-12-99

Figure Lengend Snippet: Figure 2 Mahanine specifically down-regulates DNMT1 and DNMT3B. A. DNMT1, DNMT3A and DNMT3B cellular localization was visualized by immunofluorescent staining. PC3 cells were treated with DMSO (as control) or mahanine (10 μM) for 24 hours, following which they were fixed in methanol, incubated with the indicated antibodies, stained with Alexa Fluor 488-tagged secondary antibodies and counterstained with propidium iodide. Slides were then mounted and examined under a fluorescence microscope. The bright field images of PC3 cells treated with DMSO or mahanine (10 μM) for 24 hours are shown (right panel). B. Cytoplasmic and nuclear fractions were separated from PC3 cells treated with DMSO or 10 μM mahanine for 24 hours. The isolated fractions were subjected to Western blot analysis to assess DNMT expression. The fold change in the expression of the respective DNMTs as compared to the control is indicated at the bottom of each immunoblot. Nucleolin and β-actin were used as loading controls for the nuclear and cytoplasmic fractions, respectively. C. PC3 and LNCaP cells were treated as indicated with DMSO or mahanine following which cells were lysed and the extracts were subjected to Western blot analysis to detect DNMT1, DNMT3B and DNMT3A protein levels (left). Quantitative estimations of the relative levels of DNMT1, DNMT3A and DNMT3B proteins were determined by densitometric measurements of immunoblots from three independent experiments after normalization with β-actin (right). Columns, mean; bars, SEM. *p < 0.05, significantly different from control. D. PC3 and LNCaP cells were treated with DMSO or 10 and 20 μM mahanine, respectively for 24 hours. Subsequently, cells were harvested for RT-PCR analysis to measure DNMT1 and DNMT3B expression. GAPDH was used as an internal control.

Article Snippet: PC3 cells were transfected with DNMT shRNAs (OriGene Technologies Inc., Rockville, MD) and RASS F1A expression vector [34].

Techniques: Staining, Control, Incubation, Fluorescence, Microscopy, Isolation, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular cancer

Article Title: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

doi: 10.1186/1476-4598-12-99

Figure Lengend Snippet: Figure 3 Mahanine degrades DNMTs via the ubiquitin-proteasomal pathway. A. PC3 cells were treated with 10 μM mahanine and 20 μM Z-VAD-FMK for 24 hours after which cellular protein lysates were subjected to Western blot analysis to detect DNMT1 and DNMT3B protein levels, β-actin was used as a loading control. B. Chymotrypsin-like proteasomal activity was measured in PC3 cells treated as indicated with mahanine and MG132 for 24 hours. Columns, mean; bars, SEM. *p < 0.05, significantly different from DMSO control. C. LNCaP and PC3 cells were treated with the indicated doses of mahanine for 24 hours with or without MG132 (5 μM). Cell lysates were analyzed for DNMT1 and DNMT3B expression by Western blot. β-actin was used as a loading control. D. PC3 cells were treated with MG132 (5 μM) in the absence and presence of mahanine (10 μM) for 24 hours. Cell lysates were subjected to immunoprecipitation (IP) of DNMT1 or DNMT3B and immunoblotted (IB) for poly-ubiquitin, DNMT1 and DNMT3B.

Article Snippet: PC3 cells were transfected with DNMT shRNAs (OriGene Technologies Inc., Rockville, MD) and RASS F1A expression vector [34].

Techniques: Ubiquitin Proteomics, Western Blot, Control, Activity Assay, Expressing, Immunoprecipitation

Journal: Molecular cancer

Article Title: Mahanine restores RASSF1A expression by down-regulating DNMT1 and DNMT3B in prostate cancer cells.

doi: 10.1186/1476-4598-12-99

Figure Lengend Snippet: Figure 6 Mahanine restores RASSF1A expression by degrading DNMTs via Akt. Prostate cancer cells express high levels of activated Akt, which phosphorylates and stabilizes DNMT1 and DNMT3B against proteasomal degradation. DNMTs enter the nucleus and methylate the promoter of RASSF1A gene to silence the expression of RASSF1A. Treatment of mahanine inhibits PDK1 and thereby prevents activation of Akt, which in turn compromises the stability of DNMTs, increases their ubiquitination and induces proteasomal degradation. In the absence of DNMT1 and DNMT3B, the RASSF1A promoter is demethylated and its expression is restored in prostate cancer cells. GF: Growth factor; RTK: Receptor tyrosine kinase; TFs: Transcription factors; P: Phosphorylated; M: Methylated; Ub: Ubiquitinated.

Article Snippet: PC3 cells were transfected with DNMT shRNAs (OriGene Technologies Inc., Rockville, MD) and RASS F1A expression vector [34].

