Journal: PLoS ONE

Article Title: miR-126&126* Restored Expressions Play a Tumor Suppressor Role by Directly Regulating ADAM9 and MMP7 in Melanoma

doi: 10.1371/journal.pone.0056824

Figure Lengend Snippet: A) Representative WB analysis of ADAM9 and MMP7 target genes (left); cell growth proliferation analysis (right). B) WB analysis of ADAM9 and MMP7 (left), cell growth proliferation (middle) and migration (right). Actin was utilized as internal loading control.

Article Snippet: ADAM9 and MMP7 silencing was performed by using 4 unique 29-mer shRNA constructs in retroviral GFP vector, #TG314947 for ADAM9, #TG311438 for MMP7 and #TR30013 for scrambled negative control (OriGene Technologies, Rockville, MD, USA). miRCURY locked-nucleic-acid (LNA) Power Inhibitor knockdown probes for miR-126 and -126* were obtained from Exiqon (Copenhagen, Denmark), (Product numbers #426717-00 and #426718-00).

Techniques: Migration

Journal: PLoS ONE

Article Title: miR-126&126* Restored Expressions Play a Tumor Suppressor Role by Directly Regulating ADAM9 and MMP7 in Melanoma

doi: 10.1371/journal.pone.0056824

Figure Lengend Snippet: Representative Western blot of A) ADAM9, MMP7 and OPN in normal human melanocytes (NHEM) and in a panel of melanoma cell lines, B) ADAM9 isoforms (left), MMP7 and OPN (middle) and corresponding secreted forms (right) in miR-126&126* versus empty vector-transduced Me665/1 and A375M cell lines. C) Representative Real-time PCR analysis of ADAM9 (left), MMP7 (middle) and OPN (right) mRNAs in the A375M cell line. The unresponsive short isoform of ADAM9 mRNA does not carry miR-126&126* binding sites in its 3′UTR. D) Relative expression values obtained by western blot analysis of ADAM9 (left), MMP7 (middle) and OPN (right) in A375M cells transfected with oligomers mimicking mature miR-126 or miR-126* vs non targeting (no Targ); GAPDH and actin were internal loading controls in RT-PCR and WB, respectively. Columns, of a minimum of two independent experiments; ** P <0.001; * P <0.05.

Article Snippet: ADAM9 and MMP7 silencing was performed by using 4 unique 29-mer shRNA constructs in retroviral GFP vector, #TG314947 for ADAM9, #TG311438 for MMP7 and #TR30013 for scrambled negative control (OriGene Technologies, Rockville, MD, USA). miRCURY locked-nucleic-acid (LNA) Power Inhibitor knockdown probes for miR-126 and -126* were obtained from Exiqon (Copenhagen, Denmark), (Product numbers #426717-00 and #426718-00).

Techniques: Western Blot, Plasmid Preparation, Real-time Polymerase Chain Reaction, Binding Assay, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: miR-126&126* Restored Expressions Play a Tumor Suppressor Role by Directly Regulating ADAM9 and MMP7 in Melanoma

doi: 10.1371/journal.pone.0056824

Figure Lengend Snippet: A) Luciferase reporter assays performed by transfecting a Luc reporter gene (psiCHECK2) linked to 3′-UTR of ADAM9 or MMP7 or OPN or PI3KR2 in miR- versus empty vector-transduced A375M cell lines. B) Schematic presentation of predicted miR-126 and miR-126* target sites identified in the ADAM9 3′UTR (left) and relative miR-126&126*-dependent luciferase activities (right) in presence of wild-type (WT) or mutant (mut) binding sites in the-ADAM9 3′UTR. C) The same schematic representation (left) and luciferase experiments (right) carried out on MMP7 3′UTR. Columns, of minimum of 5 experiments per group; ** P <0.001; * P <0.05.

Article Snippet: ADAM9 and MMP7 silencing was performed by using 4 unique 29-mer shRNA constructs in retroviral GFP vector, #TG314947 for ADAM9, #TG311438 for MMP7 and #TR30013 for scrambled negative control (OriGene Technologies, Rockville, MD, USA). miRCURY locked-nucleic-acid (LNA) Power Inhibitor knockdown probes for miR-126 and -126* were obtained from Exiqon (Copenhagen, Denmark), (Product numbers #426717-00 and #426718-00).

