Fig 1: DSCAM-AS1 regulated QPRT expression by sponging miRNAs. (A) miRNAs targeting DSCAM-AS1, DCTPP1 and QPRT were predicated using PITA, RNA22, miRmap, mircoT, miRanda, PicTar, and Targetscan softwares. Venn diagram was used to find miRNAs targeting both DSCAM-AS1 and DCTPP1 or QPRT. (B) miRNA-494-3p was predicted to tightly bind to DSCAM-AS1; however, the connection between miRNA-494-3p and the DCTPP1 mRNA seem to be weak. miRNA-150-5p and miRNA-2467-3p were predicted to tightly bind to both DSCAM-AS1 and QPRT mRNA. (C) PCR was performed to detect miRNA-494-3p, miRNA-150-5p and miRNA-2467-3p expression after transfection with their inhibitors and mimics. (D) PCR was performed to detect DCTPP1 or QPRT expression after transfection with miRNA-494-3p, miRNA-150-5p and miRNA-2467-3p inhibitors and mimics. (E) As per the RNA pull-down assay, both miRNA-150-5p and miRNA-2467-3p WT pulled down DSCAM-AS1 from the cell lysate (P<0.001, E), but their MT failed to do so. (F) The association between miRNA-150-5p and miRNA-2467-3p and the QPRT mRNA 3' UTR was determined using a luciferase reporter assay. Transfection with miRNA-150-5p and miRNA-2467-3p mimics decreased the WT luciferase reporter activity; however, the transfection did not decrease the MT luciferase reporter activity. *P<0.05, **P<0.01 and ***P<0.001 vs. control group.
Fig 2: DSCAM-AS1 positively regulates DCTPP1 and QPRT expression in ER-positive BC cells. (A) According to Starbase web (https://web.archive.org/web/20110201054358/http://starbase.sysu.edu.cn/clipSeq.php), DSCAM-AS1 expression was positively correlated with both DCTPP1 (P=1.59×10-4) and QPRT (P=3.44×10-14) expression in BC. (B) Remarkably, the correlation coefficient is higher in ER-positive BC (data from GSE6532 dataset) than in the total BC. DSCAM-AS1 was also up-regulated in BC tissues according to the TCGA database (C1) and our PCR results (C2). (D) Data in TCGA database further showed that DSCAM-AS1 expression was higher in ER-positive BC than that in ER-negative BC. (E) As indicated by TCGA database, higher DSCAM-AS1 expression was associated with better prognosis in patients with BC. However, up-regulated DSCAM-AS1 was conversely associated with a poor prognosis in both ER-positive and ER-negative BC patients. Notably, high expression of DSCAM-AS1 was correlated with very poor prognosis when BC-positive patients were divided by the upper quartile value of the DSCAM-AS1 expression, and not the medium value. (F) PCR assay was performed to detect DSCAM-AS1, DCTPP1 and QPRT expression after DSCAM-AS1 knockdown. (G) Western blot was performed to detect DCTPP1 and QPRT protein levels after DSCAM-AS1 knockdown. B1-B2: *P<0.05 and ***P<0.001 vs. Normal breast tissue group. (E, F) *P<0.05 and **P<0.01 vs. control group. DSC(KD): DSCAM-AS1 knockdown.
Fig 3: DSCAM-AS1 regulated DCTPP1 transcription by modulating histone acetylation. (A) The epigenetic regulation of DCTPP1 expression was analyzed using the UCSC Genome Browser Gateway. The DCTPP1 promoter was influenced by histone 3 acetylation at lysine 27. (B) As indicated by catRAPID, DSCAM-AS1 was predicted to bind to two key histone acetylases: KAT5 and EP300. The interaction matrix showed that bases 0 to 200 and 800 to 900 of DSCAM-AS1 probably have strong associations with both the KAT5 and EP300 proteins. (C) In the RIP assay, anti-KAT5, anti-EP300 antibodies, and IgG were used to separate KAT5-RNA, EP300-RNA, and IgG-RNA complexes from the cell lysate. DSCAM-AS1 was successfully detected in the KAT5-RNA and EP300-RNA complexes but not in the IgG-RNA complex. (D) In the RNA pull-down assay, bio-DSCAM and bio-DSCAM-AS1 were used to detect proteins that could bind to DSCAM mRNA and lncRNA DSCAM-AS1. Western blotting indicated that DSCAM-AS1, but not DSCAM mRNA, bound to KAT5 and EP300 proteins. (E) Results from ChIP assay showed that acetylated histone 3 bound to the DCTPP1 promoter. However, DSCAM-AS1 knockdown decreased acetylated histone 3 enrichment in the DCTPP1 promoter. (F) In FISH and immunofluorescence assays, both DSCAM-AS1 and DCTPP1 were observed in the cytoplasm and cell nucleus (F). **P<0.01 and ***P<0.001.
Fig 4: Depletion of DSCAM-AS1 and knockdown of DCTPP1 and QPRT together affected BC cell growth and invasion. Cell viability (A), apoptosis (B), migration (C), and invasion (D) assays were performed after silencing DSCAM-AS1 or DCTPP1 and QPRT together. DSCAM-AS1 depletion and knockdown of DCTPP1 and QPRT remarkably inhibited cell viability, migration, and invasion in MCF-7 and T47D cells; however, they promoted apoptosis. The bar in the pictures (D) indicates a length of 5 µm. (E) In the in vivo assay, silencing DSCAM-AS1 or DCTPP1 and QPRT together dramatically inhibited MCF-7 and T47D cell growth in nude mice. (F) The molecular mechanisms behind DSCAM-AS1 regulating DCTPP1 and QPRT. DSCAM-AS1 increased QPRT expression through sponging miRNA-150-5p and miRNA-2467-3p. In contrast, DSCAM-AS1 promoted the transcription of the DCTPP1 gene by affecting H3K27 acetylation and enhancing DCTPP1 mRNA stability through binding to the 3’UTR, which collectively resulted in the overexpression of DCTPP1. **P<0.01 and ***P<0.001 vs. control group. DSC(KD): DSCAM-AS1 knockdown; DCT(KD):DCTPP1 knockdown; QPR(KD): QPRT knockdown.
Fig 5: The regulatory effects of DCTPP1 and QPRT on BC cell growth and apoptosis. DCTPP1 and QPRT mRNA (A) and protein (B) levels in MCF-7 and T47D cells were changed after transfection with shRNA-DCTPP1, shRNA-QPRT, and DCTPP1 and QPRT expression vectors. (C) Down-regulation of DCTPP1 or QPRT was associated with reduced viability in both MCF-7 and T47D cells and increased DCTPP1 or QPRT was associated with increased cell viability. (D) The apoptosis rate of MCF-7 and T47D cells increased after knocking down either DCTPP1 or QPRT. DCTPP1 overexpression decreased the apoptosis rate of MCF-7 and T47D cells, and QPRT overexpression only marginally decreased MCF-7 and T47D apoptosis. *P<0.05, **P<0.01 and ***P<0.001 vs. control group.
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