Fig 1: DRAIC inhibits cell proliferation and migration by regulating the miR-34a-5p/ITGA6 signal axis. 293T and SH-SY5Y cells were co-transfected with miR-34a-5p mimic and ITGA6 overexpression plasmid. (a) Cell proliferation was measured by CCK-8 assay. (b) Cell migration was analyzed by Transwell assay. Data are reported as mean ± SD. **P < 0.01, versus mimic NC +vector group. ##P < 0.01 versus miR-34a-5p mimic +vector group.
Fig 2: MiR-126 directly bound to ITGA6. a The binding site between miR-126 and ITGA6 3'UTR was analyzed by miRDB website. b, c The relationship between miR-126 and ITGA6 was validated by dual-luciferase reporter assay. d, e The relationship between miR-126 and ITGA6 was validated by RIP assay. f The efficiency of miR-126 mimic and miR-126 inhibitor was examined using qRT-PCR. g, h The effects of miR-126 mimic and miR-126 inhibitor on the expression of ITGA6 was determined by qRT-PCR and western blot. i, j The expression of ITGA6 in serum-derived exosomes from NSCLC patients and healthy controls was detected by qRT-PCR and western blot. k, l The expression of ITGA6 in HBE, A549 and H460 cells was detected by qRT-PCR and western blot. *P < 0.05
Fig 3: DRAIC regulated the expression of ITGA6 in cells by sponging miR-34a-5p. (a) RNA pull-down assay was used to identify the relationship between the miR-34a-5p and DRAIC. (b) Luciferase activity experiment proved that miR-34a-5p and DRAIC had binding sites, and miR-34a-5p directly bind to ITGA6 3’UTR. (c) The expression level of miR-34a-5p was detected by qRT-PCR in 293T and SH-SY5Y cells transfected with DRAIC overexpression plasmid or interference sequence. (d, e) mRNA and protein expression levels of ITGA6 were determined by qRT-PCR and Western blot, respectively. The blue text shows the binding site of the two genes. Data are reported as mean ± SD. **P < 0.01, ***P < 0.001 versus Bio-NC or mimic NC and/or NC group. ##P < 0.01 versus DRAIC + mimic NC group.
Fig 4: Exosomal miR-126 suppressed NSCLC cell malignant behaviors in vitro by targeting ITGA6. a, b The expression of ITGA6 in serum-derived exosomes transfected with miR-126, miR-NC, miR-126 + ITGA6 or miR-126 + pcDNA was determined by qRT-PCR and western blot. A549 and H460 cells were co-cultured with these transfected exosomes. In these cells, c, d cell cycle was investigated by flow cytometry assay. e The capacity of colony formation was assessed by colony formation assay. f, g Cell proliferation was assessed by MTT assay. h, i Cell migration and cell inmivasion were determined by transwell assay. j, k The expression of E-cadherin, N-cadherin and Vimentin was detected by western blot. l Cell apoptosis was monitored by flow cytometry assay. *P < 0.05
Fig 5: Exosomal miR-126 inhibited tumor growth in vivo. a Tumor volume was recorded in nude mice after EXO-miR-126 treatment. b Tumor weight was measured in nude mice after EXO-miR-126 treatment. c The expression of miR-126 in these tumor tissues was detected by qRT-PCR. d, e The expression of ITGA6 in these tumor tissues was detected by qRT-PCR and western blot. f IHC staining assay showed the expression of Ki67 and Cleaved casp3 in tumor tissues. *P < 0.05
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