Techniques: Luciferase, Plasmid Preparation, Mutagenesis, Binding Assay

Journal: PLoS ONE

Article Title: miR-126&126* Restored Expressions Play a Tumor Suppressor Role by Directly Regulating ADAM9 and MMP7 in Melanoma

doi: 10.1371/journal.pone.0056824

Figure Lengend Snippet: Western blot analyses showing the effectiveness of stable si-ADAM9, si-MMP7 and scrambled control (SCR) transduction. B) Invasion and migration assays in si-ADAM9- or si-MMP7-infected melanoma cell lines compared with scrambled control. C) Invasion (left) and migration (right) in presence of either ADAM9 or MMP7 recombinant proteins in miR-126&126*-transduced A375M cell lines compared with control cells. Columns, mean±SD of a minimum of two independent experiments; ** P <0.001; * P <0.05.

Article Snippet: ADAM9 and MMP7 silencing was performed by using 4 unique 29-mer shRNA constructs in retroviral GFP vector, #TG314947 for ADAM9, #TG311438 for MMP7 and #TR30013 for scrambled negative control (OriGene Technologies, Rockville, MD, USA). miRCURY locked-nucleic-acid (LNA) Power Inhibitor knockdown probes for miR-126 and -126* were obtained from Exiqon (Copenhagen, Denmark), (Product numbers #426717-00 and #426718-00).

Techniques: Western Blot, Transduction, Migration, Infection, Recombinant

Journal: PLoS ONE

Article Title: miR-126&126* Restored Expressions Play a Tumor Suppressor Role by Directly Regulating ADAM9 and MMP7 in Melanoma

doi: 10.1371/journal.pone.0056824

Figure Lengend Snippet: Representative WB of A) pro-HB-EGF (bottom) and relative densitometric analysis (top) in miR-126&126*- versus empty vector-transduced Me665/1 melanoma cell line treated or not with PMA. B ) pro-HB-EGF and HB-EGF-C levels in si-ADAM9- or si-MMP7-infected melanoma compared with si-scrambled control. C) Real time PCR analysis of ADAM9 and MMP7 in the same silenced cells.

Article Snippet: ADAM9 and MMP7 silencing was performed by using 4 unique 29-mer shRNA constructs in retroviral GFP vector, #TG314947 for ADAM9, #TG311438 for MMP7 and #TR30013 for scrambled negative control (OriGene Technologies, Rockville, MD, USA). miRCURY locked-nucleic-acid (LNA) Power Inhibitor knockdown probes for miR-126 and -126* were obtained from Exiqon (Copenhagen, Denmark), (Product numbers #426717-00 and #426718-00).

Techniques: Plasmid Preparation, Infection, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Folic Acid Remodels Chromatin on Hes1 and Neurog2 Promoters during Caudal Neural Tube Development *

doi: 10.1074/jbc.M110.126714

Figure Lengend Snippet: KDM6B directly regulates histone H3K27 methylation in WT cultured neurospheres. A, transfection of scrambled negative control shRNA-RFP (TR30015, OriGene) into cells from WT neurospheres and immunostaining for KDM6B; B, transfection with KDM6B shRNA-RFP and immunostaining for KDM6B; C, transfection of scrambled negative control shRNA-RFP and immunostaining for H3K27me3; D, transfection with KDM6B shRNA-RFP and immunostaining for H3K27me3. Experiments were performed in quadruplicate. A representative of 4 separate experiments is shown. These results demonstrate that H3K27 methylation is directly regulated by KDM6B in cultured neurospheres.

Article Snippet: A scrambled negative control-shRNA-RFP from OriGene (TR30015) did not silence KDM6B levels ( A ) or increase H3K27 methylation ( C ).

Techniques: Methylation, Cell Culture, Transfection, Negative Control, shRNA, Immunostaining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Long non‐coding RNA cardiac hypertrophy‐associated regulator governs cardiac hypertrophy via regulating miR‐20b and the downstream PTEN/AKT pathway

