cdna labeling, microarray hybridization and bioinformatics analysis  (CapitalBio Corporation)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK13 upregulation-induced formation of the positive feedback loop among circCDK13, miR-212-5p/miR-449a and E2F5 contributes to prostate carcinogenesis

doi: 10.1186/s13046-020-01814-5

Figure Lengend Snippet: CDK13 is upregulated in PCa tissues and the upregulation of CDK13 promoted proliferation in PCa cells. a , RT-qPCR detected CDK13 mRNA expression in 30 pairs of PCa and benign prostatic hyperplasia (BPH). * P < 0.05 vs. BPH. b , CDK13 protein expression was measured by Western blotting in 3 pairs of randomly selected BPH (B) and PCa (P) tissues. c , Hematoxylin and eosin (HE) staining of BPH and PCa tissues. d , Immunochemistry staining of CDK13 in BPH and PCa tissues. Scale bar = 50 mm. e , The expression levels of CDK13 mRNA in BPH and PCa tissues from the GSE13507 database ( P = 0.0007). f , The expression of CDK13 was examined by RT-qPCR (up panel) or Western blotting (down panel) in human normal prostate epithelial cells (RWPE-1) and PCa cell lines (LNCaP, PC3, 22RV1 and DU145). CDK13 expression was significantly increased in PC3 and 22RV1 cell lines. * P < 0.05 vs. RWPE-1. g and h , Cell viability was measured by MTS assay (G) and colony formation assays (H) in PC3 and 22RV1 cell lines after transfected with indicated vectors. * P < 0.05, and ** P < 0.01 vs. their respective empty vector. i , PC3 and 22RV1 cells were transfected with shCDK13 or control vector, cell apoptosis was detected by AnnexinV/PI flow cytometry. Right panel shows the apoptosis rate of three independent experiments. ** P < 0.001 vs. pLKO. pWPI is a control vector of oeCDK13 and pLKO is the control vector of shCDK13

Article Snippet: Microarray hybridization analysis of mRNA expression in 2 PCa samples and 2 BPH were performed according to the manufacturer’s protocol (Arraystar, Inc., Rockville, MD, USA).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Staining, MTS Assay, Transfection, Plasmid Preparation, Control, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK13 upregulation-induced formation of the positive feedback loop among circCDK13, miR-212-5p/miR-449a and E2F5 contributes to prostate carcinogenesis

doi: 10.1186/s13046-020-01814-5

Figure Lengend Snippet: CDK13 interacts with E2F5 in PCa cells. a , Co-immunoprecipitation coupled with mass spectrometry (CoIP-MS) was performed with an anti-CDK13 in PC3 cells to detect the proteins interacting with CDK13, which are shown on the right panel. b , Heat map showing the differential expression (fold changes) of mRNAs between PCa and BPH tissues. Red color indicates several genes that are known to be transcriptionally upregulated in PCa tissues. c , CoIP analysis was used to detect the interaction between CDK13 and E2F5, and β-actin was used as a negative control. d , in situ proximity ligation (PLA) analysis detected the interaction between CDK13 and E2F5. Red color indicates PLA-positive cells. e , Immunofluorescence staining was performed to detect the expression and location of CDK13 and E2F5 in PCa and BPH tissues. Scale bars = 100 μm. f , E2F5 mRNA level was determined by RT-qPCR in 30 pairs of PCa and normal prostate tissues. ** P < 0.01 vs. normal tissues. g , The expression levels of E2F5 mRNA in BPH and PCa tissues from the GSE13507 database ( P < 0.0001). h , E2F5 protein expression was detected by Western blotting in 3 pairs of randomly selected BPH (B) and PCa (P) tissues. i , Immunohistochemistry staining detected the E2F5 protein expression in PCa and BPH tissues. j and k , the correlation between CDK13 and E2F5 mRNA expression in the PCa tissues was analyzed by Pearson correlation analysis in our clinical data ( R = 0.4928, P = 0.0049) or several other data published in TCGA database (Yu: R = 0.3959, P = 0.0001; Wallace: R = 0.3752, P = 0.0015; Glinsky: R = 0.238, P = 0.0347)

Article Snippet: Microarray hybridization analysis of mRNA expression in 2 PCa samples and 2 BPH were performed according to the manufacturer’s protocol (Arraystar, Inc., Rockville, MD, USA).

Techniques: Immunoprecipitation, Mass Spectrometry, Quantitative Proteomics, Negative Control, In Situ, Ligation, Immunofluorescence, Staining, Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK13 upregulation-induced formation of the positive feedback loop among circCDK13, miR-212-5p/miR-449a and E2F5 contributes to prostate carcinogenesis

