vector 425  (Addgene inc)

Journal: Archives of clinical and biomedical research

Article Title: miR-425-5p, a SOX2 target, regulates the expression of FOXJ3 and RAB31 and promotes the survival of GSCs

doi: 10.26502/acbr.50170100

Figure Lengend Snippet: (A) Schematic representation of the common regulated miRNA obtained by two different approaches: SOX2 expression was either down-regulated using siRNA in GSC11 cells or expressed using a lentivirus-based system in GBM1A cells. (B) GBMIAand GBM1B neurospheres were forced to differentiate and expression of candidate SOX2-regulated pre-miRNAs was measured 5 days after. (C) qRT-PCR to measure expression of transgenic SOX2 in GBM1A and GBM1B neurospheres. (D) Expression of candidate SOX2-regulated pre-miRNAs in GBM1A and GBM1B cells expressing exogenous SOX2 . (E) GBM1A and GBM1B were transduced with lentivirus expressing a scrambled miRNA sponge (SC), miR-128b sponge (128b) or miR-425-5p sponge (425-5p). Expression of pre-miRNA was measured 3 days after transduction using qRT-PCR. (F) Equal numbers of GBM1A and GBM1B neurospheres transduced with scrambled miRNA sponge (SC), miR-128b sponge (128b) or miR-425-5p sponge (425-5p) were cultured in neuropshere medium for 2 weeks and spheres >100 μm in diameter were quantified using computer-assisted image analysis. *p<0.05

Article Snippet: Lentiviral expression vectors LentimiRa-Off-hsa-miR-425-5p vector to inhibit the expression of miR-425-5p or pLenti-III-mir-GFP as a Scramble control vector were purchased from ABM (Applied Biological Materials Inc, Richmond, BC, Canada); psPAX2 packaging plasmid and pMD2.G envelope plasmid were obtained from Addgene (Addgene, Cambridge, MA).

Techniques: Expressing, Quantitative RT-PCR, Transgenic Assay, Transduction, Cell Culture

Journal: Archives of clinical and biomedical research

Article Title: miR-425-5p, a SOX2 target, regulates the expression of FOXJ3 and RAB31 and promotes the survival of GSCs

doi: 10.26502/acbr.50170100

Figure Lengend Snippet: Equal numbers of GBM1A, GBM1B, GSC11, and GSC23 cells were transduced with scrambled miRNA sponge or miR-425-5p sponge. (A) Morphology of GBM1A, GBM1B, GSC-11 and GSC-23 cells 7 days after transduction with control sponge or miR-425-5p sponge. (B) qRT-PCR to measure expression of pre-miR-425-5p 3 days after transduction with scrambled miRNA sponge or miR-425-5p sponge. *p<0.05 and **p<0.01

Article Snippet: Lentiviral expression vectors LentimiRa-Off-hsa-miR-425-5p vector to inhibit the expression of miR-425-5p or pLenti-III-mir-GFP as a Scramble control vector were purchased from ABM (Applied Biological Materials Inc, Richmond, BC, Canada); psPAX2 packaging plasmid and pMD2.G envelope plasmid were obtained from Addgene (Addgene, Cambridge, MA).

Techniques: Transduction, Quantitative RT-PCR, Expressing

Journal: Archives of clinical and biomedical research

Article Title: miR-425-5p, a SOX2 target, regulates the expression of FOXJ3 and RAB31 and promotes the survival of GSCs

doi: 10.26502/acbr.50170100

Figure Lengend Snippet: (A and B) Equal numbers of GSC23 neurospheres transduced with scrambled miRNA sponge (SC.) or miR-425-5p sponge were cultured in neuropshere medium for 1 week and were analyzed using FACS. (C) Caspase 3/7 activity was measured 1 week after equal numbers of GSC23 cells were transduced with scrambled miRNA sponge (SC.) or miR-425-5p sponge. Untransduced neurospheres (control) were used as negative control. (D) Western blot measuring AKT and p-AKT 7 days after miR-425-5p inhibition. GRB2 expression was used as a loading control. (E) GSC23 neurospheres were transduced with a scrambled miRNA sponge or or miR-425-5p sponge and stained with APC-AnnexinV and SYTOX Blue 7 days post-transduction. AnnexinV/SYTOX Blue double positive cells were measured using flew cytometry analysis. (F) Representative TEM images of GSC23 cells transduced with scrambled miRNA sponge (SC.) or miR-425-sponge. Cells were collected 7 days after transduction. Asterisk (*) marks the nucleus and white arrows marks areas of nuclear membrane rupture (Magnification l000x). **p<0.01

Article Snippet: Lentiviral expression vectors LentimiRa-Off-hsa-miR-425-5p vector to inhibit the expression of miR-425-5p or pLenti-III-mir-GFP as a Scramble control vector were purchased from ABM (Applied Biological Materials Inc, Richmond, BC, Canada); psPAX2 packaging plasmid and pMD2.G envelope plasmid were obtained from Addgene (Addgene, Cambridge, MA).