Techniques: Expressing, Activation Assay, Ubiquitin Proteomics, Methylation

Journal: The American Journal of Pathology

Article Title: miR-185 Inhibits Hepatocellular Carcinoma Growth by Targeting the DNMT1/PTEN/Akt Pathway

doi: 10.1016/j.ajpath.2014.05.004

Figure Lengend Snippet: miR-185 targets DNMT1 in HCC cells. Huh7 (A) and HepG2 (B) cells were transiently transfected with miR-185 mimic and miRNA scramble control oligonucleotide. The cell lysates were obtained for Western blot analysis to measure DNMT1 protein levels. DNMT1 protein expression was analyzed with β-actin as an internal control. The experiments were repeated five times (N = 5). Normalized fold expression of DNMT1 in Huh7 cells transfected with miR-185 mimic was compared with scramble control as depicted in bar graphs. Data are given as means ± SD. ∗P < 0.05.

Article Snippet: DNMT1 3′ untranslated region (3′-UTR) luciferase reporter plasmid was purchased from OriGene Technologies Inc. (Rockville, MD).

Techniques: Transfection, Control, Western Blot, Expressing

Journal: The American Journal of Pathology

Article Title: miR-185 Inhibits Hepatocellular Carcinoma Growth by Targeting the DNMT1/PTEN/Akt Pathway

doi: 10.1016/j.ajpath.2014.05.004

Figure Lengend Snippet: miR-185 targets DNMT1 and inhibits HCC proliferation and invasion in vitro. A: Real-time PCR quantification of miR-185 showed a significant increase in Huh7 cells stably transduced with L/miR-185 compared with cells stably transduced with L/Control. B: Western blot analysis for DNMT1 in Huh7 cells with or without stable overexpression of miR-185. Representative Western blots and average densitometry data. The level of DNMT1 protein is decreased in Huh7 cells stably expressing miR-185 (gray bars) compared with control cells (white bars). C: Cell proliferation was measured at 24, 48, and 72 hours in Huh7 stable cells after plating 1 × 103 per well in 6-well plates. miR-185 overexpression decreased cell proliferation at all the time points. D: A cell invasion assay was performed in Huh7 stable cells. Huh7 miR-185 stable cells showed an approximately 50% reduction in the number of invaded cells on the matrix membrane surface compared with control after 24 hours. The images depict invaded cells. Data are given as means ± SD. N = 5 (B). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Article Snippet: DNMT1 3′ untranslated region (3′-UTR) luciferase reporter plasmid was purchased from OriGene Technologies Inc. (Rockville, MD).

Techniques: In Vitro, Real-time Polymerase Chain Reaction, Stable Transfection, Transduction, Control, Western Blot, Over Expression, Expressing, Invasion Assay, Membrane

Journal: The American Journal of Pathology

Article Title: miR-185 Inhibits Hepatocellular Carcinoma Growth by Targeting the DNMT1/PTEN/Akt Pathway

doi: 10.1016/j.ajpath.2014.05.004

Figure Lengend Snippet: miR-185 decreases DNMT 3′-UTR luciferase reporter activity in HCC cells. A: The putative miR-185 binding site in DNMT1 3′-UTR is depicted in the top sequence pairs. The original strand of DNMT1 3′-UTR contains the 7-Bp seed sequences that bind to miR-185; the DNMT1 3′-UTR mutant strand shows deletion of the 7-Bp seed sequence. B:DNMT1 3′-UTR luciferase reporter acidity assay in Huh7 stable cells transfected with wild-type or mutant DNMT1 3′-UTR reporter construct. Data are given as means ± SD. N = 3. ∗P < 0.05.

Article Snippet: DNMT1 3′ untranslated region (3′-UTR) luciferase reporter plasmid was purchased from OriGene Technologies Inc. (Rockville, MD).

Techniques: Luciferase, Activity Assay, Binding Assay, Sequencing, Mutagenesis, Transfection, Construct

Journal: The American Journal of Pathology

Article Title: miR-185 Inhibits Hepatocellular Carcinoma Growth by Targeting the DNMT1/PTEN/Akt Pathway

doi: 10.1016/j.ajpath.2014.05.004

Figure Lengend Snippet: Restoration of DNMT1 prevents miR-185–induced inhibition of HCC cell proliferation and invasion. A: Western blot analysis for DNMT1, PTEN, p-Akt, and Akt in Huh7 stable cells with or without DNMT1 overexpression. B: Cell proliferation analysis of Huh7 stable cells with or without DNMT1 overexpression. The experiments were performed 72 hours after plating 1 × 103 per well in 6-well plates. The experiments were repeated three times. C: Cell invasion assay of Huh7 stable cells with or without DNMT1 overexpression. The numbers of invaded cells were counted 24 hours after cell plating. The images depict invaded cells. Data are given as means ± SD. N = 5 (A); N = 3 (B and C). ∗P < 0.05.

Article Snippet: DNMT1 3′ untranslated region (3′-UTR) luciferase reporter plasmid was purchased from OriGene Technologies Inc. (Rockville, MD).

Techniques: Inhibition, Western Blot, Over Expression, Invasion Assay

Journal: The American Journal of Pathology

Article Title: miR-185 Inhibits Hepatocellular Carcinoma Growth by Targeting the DNMT1/PTEN/Akt Pathway

doi: 10.1016/j.ajpath.2014.05.004

Figure Lengend Snippet: miR-185 overexpression inhibits HCC growth in SCID mice. An Huh7 stable cell suspension mixed in Matrigel solution was inoculated into SCID mice via direct liver injections. A: Gross images depict liver tumors that originated from miR-185 overexpressed or control cells. Local liver tumors that originated from miR-185–transduced Huh7 cells are significantly smaller (11-fold decrease) in tumor volume compared with control. B: Ki-67 immunostaining showed a lower proliferative rate in miR-185 overexpressed tumors compared with the control. C: Western blot analysis for DNMT1, PTEN, and p-Akt in miR-185 overexpressed or control tumors recovered from the livers of SCID mice. Normalized fold expressions for DNMT1, PTEN, and p-Akt are depicted in bar graphs. D: Schematic illustration of miR-185–induced inhibition of HCC growth. Data are given as means ± SD. N = 12 (A); N = 5 (C). ∗P < 0.05.

Article Snippet: DNMT1 3′ untranslated region (3′-UTR) luciferase reporter plasmid was purchased from OriGene Technologies Inc. (Rockville, MD).

Techniques: Over Expression, Stable Transfection, Suspension, Control, Immunostaining, Western Blot, Inhibition

Journal: The American Journal of Pathology

Article Title: miR-185 Inhibits Hepatocellular Carcinoma Growth by Targeting the DNMT1/PTEN/Akt Pathway

doi: 10.1016/j.ajpath.2014.05.004

Figure Lengend Snippet: miR-185 targets DNMT1 in HCC cells. Huh7 (A) and HepG2 (B) cells were transiently transfected with miR-185 mimic and miRNA scramble control oligonucleotide. The cell lysates were obtained for Western blot analysis to measure DNMT1 protein levels. DNMT1 protein expression was analyzed with β-actin as an internal control. The experiments were repeated five times (N = 5). Normalized fold expression of DNMT1 in Huh7 cells transfected with miR-185 mimic was compared with scramble control as depicted in bar graphs. Data are given as means ± SD. ∗P < 0.05.

Article Snippet: DNMT1 3′-UTR Luciferase Reporter Assay DNMT1 3′-UTR luciferase reporter plasmid was obtained from OriGene Technologies Inc. Mutant DNMT1 3′-UTR luciferase reporter plasmid was constructed by cloning synthetic DNMT1 3′-UTR nucleotide (with deletion of the 7 bp of hsa–miR-185 seed sequence) into the pMirTarget vector.

Techniques: Transfection, Control, Western Blot, Expressing

Journal: The American Journal of Pathology

Article Title: miR-185 Inhibits Hepatocellular Carcinoma Growth by Targeting the DNMT1/PTEN/Akt Pathway

doi: 10.1016/j.ajpath.2014.05.004

Figure Lengend Snippet: miR-185 targets DNMT1 and inhibits HCC proliferation and invasion in vitro. A: Real-time PCR quantification of miR-185 showed a significant increase in Huh7 cells stably transduced with L/miR-185 compared with cells stably transduced with L/Control. B: Western blot analysis for DNMT1 in Huh7 cells with or without stable overexpression of miR-185. Representative Western blots and average densitometry data. The level of DNMT1 protein is decreased in Huh7 cells stably expressing miR-185 (gray bars) compared with control cells (white bars). C: Cell proliferation was measured at 24, 48, and 72 hours in Huh7 stable cells after plating 1 × 103 per well in 6-well plates. miR-185 overexpression decreased cell proliferation at all the time points. D: A cell invasion assay was performed in Huh7 stable cells. Huh7 miR-185 stable cells showed an approximately 50% reduction in the number of invaded cells on the matrix membrane surface compared with control after 24 hours. The images depict invaded cells. Data are given as means ± SD. N = 5 (B). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Article Snippet: DNMT1 3′-UTR Luciferase Reporter Assay DNMT1 3′-UTR luciferase reporter plasmid was obtained from OriGene Technologies Inc. Mutant DNMT1 3′-UTR luciferase reporter plasmid was constructed by cloning synthetic DNMT1 3′-UTR nucleotide (with deletion of the 7 bp of hsa–miR-185 seed sequence) into the pMirTarget vector.

