lentiviral vectors expressing short hairpin rnas (shrnas) against brca1 (nm_007299)  (Genechem)

Journal: BioMed Research International

Article Title: Identification of Novel Breast Cancer Subtype-Specific Biomarkers by Integrating Genomics Analysis of DNA Copy Number Aberrations and miRNA-mRNA Dual Expression Profiling

doi: 10.1155/2015/746970

Figure Lengend Snippet: BRCA1 facilitates miR-143 and miR-145 processing. In order to confirm whether BRCA1 facilitates miR-143 and miR-145 processing, BRCA1 overexpression and siBRCA1 expression plasmids were transfected into MCF-7 cells. The empty vector or scramble siRNA expression vector served as controls. (a) BRCA1 mRNA level and (b) protein level were examined by qPCR and Western blot, respectively, in stable transfected cells, normalized by beta-actin ( ∗ P < 0.05 as compared with mock control; n = 3). Then, the expression levels of the primary (pri), precursor (pre), and mature (mat) forms of the indicated miRNAs were examined in human BRCA1 (c) overexpressed and (d) knock-down MCF-7 cells using qRT-PCR analysis. Pri- and pre-miRNAs were normalized by beta-actin, and mature miRNA was normalized by U6 snRNA ( ∗ P < 0.05 as compared with mock control; n = 3). Meanwhile, in vivo monitoring assay of pri-miRNA processing in (e) BRCA1 overexpression or (f) knock-down MCF-7 cells carrying miR-143 or miR-145 at the 3′ untranslated region of the luciferase gene. The intensities were normalized by Renilla luciferase and are shown as fold induction as compared with an empty pmirGLO vector ( ∗ P < 0.05; n = 3). Error bars represent standard deviation.

Article Snippet: MCF-7 cells were transfected with a pCMV6-XL4 vector containing the full-length BRCA1 gene purchased from Origene (Beijing, China) (empty vector as control) or pSilencer siRNA expression vector (Ambion) containing BRCA1 specific shRNA expression cassette (scramble shRNA expression vector as siRNA control).

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, In Vivo, Luciferase, Standard Deviation

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: Down‐regulation of BRCA1 in EBV‐associated NPCs. (A) The expression of BRCA1, ATM and PARP1 proteins in immortalized normal NP (NP69), four NPC cell lines and four NPC patient‐derived xenografts (PDXs) were analysed by immunoblotting. Actin was probed as the loading control. (B) The expression levels of BRCA1 mRNA in the cell lines were measured using RT‐qPCR. The relative BRCA1 mRNA expression was calculated using 2(∆∆−Ct) method, and the expression in NP69 was set as 1 for comparison. The data shown is the mean + SD. (C) The whiskers 10‐90 percentiles plot shows the relative BRCA1 mRNA expression in primary samples. The BRCA1 mRNA was significantly up‐regulated in NPCs (n = 55) when compared with the NPs (n = 22). (D) Immunohistochemistry staining of BRCA1 protein in primary samples (number of NPs = 30 and NPCs = 41). The representative images of negative and positive BRCA1 stain in NP and NPC specimens are shown (original magnification X400). (E) The dot plot shows the total expression levels of miR‐BART2‐3p, BART12, BART17‐5p and BART19‐3p in 20 NPC biopsies, in which the BRCA1 protein expression status was analysed in IHC. The expression of miR‐BARTs was normalized to EBNA1. The median values of each group are shown by the dash line and the Mann‐Whitney test was used for the statistical analysis

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Expressing, Derivative Assay, Western Blot, Control, Quantitative RT-PCR, Comparison, Immunohistochemistry, Staining, MANN-WHITNEY

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: The BRCA1 is the potential target of miR‐BARTs. (A) The relative luciferase activity of the reporter plasmids harbouring a full length of BRCA1‐3’UTR (sFL‐3’UTR) or a full length of BRCA1‐3’UTR in reversed orientation (asFL‐3’UTR) was co‐transfected together with the indicated miRNAs. The luciferase signal with the co‐transfection of negative miRNA mimic control (miR‐NEG) was set at 1 for comparison. (B) The direct interaction between the putative binding sites on BRCA1 and miR‐BARTs were demonstrated in the reporter assays. The firefly luciferase reporter activity was normalized to the Renilla luciferase control. The data shown is the mean + SD from three independent experiments. The result with the co‐transfection of miR‐NEG and pMIR‐CTL was set at 1. pMIR‐CTL = pMIR‐REPORTTM vectors containing unrelated sequences; pMIR‐B = pMIR‐REPORTTM vector harbouring the predicted miR‐BART binding site, pMIR‐CDS = predicted binding site on CDS (Table ). B2‐3p = BART2‐3p; B12 = BART12; B17‐5p = BART17‐5p; B19‐3p = BART19‐3p. * P < 0.05, ** P < 0.001