Techniques: Transduction, Cell Culture, Activity Assay, Negative Control, Western Blot, Inhibition, Expressing, Staining, Cytometry

Journal: Archives of clinical and biomedical research

Article Title: miR-425-5p, a SOX2 target, regulates the expression of FOXJ3 and RAB31 and promotes the survival of GSCs

doi: 10.26502/acbr.50170100

Figure Lengend Snippet: miR-425-5p expression and survival data was retrieved from the TCGA database using the BETASTASIS portal ( http://www.betastasis.com ). (A) miR-425-5p levels are enriched in GBM compared to normal brain. (B) Positive correlation between miR-425-5p and SOX2 expression in GBM. Pearson coefficient analysis was applied to establish correlations from gene expression data. (C) Evaluation of the expression of miR-425-5p in 11 glioblastoma samples using qRT-PCR analysis. (D) Correlation between SOX2 mRNA expression and miR-425-5p expression in 11 glioblastoma samples. Scatter plot illustrate the correlation between SOX2 and miR-425-5p expression levels. Linear regression analysis was used to establish correlation. (E) The putative promoter region of miR-425-5p has multiple SOX2 binding sites (red rectangles) predicted by PROMO search tool. Blue arrows indicate the region for which primer were designed for PCR analysis (Reg. 1) (top panel). DNA purified from chromatin immunoprecipitation was analyzed by qRT-PCR using primer pairs designed to amplify fragments containing SOX2 binding sites (Reg. 1) and primers targeting promoter region lacking SOX2 binding sites with the putative miR-425-5p promoter (Ctrl.) in GBM1A neurospheres expressing exogenous SOX2. (F) Kaplan–Meier survival analysis in nude mice after orthotopic implantation of GSC11 tumor cells transduced with miR-425-5p sponge or scrambled miRNA sponge (SC.). P values were determined using a log-rank test. *p<0.05

Article Snippet: Lentiviral expression vectors LentimiRa-Off-hsa-miR-425-5p vector to inhibit the expression of miR-425-5p or pLenti-III-mir-GFP as a Scramble control vector were purchased from ABM (Applied Biological Materials Inc, Richmond, BC, Canada); psPAX2 packaging plasmid and pMD2.G envelope plasmid were obtained from Addgene (Addgene, Cambridge, MA).

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Purification, Chromatin Immunoprecipitation, Transduction

Journal: Archives of clinical and biomedical research

Article Title: miR-425-5p, a SOX2 target, regulates the expression of FOXJ3 and RAB31 and promotes the survival of GSCs

doi: 10.26502/acbr.50170100

Figure Lengend Snippet: (A) Bioinformatics analysis to identify miR-425-5p targets using 3 different prediction algorithms. (B) qRT-PCR to measure gene expression of high-confidence miR-425-5p targets in GBM1A and GBM1B neurospheres expressing transgenic SOX2. (C) Expression of F0XJ3 and RAB31 genes following GSC forced differentiation. (D) Expression of FOXJ3 and RAB31 genes after SOX2 depletion using siRNA in GSCs. (E) Bioinformatic analysis using Target Scan portal showing miR-425-5p binding sites on the FOXJ3 and RAB31 3′UTRs are conserved among several species. (F) qRT-PCR to measure FOXJ3 and RAB31 gene expression in two distinct GSC isolates 3 days after miR-425-5p inhibition. *p<0.05

Article Snippet: Lentiviral expression vectors LentimiRa-Off-hsa-miR-425-5p vector to inhibit the expression of miR-425-5p or pLenti-III-mir-GFP as a Scramble control vector were purchased from ABM (Applied Biological Materials Inc, Richmond, BC, Canada); psPAX2 packaging plasmid and pMD2.G envelope plasmid were obtained from Addgene (Addgene, Cambridge, MA).

Techniques: Quantitative RT-PCR, Expressing, Transgenic Assay, Binding Assay, Inhibition