Fig 1: MEG3/miR-376a loop regulates tube formation. (A) TargetScan prediction genes were collected from the online prediction website. Predicted candidate genes were then analyzed according to the PANTHER classification system. The listed genes are associated with the process of angiogenesis. (B) qRT-PCR analysis depicting the changes in angiogenesis-related genes in ODMECs treated with control mimics and miR-376a mimics. (C) Prediction of miR-376a target sequences in the 3′-UTR of the RASA1 gene and its mutant. (D) Luciferase assay after co-transfection of ODMECs with the wild-type or mutant RASA1 reporter and miR-376a mimics or the control mimics. (E) qRT-PCR analysis of RASA1 expression in ODMECs treated with MEG3 or miR-376a mimics. (F) Tube formation by ODMECs treated with MEG3, si-control, or si-RASA1. Scale bar = 100 μm. (G) Quantified data from F.
Fig 2: Expression of miRNA target mRNAs and proteins. (A) Relative expression levels of HGS and VEGFR2 in LoVo-CM-treated HUVECs relative to SW620-CM. (B) Relative expression levels of HGS and VEGFR2 in LoVo-CM-treated HUVECs relative to DMH4 treatment. The mRNA levels were normalized GAPDH, and the results are a summary of two technical repeats from three biological replicates. (C) Upper panel: western blots of HGS. Lower panel: western blots of GAPDH for loading control with for HGS. (D and E) Relative expression levels of PIK3R1 and RASA1 in (E) LoVo-CM-treated HUVECs relative to SW620-CM and (F) in LoVo-CM-treated HUVECs relative to DMH4 treatment. (F) Western blots of RASA1 and VEGFR2. Lower panel: western blots of GAPDH for loading control with for VEGFR2 and RASA1. (G and H) Graphs showing the relative expression levels of indicated protein targets, which are normalized to the GAPDH. Error bars, SEM. miRNA, miR, microRNA; HGS, hepatocyte growth factor-regulated tyrosine kinase substrate; VEGFR2, vascular endothelial growth factor receptor 2; HUVEC, human umbilical vein endothelial cell; CM, conditioned medium; RASA1, RAS P21 protein activator 1.
Fig 3: miR-335-5p inhibits CRC cell migration, invasion, and EMT by targeting RASA1(A) Prediction of target genes of miR-335-5p using three computational target prediction algorithms (TargetScan 50, miRDB, and miRTarBase). (B) Correlation analysis of miR-335-5p and RASA1 mRNA expression levels in CRC tissues based on TCGA data. ∗p < 0.05. (C) Wild-type (WT) and mutant (mut) binding sites of miR-335-5p in the 3′ UTR of RASA1 and associated luciferase assay. SW480 cells were co-transfected with either negative control (NC) or miR-335-5p mimics and luciferase reporter plasmids, including RASA1 3′ UTR WT or RASA1 3′ UTR mut. Data are presented as mean ± SD (n = 3). ∗p < 0.05. (D) Expression of RASA1 mRNA was determined by quantitative RT-PCR in SW480 and SW620 cells transfected with NC or miR-335-5p mimics. ∗∗p < 0.01. (E) Western blot analysis of RASA1 and Ras protein expression in SW480 and SW620 cells transfected with NC or miR-335-5p mimics. (F) Western blot analysis of E-cadherin and vimentin protein expression in SW480 and SW620 cells transfected with NC or miR-335-5p mimics. (G) Determination of the migration and invasion abilities of SW480 and SW620 cells transfected with NC or miR-335-5p mimics using a Transwell assay. Data are presented as mean ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01. Scale bars, 100 μm. (H) Determination of the migration and invasion abilities of SW480 and SW620 cells transfected with NC or miR-335-5p inhibitor using a Transwell assay. Data are presented as mean ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01. Scale bars, 100 μm.
Fig 4: Upregulation of RASA1 suppresses cell growth and enhances apoptosis. (A) Expression of RASA1 mRNA was enhanced following overexpression of RASA1. (B) Expression of RASA1 was increased following upregulation of RASA1 compared with the negative control. (C) Cell proliferation was investigated by performing Cell Counting Kit-8 assays, and it was demonstrated that overexpression of RASA1 significantly suppresses cell growth. (D) Cell apoptosis was analyzed by flow cytometry, and overexpression of RASA1 was revealed to significantly enhance apoptosis in human umbilical vein endothelial cells. *P<0.05; **P<0.01; ***P<0.001. RASA1, Ras p21 protein activator 1; OD, optical density; APC, allophycocyanin.
Fig 5: Exosome-transmitted miRNA-335-5p promotes CRC cell invasion, metastasis, and EMT, as well as decreases the level of RASA1(A) Determination of the migration and invasion abilities of SW480 cells treated with NC-exo (SW480-exo transfected with NC miRNA) or miR-335-5p mimics-exo (SW480-exo transfected with miR-335-5p mimics) using a Transwell assay. Data are presented as mean ± SD (n = 3). ∗∗p < 0.01, ∗∗∗p < 0.001. Scale bars, 100 μm. (B) Determination of the migration and invasion abilities of SW620 cells treated with NC-exo or miR-335-5p mimics-exo using a Transwell assay. Data are presented as mean ± SD (n = 3). ∗∗p < 0.01. Scale bars, 100 μm. (C) Western blot analysis of RASA1 and Ras protein expression levels in SW480 and SW620 cells treated with NC-exo or miR-335-5p mimics-exo. (D) Western blot analysis of E-cadherin and vimentin protein expression levels in SW480 and SW620 cells treated with NC-exo or miR-335-5p mimics-exo. (E) Bioluminescence assay evaluating tumor metastases in mice. SW480 cells transduced with a lentiviral vector encoding the firefly luciferase gene (3 × 106 cells per mouse, n = 6) were injected into the tail vein of NOD/SCID mice, and the in vivo bioluminescent signal was quantified at the 9th week. Representative in vivo images of NC-exo or miR-335-5p mimics-exo treated mice are shown. Data are presented as mean ± SD (n = 6). ∗∗p < 0.01. (F) Representative photographs of macroscopic lung appearance. The number of metastatic tumor nodules for each group is shown on the right panel. Data are presented as mean ± SD (n = 6). ∗∗p < 0.01.
Supplier Page from Abcam for Anti-RASA1 antibody [EP536Y]