breast cancer target cells  (ATCC)

Journal: American Journal of Cancer Research

Article Title: Profiling the epigenetic interplay of lncRNA RUNXOR and oncogenic RUNX1 in breast cancer cells by gene in situ cis-activation

doi:

Figure Lengend Snippet: The RUNX1-RUNXOR epigenetic interplay in breast cancer cells. A. Structure of the RUNX1/RUNXOR locus. RUNX1 is controlled by two promoters: distal promoter P1 and proximal promoter P2, which are separated by 150 kb. RUNXOR is transcribed from a promoter 3.8 kb upstream of the RUNX1 distal promoter P1. These two genes are transcribed in the same direction and share most of the sequence (colored red and green, respectively). Black block: RUNX1 exon 1-9; blue ovals: RUNX1 enhancer; purple oval: RUNX1 silencer, RE1, RE2: enhancers 1-2; pRUNX1: RUNX1 promoters; pRUNXOR: RUNXOR lncRNA promoter. B. Positive correlation between RUNX1 mRNA and RUNXOR lncRNA in cancer cells. Dot plot showed the relative levels of RUNX1 and RUNXOR in cell lines of different tissue origins calculated as (1/ave Ct) ×100). Pearson correlation analysis was performed using PRISM 6. C. RUNXOR lncRNA-mediated epigenetic regulation of oncogenic RUNX1 mRNA gene in breast cancer cells.

Article Snippet: RUNXOR targeting Human breast cancer MCF7 cells, purchased from ATCC, were co-transfected with cas9-gRNA vector and donor vector simultaneously using Lipofectamine 3000 Reagent.

Techniques: Sequencing, Blocking Assay

Journal: American Journal of Cancer Research

Article Title: Profiling the epigenetic interplay of lncRNA RUNXOR and oncogenic RUNX1 in breast cancer cells by gene in situ cis-activation

doi:

Figure Lengend Snippet: The cis-overexpression of RUNXOR by CRISPR Cas9 knock-in system. Activation of the endogenous RUNXOR lncRNA gene by the “gene in situ cis-activation” system. A. Schematic diagram illustrating the gene in situ cis-activation strategy. Cas9: CRISPR Cas9; gRNA: Cas9 guiding RNA; pCMV: CMV promoter; pEF1: EF1 promoter; pRUNXOR: RUNXOR promoter; pH1: RNA polymerase III H1 promoter; Cre: Cre recombinase; loxP: the locus of X-over P1 recombination site recognized by Cre; Arm 1, Arm2: the genomic sequences used for Cas9-mediated recombination. Under the guidance of gRNA, Cas9 mediated genomic recombination at the RUNXOR promoter loci, resulting in the insertion of the strong CMV promoter in front of RUNXOR. After transfection, cell clones positive for puromycin and negative for ganciclovir were selected and validated by sequencing. The chosen clones were then transiently transfected with a vector expressing Cre recombinase to eliminate the puromycin/GFP selection cassette. B. Initial screening of targeted clones by PCR. Positive clones were selected by targeting primers located in the targeting vector and the RUNXOR region beyond Arm 1 and Arm 2, respectively. Primers were designed from the RUNXOR arm, selection marker, and genomic regions. Arm 1: JH5859/JH5860; Arm 2: JH745/JH5826. Using these pairs of primers, only the targeted clones can be amplified. Different lanes represent different clones. C. Cis-overexpression of RUNXOR lncRNA in the selected knock-in clones. Total mRNAs extracted from the aforementioned clones were subjected to RT-qPCR. The control was set as 1 for standardization. The data were shown as mean ± S.E.M of three independent experiments. Results were considered significant if P<0.05. *P<0.05, **P<0.01; ***P<0.001.

Article Snippet: RUNXOR targeting Human breast cancer MCF7 cells, purchased from ATCC, were co-transfected with cas9-gRNA vector and donor vector simultaneously using Lipofectamine 3000 Reagent.

Techniques: Over Expression, CRISPR, Knock-In, Activation Assay, In Situ, Genomic Sequencing, Transfection, Clone Assay, Sequencing, Plasmid Preparation, Expressing, Selection, Marker, Amplification, Quantitative RT-PCR, Control, IF-P

Journal: American Journal of Cancer Research

Article Title: Profiling the epigenetic interplay of lncRNA RUNXOR and oncogenic RUNX1 in breast cancer cells by gene in situ cis-activation

doi:

Figure Lengend Snippet: Cis-overexpression of RUNXOR specifically activates the RUNX1 P1 transcripts. A. Expression of RUNX1 mRNAs in the RUNXOR cis-overexpressing cell clone. Total mRNA was extracted for RT-qPCR. Ctrl: MCF7 control cells; C2: MCF7 clone cells with CMV/puromycin/GFP random insertion; A3: The RUNXOR knock-in MCF7 clone cells. Note the activation of RUNX1 in the RUNXOR-overexpressing A3 cells. B. Western blot analysis of RUNX1. The protein level of RUNX1 was measured by Western blot. Beta-ACTIN was used as the loading control. C. Distinct expression of RUNX1 isoforms. The expression of RUNX1 isoforms was quantitated by qPCR using primers specific for each promoter. RUNX1 P1 represents the transcripts from the P1 promoter. RUNX1 P2 represents the transcript from the P2 promoter. All results were performed in triplicates and were shown as mean ± S.E.M of three independent experiments. The values of the control (Ctrl) were set as 1 for standardization. Results were considered significant if P<0.05. *P<0.05, **P<0.01.

