Journal: Journal of Breast Cancer
Article Title: c-Myc-Induced Long Non-Coding RNA Small Nucleolar RNA Host Gene 7 Regulates Glycolysis in Breast Cancer
Figure Lengend Snippet: LncRNA- SNHG7 is a direct transcriptional target of c-Myc. (A, B) MCF-7 and MDA-MB-231 cells were transduced with sh-ctrl or sh-c-Myc lentivirus, pCDH or pCDH-c-Myc lentivirus. Western blot analysis of c-Myc and β-actin. LncRNA- SNHG7 level was assessed by quantitative RT-PCR. (C) Schematic illustration of the consensus c-Myc binding sites in lncRNA- SNHG7 gene promoter. The wild-type and mutant binding sites are shown in the open boxes. The corresponding pGL3-based luciferase reporter constructs generated are shown. (D, E) MCF-7 were transduced with sh-ctrl or sh-c-Myc lentivirus, pCDH or pCDH-c-Myc lentivirus followed by transfection with the indicated pGL3-based luciferase reporter constructs. The reporter activity was measured 24 hours post-transfection, and plotted after normalizing with the Renilla luciferase activity. Western blot analysis of c-Myc and β-actin. (F) MCF-7 cells lysates were analyzed by ChIP assay using anti-c-Myc or IgG rabbit antibody. The ChIP products were amplified by semi-quantitative RT-PCR. (G) Correlation analyses conducted between c-Myc and lncRNA- SNHG7 in breast cancers (n = 50 biologically independent samples). Pearson correlation coefficients ( r ) and p -values. Data were presented as mean ± standard deviation. lncRNA = long non-coding RNA; sh = short hairpin; RT-PCR = real-time polymerase chain reaction; NS = not significant; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; IgG = immunoglobulin G. * p
Article Snippet: Primary antibodies used in the current study were: anti-HK2 (Thermo Fisher), anti-GPI (Thermo Fisher), anti-PFKL (Thermo Fisher), anti-ALDB (Thermo Fisher), anti-TPI1 (Sigma Aldrich), anti-PGK1 (Thermo Fisher), anti-PGAM1 (Sigma Aldrich), anti-ENO1 (Sigma Aldrich), anti-PKM2 (Sigma Aldrich), anti-lactate dehydrogenase A (LDHA; Thermo Fisher), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Thermo Fisher), and anti-β actin (Sigma Aldrich).
Techniques: Multiple Displacement Amplification, Transduction, Western Blot, Quantitative RT-PCR, Binding Assay, Mutagenesis, Luciferase, Construct, Generated, Transfection, Activity Assay, Chromatin Immunoprecipitation, Amplification, Standard Deviation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction