Fig 1: miR-20b-5p Promoted Tumor Growth and Metastasis In Vivo(A) miR-20b-5p expression levels in stable overexpressing KYSE180 cells (Lenti-mimic) and control cells (Lenti-vector). (B–D) Stable miR-20b-5p-overexpressing KYSE180 cells were subcutaneously injected into nude mice to form solid tumors, and representative images of tumor volumes and weights were analyzed by in vivo luciferase imaging on the last day of analysis (n = 5 for each group). (E and F) The metastatic nodules were observed in the lungs, brains, livers and bones of mice treated with stable miR-20b-5p-overexpressing KYSE180 cells or control vector cells by the vein injection method. (G–I) KYSE180 cells were subcutaneously injected into nude mice to form solid tumors and synchronously treated with miR-20b-5p antagomir or miRNA antagomir negative control (n = 5 for each group); a 10-nmol miR-20b-5p antagomir as well as the miRNA-negative control in 0.1 mL of saline buffer was locally injected into nude mice to treat tumor mass once every 5 days; tumor volumes (H) and weights (I) in nude mice are shown. (J) Immunohistochemical staining of Ki67, RB1, and TP53INP1 in tumor tissues dissected from nude mice treated with miR-20b-5p antagomir or the miRNA-negative control. (K) RB1 and TP53INP1 protein expression were measured by immunohistochemical staining in 103 ESCC samples and pair-matched esophageal tissues. @p < 0.05, @@p < 0.01.
Fig 2: Generation and identify of Biallelic RB1-mutated (RB1Mut/Mut) hESC lines(A) Bright field image shows the survived single-clones after resistance selection can be picked by mechanically cross-hatch the colony.(B) The location of the primers used to identify the recombination.
Fig 3: A Rescue Assay Was Performed to Confirm that RB1 and TP53INP1 Were the Functional Targets of miR-20b-5p(A and B) Protein and mRNA levels of RB1 (A) and TP53INP1 (B) in KYSE30 and KYSE180 cell lines cotransfected with miR-20b-5p mimic and pEGFP-C1 plasmid containing RB1 and TP53INP1 CDS sequence. (C–F) Transwell assay of cells cotransfected with miR-20b-5p mimic and RB1 or TP53INP1 plasmids. (G and H) The expression of RB1 (G) and TP53INP1 (H) at the mRNA and protein level after siRNA silencing in TE1 or EC109 cells. (I and J) Transwell assay after transfection with RB1 (I) or TP53INP1 (J) siRNA in TE1 or EC109 cells, respectively. Data are presented as the mean value ± SD from triplicate experiments. @p < 0.05, @@p < 0.01.
Fig 4: RB1 and TP53INP1 Were Two Direct Target Genes of miR-20b-5p(A and B) RB1 and TP53INP1 were identified as potential regulatory targets of miR-20b-5p by considering the downregulation of genes using prediction tools and qRT-PCR. (C and D) The expression levels of RB1 (C) and TP53INP1 (D) mRNA and protein were measured by qRT-PCR and western blot analysis using GAPDH as the loading control after transfection with miR-20b-5p mimic in the KYSE30 and KYSE180 cell lines, respectively. (E and F) The expression levels of RB1 (E) and TP53INP1 (F) mRNA and protein were measured by qRT-PCR and western blot analysis using GAPDH as the loading control after transfection with miR-20b-5p inhibitors in the TE1 and EC109 cell lines, respectively. (G–I) Dual-luciferase reporter assay. The relative luciferase activity was normalized to the Renilla luciferase activity assay after cotransfection with miR-20b-5p mimic and miR-RB-REPORT constructs containing WT or MUT RB1 and the TP53INP1 3′ UTR region in KYSE30 and KYSE180 cell lines. Data are presented as the mean value ± SD from triplicate experiments. @@p < 0.01.
Fig 5: Chromosome shortening during the metaphase upon the knockdown of cohesin components. a Venn diagram for identification of up- or downregulated genes from siRNA-mediated knockdown of SMC3, RAD21, and REC8 in ESCs. Data were adjusted with P < 0.1 and fold-change > 1.5. (Additional file 2: Table S1). b Comparison of gene expression of RB. RB transcript levels from qPCR and RNA sequencing experiments were analyzed to evaluate RB gene expression. The error bars are the mean ± SD (n = 3 for qPCR). The RB expression levels measured by RNA-Seq experiments are represented as average values of two biological replicates. c Analysis of RB protein levels following transfection with a siRNA pool against SMC3, REC8, RAD21, and STAG3 in ESCs. a-tubulin was used as a loading control. d Quantification of RB expression shown in c using Image Lab 6.0 software (Bio-Rad). Results are illustrated as mean ± SD value (n = 3). e Immunoprecipitation (IP) analysis for physical interaction between RB and CAP-D3 in ESCs. (i–iii) Western blotting; (iv) quantification of RB and CAP-D3 levels from IP results. The error bars are the mean ± SD from three independent biological replicates. f Immunofluorescence analysis of chromosome compaction in ESCs during the metaphase stage. Chromosomes were stained with anti-RB antibody, anti-CAP-D3, and DAPI. Ctrl, siControl; pRB, pEF1a-RB expression vector; siSMC3, siRAD21, siREC8, STAG3, and siCohesin/siRB1 indicate siRNA treatment against SMC3, RAD21, REC8, STAG3, and cohesin/RB1 respectively. g,h Intensity of RB and CAP-D3 protein signals per a chromosome in ESCs. a.u., arbitrary unit. The data are reported as mean ± SD values from three biological replicates (n > 100 for condition). Statistics: Paired two-tailed t-test; ns, not significant; ***P < 0.001
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