Article Snippet: RUNXOR targeting Human breast cancer MCF7 cells, purchased from ATCC, were co-transfected with cas9-gRNA vector and donor vector simultaneously using Lipofectamine 3000 Reagent.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Control, Knock-In, Activation Assay, Western Blot, IF-P

Journal: American Journal of Cancer Research

Article Title: Profiling the epigenetic interplay of lncRNA RUNXOR and oncogenic RUNX1 in breast cancer cells by gene in situ cis-activation

doi:

Figure Lengend Snippet: Changes of RUNX1 local chromatin structures upon RUNXOR overexpression. RUNXOR triggers the alteration of RUNX1 local chromatin structures. A. Location of the 3C PCR primer sets used to quantitate the intrachromosomal loop between the cis-regulating elements within the RUNX1 locus. B. Quantitation of the 3C products by qPCR. ERCC3, a housekeeping gene, was used as the 3C internal control for normalization of the discrepancies of digestion/ligation efficiency and DNA quantities between the control and A3 groups. Q-PCR was performed in triplicate and shown as mean ± S.E.M. C. Confirmation of the 3C products by DNA sequencing. Red arrows: the site of ligated Mbo1 restriction enzyme site flanked by genomic DNAs from two remote areas, respectively.

Article Snippet: RUNXOR targeting Human breast cancer MCF7 cells, purchased from ATCC, were co-transfected with cas9-gRNA vector and donor vector simultaneously using Lipofectamine 3000 Reagent.

Techniques: Over Expression, Quantitation Assay, Control, Ligation, DNA Sequencing

Journal: American Journal of Cancer Research

Article Title: Profiling the epigenetic interplay of lncRNA RUNXOR and oncogenic RUNX1 in breast cancer cells by gene in situ cis-activation

doi:

Figure Lengend Snippet: RUNXOR overexpression changes the methylation status of RUNX1. RUNXOR changes the methylation status of RUNX1. A. Schematic diagram of the RUNX1/RUNXOR locus and the primers used for methylation analysis. B. DNA CpG methylation status of the RUNX1 P1 promoter, RE1 and P2 promoter. P1 promoter: DNA methylation was decreased from 75% to 36.7% in the RUNX1 P1 promoter after RUNXOR overexpression. RE1: There was a significant demethylation in the A3 group (46.7%) as compared with the control group (95%). P2 promoter: Genomic DNA was hypomethylated in both groups. Open circles: unmethylated CpGs; solid circles: methylated CpGs.

Article Snippet: RUNXOR targeting Human breast cancer MCF7 cells, purchased from ATCC, were co-transfected with cas9-gRNA vector and donor vector simultaneously using Lipofectamine 3000 Reagent.

Techniques: Over Expression, Methylation, CpG Methylation Assay, DNA Methylation Assay, Control

Journal: American Journal of Cancer Research

Article Title: Profiling the epigenetic interplay of lncRNA RUNXOR and oncogenic RUNX1 in breast cancer cells by gene in situ cis-activation

doi:

Figure Lengend Snippet: Alteration of histone methylation in the RUNX1 distal promoter P1. RUNXOR alters histone methylation in the RUNX1 distal promoter P1. The chromatin complex was fixed with formaldehyde and was immunoprecipitated with antibodies targeting H3K4me3 (A), H3K9me2 (B) and H3K27me3 (C), respectively. DNA from the pulled down chromatin was purified and subjected to qPCR using primers that targeted the RUNX1 P1. Results are shown as relative to control for standardization. P<0.05 was regarded as significant. **P<0.01.

Article Snippet: RUNXOR targeting Human breast cancer MCF7 cells, purchased from ATCC, were co-transfected with cas9-gRNA vector and donor vector simultaneously using Lipofectamine 3000 Reagent.

Techniques: Methylation, Immunoprecipitation, Purification, Control

Journal: American Journal of Cancer Research

Article Title: Profiling the epigenetic interplay of lncRNA RUNXOR and oncogenic RUNX1 in breast cancer cells by gene in situ cis-activation

doi:

Figure Lengend Snippet: Phenotypic characterization of MCF7 cells upon RUNXOR cis-overexpression. A. Apoptotic analysis. Cells stained by Annexin V and 7-AAD were subjected for analysis using flow cytometer. Apoptotic cells were characterized as Annexin V positive/7-AAD negative (marked as Q3 at the fourth quadrant), and comparisons made were in triplicates. B. Cell cycle analysis. Cells stained with propidium iodide were analyzed using flow cytometry and data processing was performed using ModFit LT.

Article Snippet: RUNXOR targeting Human breast cancer MCF7 cells, purchased from ATCC, were co-transfected with cas9-gRNA vector and donor vector simultaneously using Lipofectamine 3000 Reagent.

Techniques: Over Expression, Staining, Flow Cytometry, Cell Cycle Assay