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Luciferase, Activity Assay, Transfection, Cotransfection, Control, Comparison, Binding Assay, Plasmid Preparation

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: Regulation of BRCA1 expression by miR‐BARTs (A) Western blot of BRCA1 in NPC cell lines. Actin was probed as the protein‐loading control, and the expression level was compared with NP69 (set as 1). (B) The total expression of viral BART2‐3p, BART12, BART17‐5p and BART19‐3p (upper panel) and the expression of cellular miR‐146a (lower panel) in the cell lines were assayed by RT‐qPCR. The expression values of total miR‐BARTs and miR‐146a were calculated using the 2(‐∆Ct) and 2(∆∆‐Ct) methods, respectively. The analysis of each sample was performed in triplicate with mean + SD shown. (C) In the EBV‐negative epithelial cells, the BRCA1 level was suppressed by the transfection of the indicated miRNA mimics, BART2‐3p (B2‐3p), BART12 (BT12), BART17‐5p (BT17‐5p) and BART19‐3p (BT19‐3p). (D) The BRCA1 protein expression in C666‐1 cells was regained by suppressing the endogenous miR‐BARTs activities with specific miR‐BART inhibitors for 48 h. The negative control mimic/inhibitor (Inh‐Ctl) transfection was used for comparison. Either actin or vinculin was probed as the loading control

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR, Transfection, Negative Control, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: The CDDP and DOX sensitivity in HK1 and NP69 cells. (A) Western blot of p53 and p21 in NPC cell lines were analysed. (B) Transfection of either BRCA1‐specific siRNA, BART17‐5p or BART19‐3p mimics increased CDDP‐ and DOX‐mediated S phase or G2/M phase cell‐cycle arrest in the HK1 and NP69 cells. The transfected cells were incubated with either the control buffer or the indicated chemotherapeutic agent for 24 h. Subsequently, the cells were fixed for DNA content analysis with BD FACSCalibur flow cytometry system. (C) Protein lysate from the treated cells were harvested for phosphor‐CHK1 (p‐CHK1) expression analysis. (D) The suppression of BRCA1 sensitized HK1 cells to CDDP and DOX treatment. HK1 cells were transfected with BRCA1‐specific siRNAs (si‐BRCA1) or siRNA control (si‐NEG) and the protein lysates were collected for BRCA1 expression analysis 24 h after transfection (left panel). The transfected HK1 cells were incubated with different concentrations of CDDP or DOX for 48 h before CCK‐8 analysis. The IC50 value was determined by fitting a sigmoidal dose‐response curve to the data using GraphPad Prism 5 program. Sum‐of‐squares F‐test was used as the comparison method (right panel). (E) Clonogenic survival assays. Approximately 500 or 1000 transfected cells were seeded into the 6‐well plate and treated with CDDP or DOX for 24 h. The cells were cultured for 14‐18 d in normal medium before staining, and colonies containing more than 30 cells were counted. The number of colonies generated from the mock treatment was compared (set as 100%). All the experiments were performed in triplicate and the Student's t ‐test was conducted, compared with the control transfected cells. * P < 0.05; ** P < 0.01

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Western Blot, Transfection, Incubation, Control, Flow Cytometry, Expressing, CCK-8 Assay, Comparison, Cell Culture, Staining, Generated

Journal: Journal of Cellular and Molecular Medicine

Article Title: EBV–encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1

doi: 10.1111/jcmm.16007

Figure Lengend Snippet: The EBV‐miRNAs impair cisplatin‐ and doxorubicin‐induced DNA damage response in nasopharyngeal epithelial cells. The representative images of the RAD51 foci staining in HK1 cells (upper left panel) and NP69 cells (upper right panel) are shown. The cells transfected with either siRNA control (si‐NEG), BRCA1‐specific siRNA (si‐BRCA1) or miR‐BARTs mimics were treated with cisplatin (CDDP) and doxorubicin (DOX), followed by immunostaining with the RAD51 antibody. At least 100 nuclei were randomly selected for counting, and the cells containing more than five apparent RAD51 foci in the nucleus were considered positive. The percentage of the RAD51‐positive cells with mean + SD from three independent experiments are shown in the lower panel. Student's t ‐test was used to compare them with the control transfected cells (miR‐NEG) in each set of experiments. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).

