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. 2019 Dec 19;48(3):1175–1191. doi: 10.1093/nar/gkz1149

Figure 6.

Figure 6.

Interaction of PTTG3P ncRNA with FOXM1 augments BUB1B transcriptional activity to enhance cell proliferation and drug resistance. (A) 1543 DEGs were shared between the upregulated genes in PTTG3P-overexpressing A549 cells and the downregulated genes in PTTG3P-knockdown H1299 cells (GSE114826), which were further analyzed with GSEA and WebGestalt. (B) Genes in the PTTG3P-associated networks in five LUAD datasets that were ranked according to their kWithin value from the WGCNA were aligned with the common DEGs in the deposited transcriptomes in GSE114826 dataset. The GSE19188 dataset was excluded because no PTTG3P-associated module was constructed. Heat maps were individually generated on the basis of the Z-score by Heatmapper and show the upregulated (red) and downregulated (green) genes. A red arrow indicates the highest concordant gene in each dataset. (C) BUB1B expression was examined at the RNA level by real-time qPCR, and (D) its protein level was determined by western blotting analysis in H1299 and A549 cells. (E) Proliferation assays and (F) cell viability assays were performed after treatment with clinical drugs in PTTG3P knockdown H1299 cells overexpressing BUB1B with an EGFP-tag (sgPTTG3P-E-BUB1B) or in PTTG3P-overexpressing A549 cells with shBUB1B knockdown (PTTG3P-shBUB1B). (G) The cellular distribution of PTTG3P ncRNA detected by fluorescence RNA-ISH and the RNA expression of PTTG3P in the cytoplasmic, nucleoplasmic, and chromatin fractions were examined by real-time qPCR in both of H1299 and A549 cell lines. GAPDH-exon and NEAT1 RT-qPCR amplicons served as RNA markers for the cellular and nuclear fractions, respectively. (H) The transcriptional activities of different promoter regions of BUB1B were detected in A549 transfectants with luciferase reporter assays. (I) The binding region of PTTG3P ncRNA/FOXM1 versus FOXM1 alone on the BUB1B promoter was determined by a chromatin immunoprecipitation assay with an anti-FOXM1 antibody and real-time qPCR. (J) Western blotting analysis of FOXM1 obtained from the RNA pull-down assay was performed in nocodazole-synchronized H1299 cells. (K) PTTG3P ncRNA was immunoprecipitated with an anti-FOXM1 antibody and examined by real-time qPCR for an RNA-IP assay. (L) Chromatin isolation by RNA purification (ChIRP) in the presence of FOXM1 was conducted with an antisense PTTG3P DNA probe (PTTG3P-asDNA probe) and a LacZ antisense probe (LacZ-asDNA probe) for a comparison of their binding to the promoters of BUB1B and GAPDH with a ChIRP-qPCR assay. GAPDH and a LacZ-asDNA probe served as negative controls; mean ± SEM, n = 3. *P< 0.05, **P< 0.005, ***P< 0.0005.