Techniques: In Vitro, Real-time Polymerase Chain Reaction, Stable Transfection, Transduction, Control, Western Blot, Over Expression, Expressing, Invasion Assay, Membrane

Journal: The American Journal of Pathology

Article Title: miR-185 Inhibits Hepatocellular Carcinoma Growth by Targeting the DNMT1/PTEN/Akt Pathway

doi: 10.1016/j.ajpath.2014.05.004

Figure Lengend Snippet: miR-185 decreases DNMT 3′-UTR luciferase reporter activity in HCC cells. A: The putative miR-185 binding site in DNMT1 3′-UTR is depicted in the top sequence pairs. The original strand of DNMT1 3′-UTR contains the 7-Bp seed sequences that bind to miR-185; the DNMT1 3′-UTR mutant strand shows deletion of the 7-Bp seed sequence. B:DNMT1 3′-UTR luciferase reporter acidity assay in Huh7 stable cells transfected with wild-type or mutant DNMT1 3′-UTR reporter construct. Data are given as means ± SD. N = 3. ∗P < 0.05.

Article Snippet: DNMT1 3′-UTR Luciferase Reporter Assay DNMT1 3′-UTR luciferase reporter plasmid was obtained from OriGene Technologies Inc. Mutant DNMT1 3′-UTR luciferase reporter plasmid was constructed by cloning synthetic DNMT1 3′-UTR nucleotide (with deletion of the 7 bp of hsa–miR-185 seed sequence) into the pMirTarget vector.

Techniques: Luciferase, Activity Assay, Binding Assay, Sequencing, Mutagenesis, Transfection, Construct

Journal: The American Journal of Pathology

Article Title: miR-185 Inhibits Hepatocellular Carcinoma Growth by Targeting the DNMT1/PTEN/Akt Pathway

doi: 10.1016/j.ajpath.2014.05.004

Figure Lengend Snippet: Restoration of DNMT1 prevents miR-185–induced inhibition of HCC cell proliferation and invasion. A: Western blot analysis for DNMT1, PTEN, p-Akt, and Akt in Huh7 stable cells with or without DNMT1 overexpression. B: Cell proliferation analysis of Huh7 stable cells with or without DNMT1 overexpression. The experiments were performed 72 hours after plating 1 × 103 per well in 6-well plates. The experiments were repeated three times. C: Cell invasion assay of Huh7 stable cells with or without DNMT1 overexpression. The numbers of invaded cells were counted 24 hours after cell plating. The images depict invaded cells. Data are given as means ± SD. N = 5 (A); N = 3 (B and C). ∗P < 0.05.

Article Snippet: DNMT1 3′-UTR Luciferase Reporter Assay DNMT1 3′-UTR luciferase reporter plasmid was obtained from OriGene Technologies Inc. Mutant DNMT1 3′-UTR luciferase reporter plasmid was constructed by cloning synthetic DNMT1 3′-UTR nucleotide (with deletion of the 7 bp of hsa–miR-185 seed sequence) into the pMirTarget vector.

Techniques: Inhibition, Western Blot, Over Expression, Invasion Assay

Journal: The American Journal of Pathology

Article Title: miR-185 Inhibits Hepatocellular Carcinoma Growth by Targeting the DNMT1/PTEN/Akt Pathway

doi: 10.1016/j.ajpath.2014.05.004

Figure Lengend Snippet: miR-185 overexpression inhibits HCC growth in SCID mice. An Huh7 stable cell suspension mixed in Matrigel solution was inoculated into SCID mice via direct liver injections. A: Gross images depict liver tumors that originated from miR-185 overexpressed or control cells. Local liver tumors that originated from miR-185–transduced Huh7 cells are significantly smaller (11-fold decrease) in tumor volume compared with control. B: Ki-67 immunostaining showed a lower proliferative rate in miR-185 overexpressed tumors compared with the control. C: Western blot analysis for DNMT1, PTEN, and p-Akt in miR-185 overexpressed or control tumors recovered from the livers of SCID mice. Normalized fold expressions for DNMT1, PTEN, and p-Akt are depicted in bar graphs. D: Schematic illustration of miR-185–induced inhibition of HCC growth. Data are given as means ± SD. N = 12 (A); N = 5 (C). ∗P < 0.05.

Article Snippet: DNMT1 3′-UTR Luciferase Reporter Assay DNMT1 3′-UTR luciferase reporter plasmid was obtained from OriGene Technologies Inc. Mutant DNMT1 3′-UTR luciferase reporter plasmid was constructed by cloning synthetic DNMT1 3′-UTR nucleotide (with deletion of the 7 bp of hsa–miR-185 seed sequence) into the pMirTarget vector.

Techniques: Over Expression, Stable Transfection, Suspension, Control, Immunostaining, Western Blot, Inhibition