doi: 10.1186/s13046-020-01814-5

Figure Lengend Snippet: Transcriptional activation of endogenous CDK13 upregulates E2F5 expression by promoting circCDK13 formation. a , PC3 cells were transfected with CDK13 sgRNA, oeCDK13 or their respective control vector, and then Western blot analysis detected the CDK13 and E2F5 expression. b , PC3 cells were treated as in (A), RT-qPCR detected E2F5 mRNA expression. c and d , PC3 cells were transfected with shCDK13 or pLKO or treated with CDK13 inhibitor THZ531, and then Western blot (C) and RT-qPCR (D) analysis detected the CDK13 and E2F5 expression. e and f , PC3 cells were transfected with CDK13 sgRNA or NC sgRNA and treated with actinomycin D (Act D). Western blotting (E) detected E2F5 protein level, and RT-qPCR (F) detected the expression of circRNAs (hsa_circ_0001699, hsa_circ_0079929, hsa_circ_0079933 and hsa_circ_0079939). * P < 0.05 vs. NC sgRNA+DMSO; # p <0.05 vs. CDK13 sgRNA+DMSO. g , PCR was used to detect hsa_circ_0079929 (termed circCDK13) in PCa tissues by using convergent or divergent primers. Divergent primers amplify circCDK13 in cDNA but not in genomic DNA (gDNA). GAPDH was used as linear control. h , RT-PCR amplified full-length circCDK13 in PC3 cells, and the amplified products were confirmed by agarose gel electrophoresis. i , Sanger sequencing confirmed head-to-tail splicing of circCDK13. j , RT-qPCR detected the expression of circCDK13 in PCa and BPH tissues. ** P < 0.01 vs. BPH. k , The correlation between circCDK13 and E2F5 mRNA expression in our clinical data was analyzed by Pearson correlation analysis ( R = 0.3976, P = 0.0296). l , PC3 and 22RV1 cells were transfected with si-circCDK13, si-linear CDK13 or si-Ctl. RT-qPCR detected circCDK13 and CDK13 mRNA expression. Bars are mean ± SEM of triplicate samples. * P < 0.05, ** P < 0.01 vs. si-Ctl. m , RT-qPCR examined the expression of CDK13 mRNA and circCDK13 in PC3 and 22RV1 cells transfected with circCDK13 or empty vector. * P < 0.05, ** P < 0.01 vs. empty vector. n , Western blotting detected the E2F5 expression in PC3 and 22RV1 cells transfected with si-circCDK13, circCDK13 or their respective control. o , PC3 and 22RV1 cells were transfected with circCDK13 and shE2F5 either alone or together, and then colony formation assay was performed to detect the cell proliferation. * P < 0.05 vs. pLKO+pWPI, # p <0.05 vs. circCDK13 + pLKO

Article Snippet: Microarray hybridization analysis of mRNA expression in 2 PCa samples and 2 BPH were performed according to the manufacturer’s protocol (Arraystar, Inc., Rockville, MD, USA).

Techniques: Activation Assay, Expressing, Transfection, Control, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Sequencing, Colony Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK13 upregulation-induced formation of the positive feedback loop among circCDK13, miR-212-5p/miR-449a and E2F5 contributes to prostate carcinogenesis

doi: 10.1186/s13046-020-01814-5

Figure Lengend Snippet: circCDK13 upregulates E2F5 protein level by sequestering miR-221-5p/449a a , RT-qPCR detected the expression of E2F5 mRNA in PC3 cells transfected with circCDK13, si-circCDK13 or their respective control. b , Venn diagram displaying potential microRNAs associated with circCDK13 sequence from three online target-prediction programs. c , circCDK13 or empty vector was transfected into PC3 cells, RT-qPCR detected the pulled down efficiency of circCDK13 by biotinylated probes against circCDK13. * P < 0.05, ** P < 0.01 vs. con probe or empty vector. d , RT-qPCR detected the level of indicated miRNAs pulled down by biotinylated probes against circCDK13. * P < 0.05, ** P < 0.01 vs. con probe. e , Biotin-labeled E2F5 3′-UTR RNA was transfected into PC3 cells followed by a biotin pull-down assay using Streptavidin-coupled Dynabeads. The miRNAs were extracted from the sedimented beads, and the relative levels of 11 candidate miRNAs were detected by RT-qPCR. * P < 0.05, ** P < 0.01 vs. control E2F5 3′-UTR. f , PC3 cells were co-transfected with miR-449a mimic, miR-212-5p mimic or both and circCDK13-directed luciferase reporter. Luciferase activity was measured by dual-luciferase reporter assays. * P < 0.05, ** P < 0.01 vs. miR-NC. g , RNA in situ hybridization detected the co-localization between miR-449a or miR-212-5p with circCDK13 (arrowheads) in PC3 cells co-transfected with circCDK13 expression vector and miR-449a or miR-212-5p mimic. Nuclei were counterstained with DAPI. Scale bars = 25 μm. h , PC3 cells were co-transfected with miR-449a, miR-212-5p or control mimic (miR-NC) and wild type or mutated (mut) E2F5 3’UTR-directed luciferase reporter. Luciferase activity was measured by dual-luciferase reporter assays. Graph bars are mean ± SEM of 3 independent experiments. * P < 0.05, ** P < 0.01 vs. miR-NC or E2F5 3’UTR mut. i , PC3 and 22RV1 cells were transfected with miR-212-5p, miR-449a mimic or both, and then Western blotting detected the CDK13 and E2F5 expression.

Article Snippet: Microarray hybridization analysis of mRNA expression in 2 PCa samples and 2 BPH were performed according to the manufacturer’s protocol (Arraystar, Inc., Rockville, MD, USA).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Control, Sequencing, Plasmid Preparation, Labeling, Pull Down Assay, Luciferase, Activity Assay, RNA In Situ Hybridization, Western Blot