Fig 1: LncRNA MBNL1-AS1 may affect NSCLC via TGFBR2 by sponging miR-301b-3p. a Heat map of the 50 DEGs retrieved from GSE101929. The abscissa indicated the sample number and the ordinate indicated the DEG. The upper right histogram indicated color gradation; each rectangle in the figure corresponded to the level of a gene in a sample. b Subcellular localization of lncRNA MBNL1-AS1 in LncATLAS. CN RCI refers to the ratio of lncRNA expression in cytoplasm and nucleus. FPKM refers to fragments per kilobase of exon model per million mapped fragments. c Comparisons of lncRNA MBNL1-AS1-targeted miRNAs in RAID database and differential miRNAs in GSE102286. d Expression of miR-301b, miR-30d, miR-218, and miR-30a in GSE102286 chip. Blue indicated the adjacent normal tissues and red indicated NSCLC tissues. e Comparisons of miR-301b-3p-targeted genes in mirDIP, miRDB, miRSearch, RNA22, and miRTarBase databases and DEGs from GSE33532 chips. f TGFBR2 expression in GSE33532 chips. DEGs, differentially expressed genes; MBNL1-AS1, muscleblind-like 1-antisense RNA 1; NSCLC, non-small cell lung cancer; TGFBR2, transforming growth factor beta receptor II
Fig 2: LncRNA MBNL1-AS1 and miR-301b-3p regulate the TGF-β pathway to modulate the biological activity of A549-SP+. a, b Levels of TGFBR2, smad2/3, and p-smad2/3 determined using western blot analysis. c Levels of Oct4 and ABCG2 in A549-SP+ assessed using immunofluorescence staining; *p < 0.05 vs. A549 cells treated with NC. Measurement data were depicted as mean ± standard deviation. Comparisons among multiple groups were analyzed using one-way ANOVA with Tukey post hoc test used. The experiment was repeated three times
Fig 3: LncRNA MBNL1-AS1 increases TGFBR2 expression via competitive combination with miR-301b-3p. a Sequence complementarity between miR-301b-3p and TGFBR2 3'-UTR detected by bioinformatics analysis. b mRNA and protein levels of TGFBR2 in NSCLC tissues and adjacent normal tissues measured using RT-qPCR and western blot analysis (n = 56). c Luciferase activity after co-transfection. d Binding situation between miR-301b-3p and TGFBR2 verified using RNA pull-down. e TGFBR2 mRNA level in A549-SP+ measured using RT-qPCR. f TGFBR2 protein level in A549-SP+ measured using western blot analysis. NC group: cells treated with NC. miR-301b-3p-inhibitor group: cells treated with miR-301b-3p inhibition. miR-301b-3p-mimic group: cells treated with miR-301b-3p overexpression. miR-NC + oe-lncRNA MBNL1-AS1 group: cells treated with lncRNA MBNL1-AS1 overexpression and miR-NC. miR-301b-3p-mimic + oe-lncRNA MBNL1-AS1 group: cells treated with lncRNA MBNL1-AS1 overexpression and miR-301b-3p overexpression; *p < 0.05 vs. the adjacent normal tissues, co-transfection with miR-NC and TGFBR2-wt, A549 cells treated with Bio-NC, or A549 cells treated with NC. Measurement data were depicted as mean ± standard deviation. b Data was analyzed using the paired t test. c, d Comparisons between two groups were analyzed using unpaired t test. e Comparisons among multiple groups analyzed using one-way ANOVA with Tukey post hoc test used. The experiment was repeated three times. Bio, biotin
Fig 4: Potential mechanism of lncRNA MBNL1-AS1 involved in the biological activity of NSCLC CSCs via regulation of TGFBR2. In NSCLC CSCs, lncRNA MBNL1-AS1 sponged to miR-301b-3p that negatively regulated TGFBR2, and downregulation of lncRNA MBNL1-AS1 weakened the adsorption capacity, increased miR-301b-3p expression, and reduced TGFBR2 level, thus promoting the proliferation, migration, invasion, drug resistance, and sphere formation yet suppressing the activation of TGF-ß pathway. By contrast, lncRNA MBNL1-AS1 overexpression strengthened the adsorption capacity of miR-301b-3p, accompanied by elevated TGFBR2 expression, hence resulting in a decline of the proliferation, migration, invasion, drug resistance, and sphere formation yet an increase in activation of TGF-ß pathway
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