Techniques: Staining, Transfection, Control, Immunostaining

Journal: International Journal of Molecular Epidemiology and Genetics

Article Title: Effects of 3300 del A-1061 Ter BRCA1 frameshift mutation and calcium propionate on oxidative stress and breast carcinogenesis

doi:

Figure Lengend Snippet: The percentage of inhibition activity of SOD induced by 3300 del A-1061 Ter BRCA1 frameshift mutation in HCC1937 cell line.

Article Snippet: BRCA1 ) consisting of the full-length BRCA1 cDNA within the pcDNA3 mammalian expression vector (GeneArt ® gene synthesis service, Invitrogen, Germany) and 3300 del A-1061 Ter BRCA1 expression vector (GeneArt ® gene synthesis service, Invitrogen, Germany) were used in this study.

Techniques: Inhibition, Activity Assay, Mutagenesis

Journal: International Journal of Molecular Epidemiology and Genetics

Article Title: Effects of 3300 del A-1061 Ter BRCA1 frameshift mutation and calcium propionate on oxidative stress and breast carcinogenesis

doi:

Figure Lengend Snippet: The percentage of inhibition activity of SOD induced by 3300 del A-1061 Ter BRCA1 frameshift mutation and calcium propionate conc. 0.5, 1.0, 1.5 and 2.0 mg/mL in SKBR3 cell line

Article Snippet: BRCA1 ) consisting of the full-length BRCA1 cDNA within the pcDNA3 mammalian expression vector (GeneArt ® gene synthesis service, Invitrogen, Germany) and 3300 del A-1061 Ter BRCA1 expression vector (GeneArt ® gene synthesis service, Invitrogen, Germany) were used in this study.

Techniques: Inhibition, Activity Assay, Mutagenesis

Journal: International Journal of Molecular Epidemiology and Genetics

Article Title: Effects of 3300 del A-1061 Ter BRCA1 frameshift mutation and calcium propionate on oxidative stress and breast carcinogenesis

doi:

Figure Lengend Snippet: The percentage of inhibition activity of SOD induced by 3300 del A-1061 Ter BRCA1 frameshift mutation in HCC1937 cell line.

Article Snippet: BRCA1 ) consisting of the full-length BRCA1 cDNA within the pcDNA3 mammalian expression vector (GeneArt ® gene synthesis service, Invitrogen, Germany) and 3300 del A-1061 Ter BRCA1 expression vector (GeneArt ® gene synthesis service, Invitrogen, Germany) were used in this study.

Techniques: Inhibition, Activity Assay, Mutagenesis

Journal: International Journal of Molecular Epidemiology and Genetics

Article Title: Effects of 3300 del A-1061 Ter BRCA1 frameshift mutation and calcium propionate on oxidative stress and breast carcinogenesis

doi:

Figure Lengend Snippet: The percentage of inhibition activity of SOD induced by 3300 del A-1061 Ter BRCA1 frameshift mutation and calcium propionate conc. 0.5, 1.0, 1.5 and 2.0 mg/mL in SKBR3 cell line

Article Snippet: BRCA1 ) consisting of the full-length BRCA1 cDNA within the pcDNA3 mammalian expression vector (GeneArt ® gene synthesis service, Invitrogen, Germany) and 3300 del A-1061 Ter BRCA1 expression vector (GeneArt ® gene synthesis service, Invitrogen, Germany) were used in this study.

Techniques: Inhibition, Activity Assay, Mutagenesis

Journal: Frontiers in Endocrinology

Article Title: Identification of BRCA1 As a Potential Biomarker for Insulin-Like Growth Factor-1 Receptor Targeted Therapy in Breast Cancer

doi: 10.3389/fendo.2017.00148

Figure Lengend Snippet: Insulin-like growth factor-1 receptor (IGF1R) gene expression in wild-type- and mutant-BRCA1-containing breast cancer cells. (A) Confluent cultures of wild-type BRCA1-expressing MCF7, MCF10A, HB2, and MDA-MB-231, and mutant BRCA1-expressing HCC1937 cells, were harvested and total protein and RNA was extracted. The bar graphs represent the IGF1R and BRCA1 mRNA levels in the various cell lines, as measured by RT-qPCR. An arbitrary value of 1 in the y -axis was given to the mRNA levels in HCC1937 cells. Bars represent mean ± SEM of three independent experiments (* p < 0.05 versus HCC1937; ** p < 0.01 versus HCC1937). Equal amounts of protein (50 μg) were separated by 6 and 10% SDS-PAGE, transferred to nitrocellulose filters and blotted with anti-BRCA1 or anti-total IGF1R antibodies, respectively. The positions of the ~220-kDa BRCA1, ~97-kDa IGF1R β-subunit, 42-kDa β-actin, and 100-kDa Cbl bands are indicated. (B) Effect of BRCA1 expression on endogenous IGF1R levels. HCC1937 cells were seeded in 10-cm plates at a density of 1 × 10 6 cells per plate. After 24 h, cells were transiently transfected with 10 μg of the pcDNA3-BRCA1 expression vector, or empty vector, using the jetPRIME reagent. After 48 h, cells were harvested, and levels of BRCA1 and endogenous IGF1R were assessed by Western blotting. Tubulin was used as a loading control.

