Fig 1: 786-O-EVs carry MALAT1 to regulate transcription factor ETS1 and reduce TFCP2L1 activity. (A) Prediction of MALAT1 regulatory transcription factors in KIRC and KIRP; the two circles in the figure represent the predicted transcription factors in KIRC and KIRP, and the middle part represents the intersections of the two sets of data. (B) Network diagram of interaction analysis between candidate transcription factors and genes known related to RCC; the diamonds represent the screened four candidate transcription factors, and the circles represent RCC-related genes obtained from the DisGeNET database (https://www.disgenet.org/); the darker color of the graph represents higher core degree of the gene in the network diagram and more interaction genes. (C) Prediction of ETS1 candidate target genes; the circle on the right side represents the downregulated genes in the GSE16441 chip, and the left side shows the prediction results of JASPAR database (http://jaspar.genereg.net/) on ETS1 target gene, and the middle part represents the intersection of the two groups of data. (D) FISH was used to detect subcellular localization of MALAT1. (E) Dual-luciferase reporter gene assay was used to verify the effect of MALAT1 on TFCP2L1 promoter activity. (F) RIP was used to detect the binding of MALAT1 to transcription factor ETS1. (G and H) RNA pull-down was used to detect the binding relationship between ETS1 and TFCP2L1 promoter. (I) qPCR was used to detect the mRNA expression of TFCP2L1. Each experiment was repeated three times independently. Data in panel E were analyzed using one-way ANOVA, and data in panel F were analyzed by independent t-test and data in panels H and I were analyzed using two-way ANOVA, followed by Tukey's multiple comparisons test. **P<0.01. EVs, extracellular vesicles; MALAT1, metastasis-associated lung adenocarcinoma transcript 1; RCC, renal cell carcinoma; TFCP2L1, transcription factor CP2 like 1; ETS1, ETS proto-oncogene 1, transcription factor.
Fig 2: LncRNA PAXIP1-AS1/ETS1/KIF14 axis affects the development of glioma in vivo. a, The representative images and volume quantitation of xenograft tumors. b, The weight quantitation of xenograft tumor. c, Western blot analysis of expression of E-Cadherin, N-Cadherin, MMP-2, VEGF-A, ETS1 and KIF14 normalized to GAPDH in xenograft tumors. d, The expression of CD31 examined by immunohistochemistry to calculate MVD in xenograft tumors (200 ×). * p < 0.05 vs. the oe-NC + sh-NC group, # p < 0.05 vs. the oe-PAXIP1-AS1 + oe-ETS1 + sh-NC group. The values are measurement data and expressed as mean ± standard deviation. Comparisons among multiple groups were made using one-way ANOVA with Tukey’s post hoc test. Comparisons between time-based measurements were performed with repeated measures ANOVA, followed by Bonferroni post hoc test. N = 10
Fig 3: LncRNA PAXIP1-AS1 regulates the expression of KIF14 by recruiting the transcription factor ETS1. a, Results of bioinformatics predictions. b, KIF14 expression in the glioma-related microarray dataset GSE104291. c, Western blot analysis of KIF14 expression normalized to GAPDH in glioma tissues, * p < 0.05 vs. the normal brain tissues. D, Western blot analysis of KIF14 expression normalized to GAPDH in TJ905 cells, * p < 0.05 vs. HAs. E, Pearson’s correlation analysis between lncRNA PAXIP1-AS1 and KIF14 expression in glioma tissues (n = 44). f, The dual luciferase reporter gene assay to examine the effect of lncRNA PAXIP1-AS1 on the KIF14 promoter activity, * p < 0.05 vs. the oe-NC group, # p < 0.05 vs. the sh-NC group. g, RIP assay verified that lncRNA PAXIP1-AS1 could bind to transcription factor ETS1, * p < 0.05 vs. IgG. h, The two sites of the KIF14 DNA promoter region that transcription factor ETS1 most likely to bind to. i, The truncated KIF14 recombinant luciferase reporter vector and ETS1 expression vector were constructed for transfection into TJ905 cells for dual luciferase reporter gene assay, * p < 0.05 vs. the oe-NC group. j, The mutant KIF14 recombinant luciferase reporter vector and ETS1 expression vector were co-transfected into TJ905 cells for dual luciferase reporter gene assay, * p < 0.05 vs. the oe-NC group. k, ChIP assay for the binding ability of ETS1 at binding site 2 of KIF14 promoter region, * p < 0.05 vs. IgG. l, ChIP assay for the enrichment of KIF14 by ETS1 after silencing lncRNA PAXIP1-AS1 in TJ905 cells, # p < 0.05 vs. the sh-NC group. m, Western blot analysis for the expression of KIF14 and ETS1 normalized to GAPDH in each group, * p < 0.05 vs. the oe-NC + sh-NC group, # p < 0.05 vs. the PAXIP1-AS1 + sh-NC group. The values are measurement data and expressed as mean ± standard deviation. Comparison of normally distributed data between two paired groups with homogeneity of variance was performed using a paired t test. The unpaired t test was used to compare two sets of data from independent groups with normal distribution and homogeneity of variance. Data comparisons between multiple groups were performed using one-way ANOVA with Tukey’s post hoc test. Experiments were repeated three times independently
Fig 4: Overexpressed lncRNA PAXIP1-AS1 affects glioma cell migration, invasion, and angiogenesis by recruiting transcription factor ETS1 to upregulate KIF14 expression. a, Transwell assay to examine the migration ability of glioma cells (200 ×). b, Transwell assay to examine the invasive ability of glioma cells (200 ×). c, The tube formation assay to examine angiogenesis of HUVECs cocultured with TJ905 cells (100 ×). d, Western blot analysis for expression of E-Cadherin, N-Cadherin, MMP-2, VEGF-A, ETS1 and KIF14 normalized to GAPDH in glioma cells. * p < 0.05 vs. the oe-NC + sh-NC group, # p < 0.05 vs. the oe-PAXIP1-AS1 + oe-ETS1 + sh-NC group. The values are measurement data and expressed as mean ± standard deviation. Comparisons among multiple groups were made by one-way ANOVA with Tukey’s post hoc test. Experiments were repeated three times independently
Fig 5: Mechanism model diagram. lncRNA MALAT1 carried by 786-O-EVs promotes the invasion and migration of renal cell carcinoma cells by regulating transcription factor ETS1 and affecting TFCP2L1 activity. lncRNA, long non-coding RNA; EVs, extracellular vesicles; MALAT1, metastasis-associated lung adenocarcinoma transcript 1; RCC, renal cell carcinoma; TFCP2L1, transcription factor CP2 like 1; ETS1, ETS proto-oncogene 1, transcription factor.
Supplier Page from Abcam for Anti-ETS1 antibody