doi: 10.1111/jcmm.14641

Figure Lengend Snippet: The anti‐hypertrophic effects of cardiac hypertrophy‐associated regulator CHAR in NRVCs. A, Down‐regulation of four lncRNAs in mice with CH induced by TAC compared with that in sham‐operated animals. The four lncRNAs tested were selected based on our previous microarray analysis. ** P < .01 vs Sham; n = 10. B, Expression down‐regulation of four selected lncRNAs in NRVCs treated with AngII. ** P < .01 vs Control; n = 10. C, CHAR overexpression reversed the enlarged cell size induced by AngII in NRVCs, n = 50. D, CHAR overexpression reversed the increased mRNA levels of ANP, BNP and β‐MHC induced by AngII in NRVCs (n = 4). E, CHAR overexpression reversed the increased protein/DNA ratio (n = 6) induced by AngII in NRVCs. ** P < .01 vs Control, and ## P < .01 vs AngII. F, Knockdown of CHAR by siRNA enlarged cell size in NRVCs (n = 50), resembling the hypertrophic cell growth. G, Knockdown of CHAR by siRNA increased the mRNA levels of the hypertrophic marker genes ANP, BNP and β‐MHC in NRVCs (n = 5). H, Knockdown of CHAR by siRNA increased the protein/DNA ratio in NRVCs (n = 4). SC: the scrambled negative control RNA. ** P < .01 vs CHAR‐SC. I‐K, Knockdown of CHAR by siRNA exacerbated the hypertrophic responses induced by AngII in NRVCs with enlarged cell size (n = 50; I); elevated mRNA levels of ANP, BNP and β‐MHC (n = 4; J); and increased protein/DNA ratio (n = 4; K). ** P < .01 & * P < .05 vs Control; ## P < .01 & # P < .05 vs AngII. AngII, angiotensin II; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; CH, cardiac hypertrophy; CHAR, cardiac hypertrophy‐associated regulator; lncRNA, Long non‐coding RNA; NC, negative control; NRVC, neonatal rat ventricular cells; PTEN, Phosphatase and tensin homolog; β‐MHC, β‐myosin heavy chain

Article Snippet: The lentivirus vectors carrying a short interference RNA to CHAR (Lenti‐sh‐CHAR) for loss‐of‐function or a scramble fragment (Lenti‐sh‐Scr) for negative control, and the lentivirus vectors carrying the full‐length sequence to CHAR (Lenti‐CHAR) for gain‐of‐function or empty vector (Lenti‐Vector) for negative control were constructed by Cyagen Biosciences.

Techniques: Microarray, Expressing, Control, Over Expression, Knockdown, Marker, Negative Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Long non‐coding RNA cardiac hypertrophy‐associated regulator governs cardiac hypertrophy via regulating miR‐20b and the downstream PTEN/AKT pathway

doi: 10.1111/jcmm.14641

Figure Lengend Snippet: The pro‐hypertrophic effects of miR‐20b in NRVCs. A, Sequence alignment showing the complementarity between CHAR and miR‐20b. B, Overexpression of CHAR after transfection with CHAR‐plasmid decreased the level of endogenous miR‐20b, relative to the empty vector as a negative control in NRVCs. ** P < 0.01vs Vector; n = 3. C, Knockdown of CHAR by siRNA increased miR‐20b level in NRVCs. CHAR‐SC: scramble RNA as a negative control. ** P < .01 vs CHAR‐SC; n = 4. D, Up‐regulation of miR‐20b in TAC hearts relative to sham‐operated control mice. ** P < .01 vs Sham; n = 4. E, Up‐regulation of miR‐20b in AngII‐treated NRVCs relative to non‐treated cells. ** P < .01 vs Control; n = 4. F, Transfection of miR‐20b mimic increased cell size in NRVCs (n = 50). NC failed to affect the cell size, and co‐transfection of miR‐20b and AMO‐20b reversed the miR‐20b‐induced enlargement of cell size. G, Transfection of miR‐20b mimic increased the mRNA levels of ANP, BNP and β‐MHC in NRVCs (n = 4). NC failed to affect biomarkers, and co‐transfection with AMO‐20b reversed the miR‐20b‐induced increases. H, Transfection of miR‐20b mimic increased the protein/DNA ratio in NRVCs (n = 4). NC did not affect the ratio, and co‐transfection with AMO‐20b reversed the increase. ** P < .01 vs Control (NC) and ## P < .01 vs miR‐20b; I‐K, miR‐20b exacerbated, whereas AMO‐20b reversed the hypertrophic phenotypes in response to AngII stimulation in NRVCs, including the increased cell area (n = 50; I); mRNA levels of ANP, BNP and β‐MHC (n = 3; J); and protein/DNA ratio (n = 6; K). AMO‐20b abolished the effects of miR‐20b. * P < .05 & ** P < .01 vs Control; ## P < .01 vs AngII; aa P < 0.01 & a p < 0.05 vs AngII; and $$ P < .01 vs miR‐20b + AngII. AngII, angiotensin II; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; CHAR, cardiac hypertrophy‐associated regulator; NC, negative control; NRVC, neonatal rat ventricular cells; PTEN, Phosphatase and tensin homolog; β‐MHC, β‐myosin heavy chain

Article Snippet: The lentivirus vectors carrying a short interference RNA to CHAR (Lenti‐sh‐CHAR) for loss‐of‐function or a scramble fragment (Lenti‐sh‐Scr) for negative control, and the lentivirus vectors carrying the full‐length sequence to CHAR (Lenti‐CHAR) for gain‐of‐function or empty vector (Lenti‐Vector) for negative control were constructed by Cyagen Biosciences.

