Fig 1: miR-29b-1-5p target 3′-UTR of RAP1B and regulates cellular function of WJ-MSCs. a 3′ UTR sequences containing the predicted “seed region” target site of RAP1B and its mut-sequences were cloned into the psiCHECK2.0 vector to prove whether RAP1B was the direct target gene of miR-29b-1-5p. b Dual-luciferase reporter gene assay to detect interaction between miR-29b-1-5p and RAP1B. RAP1B-mut, RAP1B 3′UTR mutant type; RAP1B-wt, RAP1B wild type; mimics NC, empty vector control. **P < 0.001. c, d qRT-PCR was used to determine the miR-29b-1-5p expression level of WJ-MSCs transfected with mimics/inhibitor. **P < 0.001 compared to mimics/inhibitor NC group. e qRT-PCR was used to determine the RAP1B mRNA expression level of WJ-MSCs in four groups. **P < 0.001. f Western blot assay was performed to verify whether miR-29b-1-5p affected the RAP1B protein level of WJ-MSCs. g Quantitative analysis of western blot assay. Data in panel are shown as means ± standard deviation (n = 3). *P < 0.05, **P < 0.001
Fig 2: si-DLEU2 suppresses oral cancer cell growth via the MAPK pathway. (a) Western blot analysis of si-DLEU2-mediated protein levels of RAP1B, p38, and p-p38. (b) Western blot analysis of si-DLEU2-/ov-RAP1B-mediated protein levels of RAP1B, p38, and p-p38. (c, d) CCK8 analysis of the effect of si-DLEU2/ov-RAP1B coaction on the proliferation rate of Tca8113 (c) and CAL-27 (d) cells. (e, f) Transwell analysis of the effect of si-DLEU2/ov-RAP1B coaction on migration and invasion of Tca8113 (e) and CAL-27 (f) cells. ∗P < 0.05.
Fig 3: RAP1B targets miR-30a-5p and reverses the effects of si-DLEU2 in Tca8113 and CAL-27 cells. (a) Scatter plot shows differentially expressed mRNAs between the oral cancer and control groups. (b) KEGG pathway enrichment analysis. (c) The key mRNAs affecting the MAPK signaling pathway were combined with putative target genes of miR-30a-5p in the starBase database to screen for suitable mRNAs. (d) Relative expression of DLEU2 in RWPE-2 and four oral cancer cell lines (C4-2, 22RV1, Tca8113, and CAL-27). (e) The TargetScan database predicts the binding sites of miR-30a-5p and RAP1B. (f) Dual-luciferase assay analysis of the binding of RAP1B to miR-30a-5p. (g) RT-qPCR analysis of the effect of si-DLEU2 on the expression of RAP1B in Tca8113 and CAL-27 cells. (h) RT-qPCR verified the validity of overexpressing RAP1B plasmid. (i) Western blot verified the validity of overexpressing RAP1B plasmid. (j) RT-qPCR analysis of the reversal of DLEU2 expression by overexpressing RAP1B on si-DLEU2. ∗P < 0.05.
Fig 4: circ6401 acts as a sponge for miR-29b-1-5p and prevents it from inhibiting the translation of RAP1B in WJ-MSCs. a RNA FISH assay for subcellular co-localization of circ6401 and miR-29b-1-5p in WJ-MSCs. Cy3 labeled the circ6401 FISH probe, FITC labeled the miR-29b-1-5p FISH probe, and DAPI stained the cell nuclei. Scale bar, 20 μm. b 3′ UTR sequences containing the predicted “seed region” target site of circ6401 and its mut-sequences were cloned into the psiCHECK2.0 vector to prove whether circ6401 was the direct target gene of miR-29b-1-5p. c Dual-luciferase reporter gene assay to detect interaction between miR-29b-1-5p and circ6401. circ6401-mut, circ6401 3′UTR mutant; circ6401-wt, circ6401 wild type; mimics NC, empty vector control. *P < 0.05. d qRT-PCR was used to determine miR-29b-1-5p expression level of WJ-MSCs transfected with lenti-NC/lenti-circ6401. Lenti-NC, lentivirus with empty vector; lenti-circ6401, lentivirus with circ6401 overexpressing vector. *P < 0.05. e RAP1B mRNA expression in WJ-MSCs after co-transfection with circ6401 overexpression vectors and miR-29b-1-5p mimics. Data in panel are shown as means ± standard deviation (n = 3). **P < 0.001. #P < 0.05 compared to lenti/mimics NC group; ##P < 0.001 compared to lenti/mimics NC group; nsP > 0.05 compared to lenti/mimics NC group
Fig 5: miR-29b-1-5p is downregulated during endometrium repair by WJ-MSCs. a The differences in miRNA expression profiles between the two groups by a miRNA microarray analysis are presented in the form of a heat map. N1–3, non-cocultured WJ-MSCs; T1–3, cocultured WJ-MSCs. Each group consisted of three individuals. miRNAs are represented by single rows and samples by single columns. b Volcano plot of miRNA microarray analysis data. The values on the X- and Y-axes represent normalized fold changes and P values, respectively. The color scale indicates relative expression, upregulation (red) and downregulation (green). miRNAs with fold change > 2 and P ≤ 0.05 were regarded as differentially expressed. c Circos plot shows the most significantly upregulated (red) and downregulated (green) miRNA expression profiles in the entire genome. d miR-29b-1-5p expression in WJ-MSCs cocultured with or without damaged ESCs by qRT-PCR. N, non-cocultured WJ-MSCs; T, cocultured WJ-MSCs. Data in panel are shown as means ± standard deviation (n = 3). **P < 0.001. e The complementary pairing sequence between 3′UTR of RAP1B and miR-29b-1-5p
Supplier Page from Abcam for Anti-RAP1B antibody