Fig 1: hsa_circ_0003176 suppressed the proliferation, migration, and invasion of NSCLC cells by upregulating RBM5 expression. (a) The mRNA expression of RBM5 in NSCLC cell lines (A549 and H1299) transfected with hsa_circ_0003176 overexpression plasmid alone or cotransfected with shRBM5 plasmid was detected using RT-qPCR. (b) The protein expression of RBM5 NSCLC cell lines (A549 and H1299) transfected with hsa_circ_0003176 overexpression plasmid alone or cotransfected with shRBM5 plasmid was detected using western blotting. (c) The viability of NSCLC cell lines (A549 and H1299) transfected with hsa_circ_0003176 overexpression plasmid alone or cotransfected with shRBM5 plasmid was detected using CCK-8 assay. (d) The colony formation ability of NSCLC cell lines (A549 and H1299) transfected with hsa_circ_0003176 overexpression plasmid alone or cotransfected with shRBM5 plasmid was detected using colony formation assay. (e) The migration ability of NSCLC cell lines (A549 and H1299) transfected with hsa_circ_0003176 overexpression plasmid alone or cotransfected with shRBM5 plasmid was detected using wound-healing assay. (f) The invasion ability of NSCLC cell lines (A549 and H1299) transfected with hsa_circ_0003176 overexpression plasmid alone or cotransfected with shRBM5 plasmid was detected using transwell invasion assay. Mock indicated negative control corresponds to hsa_circ_0003176 overexpression plasmid, and shNC indicated negative control corresponds to shRBM5 plasmid. Data was shown as mean ± SD. N = 3 ~ 5, ∗∗p < 0.01, and ∗∗∗p < 0.001.
Fig 2: Hsa_circ_0003176 suppressed miR-185-5p expression by sponging miR-182-5p. (a) The expression of miR-182-5p in NSCLC cell lines (H1299 and A549) and normal control cell line (16HBE) was detected by RT-qPCR. N = 3. (b) The expression of miR-182-5p was detected by RT-qPCR in 10 NSCLC tissues and matched adjacent normal tissues. (c) Pearson correlation coefficient was used to analyze the correlation between the RBM5 expression and miR-182-5p expression in 10 NSCLC tissues. (d) RNA pull-down assay was used to detect the binding relationship between miR-182-5p and hsa_circ_0003176. N = 3. (e) The dual-luciferase reporter assay was used to determine the direct interaction between miR-182-5p and hsa_circ_0003176. NC mim. indicated negative control mimics, and miR mim. indicated miR-182-5p mimics. N = 6. (f) RT-qPCR was used to evaluate the expression of miR-182-5p in NSCLC cells (H1299 and A549) transfected with or without hsa_circ_0003176 overexpression plasmid. N = 3. (g) RT-qPCR was used to evaluate the effect of hsa_circ_0003176 on miR-182-5p expression in vivo. N = 6. Data was shown as mean ± SD. ∗p < 0.5, ∗∗p < 0.01, and ∗∗∗p < 0.001.
Fig 3: hsa_circ_000 3176 regulates RBM5 expression by regulating miR-182-5p. (a) The mRNA expression of RBM5 in NSCLC cell lines (H1299 and A549) and normal control cell line (16HBE) was detected by RT-qPCR. N = 3. (b) The mRNA expression of RBM5 was detected by RT-qPCR in 10 NSCLC tissues and matched adjacent normal tissues. (c) Putative binding sites of miR-182-5p with respect to RBM5 were predicated. (d) The luciferase activity of RBM5 in the NSCLC cells after cotransfection with miR-182-5p mimics or NC mimics and RBM5-WT or RBM5-MUT. (e) RT-qPCR was used to detect the expression of RBM5 in NSCLC cell lines (A549 and H1299) transfected with hsa_circ_0003176 overexpression plasmid alone or cotransfected with miR-182-5p mimics. Data was shown as mean ± SD. N = 3 ~ 6, ∗p < 0.5, and ∗∗∗p < 0.001.
Fig 4: RNA binding motif 5 is lowly expressed in colorectal cancer tissues and cells. A: The mRNA expression of RNA binding motif 5 (RBM5) in tumor tissues and normal tissues was analyzed via the Gene Expression Profiling Interactive Analysis; B: Relative mRNA expression of RBM5 in fetal human cell (FHC), HT29, HCT116, LOVO, SW480 and SW620 cells was detected by quantitative real time-polymerase chain reaction; C: Relative protein expression of RBM5 in FHC, HT29, HCT116, LOVO, SW480, and SW620 cells was detected by Western blot. RBM5: RNA binding motif 5. aP < 0.05, bP < 0.01, cP < 0.001.
Fig 5: RNA binding motif 5 overexpression represses proliferation, migration, invasion and glycolysis of colorectal cancer cells. A: Relative protein expression of RNA binding motif 5 in HCT116 and SW480 cells was detected by Western blot; B: Cell counting kit-8 (CCK-8) assay was used to assess the viability of HCT116 and SW480 cells; C: 5-ethynyl-2’-deoxyuridine assay was used to assess proliferation of HCT116 and SW480 cells; D and E: Migration and invasion of HCT116 and SW480 cells were evaluated by transwell assays; F: Glucose consumption, lactic acid production, and adenosine-triphosphate production were detected by corresponding kits; G: Protein levels of hexokinase 2 and lactate dehydrogenase A were detected by Western blot in HCT116 and SW480 cells. RBM5: RNA binding motif 5; EDU: 5-ethynyl-2’-deoxyuridine; HK2: Hexokinase 2; LDHA: Lactate dehydrogenase A. bP < 0.01, cP < 0.001.
Supplier Page from Abcam for Anti-RBM5 antibody