Article Snippet: To generate wild-type BRCA1-expressing HCC1937 cells, naïve HCC1937 cells were transiently transfected with 10 μg of a pcDNA3-BRCA1 expression vector, or empty pcDNA3 vector (Invitrogen, Carlsbad, CA, USA), using the jetPRIME ® reagent (Polyplus Transfection, Illkirch, France).

Techniques: Expressing, Mutagenesis, Quantitative RT-PCR, SDS Page, Transfection, Plasmid Preparation, Western Blot

Journal: Frontiers in Endocrinology

Article Title: Identification of BRCA1 As a Potential Biomarker for Insulin-Like Growth Factor-1 Receptor Targeted Therapy in Breast Cancer

doi: 10.3389/fendo.2017.00148

Figure Lengend Snippet: Effect of BRCA1 silencing on the anti-proliferative activity of MK-0646. (A) MCF7, MCF10A, and HB2 cells were seeded in 10-cm plates at a density of 1 × 10 6 cells per plate. After 24 h, cells were infected with 3 μg of the lentivirus vector pGIPZ encoding BRCA1 shRNA or empty vector (NS). Levels of BRCA1 mRNA and protein in untransfected cells, empty vector-transfected cells, and a number of selected clones were measured by RT-qPCR (bar graphs) and Western blots, respectively. Figures above the bars denote arbitrary units of absorbance. Equal loading was confirmed by Cbl or tubulin measurement. Bars represent mean ± SEM of three independent experiments (** p < 0.01 versus respective control). (B) Untransfected MCF7 cells, empty vector-transfected cells (NS), and MCF7-BRCA1 knockdown cells (C5 and C8 clones) were plated in 96-well plates at a density of 3 × 10 3 cells/well. The cells were serum starved for 24 h, after which the medium was changed to serum-free media, including or lacking MK-0646 antibody (MK, 10 μg/ml). Cell proliferation was examined by XTT assays. Bars represent mean ± SEM of three independent experiments (* p < 0.05 versus respective control). (C) Wild-type BRCA1-containing MCF7 and MDA-MB-231 cells, and mutant BRCA1-containing HCC1937 cells, were seeded in 96-well plates at a density of 3 × 10 3 cells/well. The effect of MK-0646 on cell proliferation was assessed as described above. Bars represent mean ± SEM of three independent experiments (* p < 0.05 versus control).

Article Snippet: To generate wild-type BRCA1-expressing HCC1937 cells, naïve HCC1937 cells were transiently transfected with 10 μg of a pcDNA3-BRCA1 expression vector, or empty pcDNA3 vector (Invitrogen, Carlsbad, CA, USA), using the jetPRIME ® reagent (Polyplus Transfection, Illkirch, France).

Techniques: Activity Assay, Infection, Plasmid Preparation, shRNA, Transfection, Clone Assay, Quantitative RT-PCR, Western Blot, Mutagenesis

Journal: Frontiers in Endocrinology

Article Title: Identification of BRCA1 As a Potential Biomarker for Insulin-Like Growth Factor-1 Receptor Targeted Therapy in Breast Cancer

doi: 10.3389/fendo.2017.00148

Figure Lengend Snippet: Effect of BRCA1 status on the synergistic activity of MK-0646. MCF7 and HCC1937 cells were plated in 12-well plates at a density of 25 × 10 3 cells/well. After 24 h, the medium was changed to 5% charcoal-treated fetal bovine serum, including or lacking MK-0646 (MK, 10 μg/ml) with (open bars) or without (solid bars) etoposide (2 μM). Control cells were incubated for the same period of time in the absence of the antibody. Cell proliferation was examined by XTT assays. A value of 100% was given to the viability of untreated cells. Bars represent mean ± SEM of three independent experiments (* p < 0.05 versus no etoposide).

Article Snippet: To generate wild-type BRCA1-expressing HCC1937 cells, naïve HCC1937 cells were transiently transfected with 10 μg of a pcDNA3-BRCA1 expression vector, or empty pcDNA3 vector (Invitrogen, Carlsbad, CA, USA), using the jetPRIME ® reagent (Polyplus Transfection, Illkirch, France).

Techniques: Activity Assay, Incubation