Techniques: Sequencing, Over Expression, Transfection, Plasmid Preparation, Negative Control, Knockdown, Control, Cotransfection

Journal: Journal of Cellular and Molecular Medicine

Article Title: Long non‐coding RNA cardiac hypertrophy‐associated regulator governs cardiac hypertrophy via regulating miR‐20b and the downstream PTEN/AKT pathway

doi: 10.1111/jcmm.14641

Figure Lengend Snippet: Roles of miR‐20b and PTEN/AKT in mediating the anti‐hypertrophic action of CHAR. A‐C, Knockdown of endogenous miR‐20b by AMO‐20b abrogated the pro‐hypertrophic effects of CHAR silencing by CHAR‐siRNA, as indicated by the changes of cell area (n = 50; A); ANP, BNP and β‐MHC mRNA levels (n = 4; B); and protein/DNA ratio (n = 4; C) in NRVCs. ** P < .01 vs Control and ## P < .01 & # P < .05 vs CHAR‐siRNA. D‐F, miR‐20b mitigated the anti‐hypertrophic effects of CHAR in NRVCs incubated with AngII, as indicated by the changes of cell surface area (n = 50; D); mRNA levels of ANP, BNP and β‐MHC (n = 4; E); and protein/DNA ratio (n = 4; F). ** P < .01 vs Control; ## P < .01 vs AngII; aa P < 0.01 vs AngII + lncRNA. G, H, CHAR countered the AngII‐induced expression down‐regulation of PTEN at mRNA (n = 3; G) and protein levels (n = 5; H) in NRVCs. ** P < .01 vs Control; ## P < .01 vs AngII. I, J, CHAR silencing down‐regulated PTEN expression at mRNA (n = 4; I) and protein levels (n = 6; J) in NRVCs, resembling the effects of AngII. ** P < .01 & * P < .05 vs Control (SC). K, CHAR overexpression reversed the AngII‐induced up‐regulation of p‐AKT in NRVCs. ** P < .01 vs Control; ## P < .01 vs AngII, n = 6. L, CHAR silencing up‐regulated p‐AKT in NRVCs, resembling the effect of AngII. ** P < .01 vs Control (SC); n = 6. M, N, AKT inhibitor MK2206 abolished the pro‐hypertrophic effects of CHAR silencing by siRNA as indicated by the change of cell area (n = 50; M) and protein/DNA ratio (n = 4; N). ** P < .01 vs Control (SC), and ## P < .01 vs CHAR‐siRNA. AngII, angiotensin II; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; CHAR, cardiac hypertrophy‐associated regulator; lncRNA, Long non‐coding RNA; NRVC, neonatal rat ventricular cells; PTEN, Phosphatase and tensin homolog; β‐MHC, β‐myosin heavy chain

Article Snippet: The lentivirus vectors carrying a short interference RNA to CHAR (Lenti‐sh‐CHAR) for loss‐of‐function or a scramble fragment (Lenti‐sh‐Scr) for negative control, and the lentivirus vectors carrying the full‐length sequence to CHAR (Lenti‐CHAR) for gain‐of‐function or empty vector (Lenti‐Vector) for negative control were constructed by Cyagen Biosciences.

Techniques: Knockdown, Control, Incubation, Expressing, Over Expression

Journal: Journal of Cellular and Molecular Medicine

Article Title: Long non‐coding RNA cardiac hypertrophy‐associated regulator governs cardiac hypertrophy via regulating miR‐20b and the downstream PTEN/AKT pathway

doi: 10.1111/jcmm.14641

Figure Lengend Snippet: Schematic cartoon showing the proposed signalling pathway linking lncRNA CHAR to CH. lncRNA CHAR is down‐regulated in pathological CH; down‐regulation of CHAR weakens its ceRNA action on miR‐20b to increase the functional level of this miRNA; up‐regulation of miR‐20b represses its target gene PTEN leading to enhanced activation of AKT and consequent exaggeration of CH. This suggests a new signalling pathway: CHAR↓ → miR‐20b↑ → PTEN↓ → p‐AKT↑ → CH. CHAR, cardiac hypertrophy‐associated regulator; lncRNA, Long non‐coding RNA; PTEN, Phosphatase and tensin homolog

Article Snippet: The lentivirus vectors carrying a short interference RNA to CHAR (Lenti‐sh‐CHAR) for loss‐of‐function or a scramble fragment (Lenti‐sh‐Scr) for negative control, and the lentivirus vectors carrying the full‐length sequence to CHAR (Lenti‐CHAR) for gain‐of‐function or empty vector (Lenti‐Vector) for negative control were constructed by Cyagen Biosciences.

Techniques: Functional Assay, Activation Assay