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. 2019 Jul 9;105(6):1630–1640. doi: 10.3324/haematol.2019.218289

Figure 8.

Figure 8.

HIF-1 directly upregulates the expression of DARS-AS1, which may, in turn, enhance the expression of HIF-1α. (A) HIF-1α or HIF-2α was knocked down or overexpressed in 293T cells. DARS-AS1 expression levels were determined by quantitative polymerase chain reaction analysis and normalized to 18S rRNA. (B) Transient transfection of a DARS-AS1 promoter [wildtype (WT) or mutant] luciferase transcriptional reporter and plasmid overexpressing HIF-1α/control vector. The luciferase values were determined from cell lysates by normalization to renilla luciferase. The pGL3 control vector containing the HRE promoter was used as a positive control. The data represent the average and standard deviation of three independent experiments. (C) Chromatin immunoprecipitation assay certified the binding of HIF-1 with the predicted two HIF response element (HRE) regions; LDHA was used as a positive control. (D) RNA immunoprecipitation experiments were performed using a Flag-antibody (exogenous HIF-1α protein with Flag-tag), and specific primers to detect DARS-AS1. (E) The expression of HIF-1α in the RPMI 8226 cells overexpressing DARS-AS1 in a hypoxic environment was analyzed by immunoblotting. (F) DARS-AS1-knockdown RPMI 8226 cells were treated with dimethylsulfoxide (DMSO, 1:1000) or MG132 (5 μM) for 6 h. The expression of HIF-1α under hypoxic conditions was analyzed by immunoblotting. (G) Proposed mode of action of DARS-AS1 in modulating myeloma tumorigenesis in a hypoxic environment. DARS-AS1 is directly regulated by HIF-1 and, in turn, promotes the expression of HIF-1α in myeloma. RBM39 mediates DARS-AS1-induced activation of mammalian target of rapamycin (mTOR) signaling. DARS-AS1 stabilizes the RBM39 by interfering with E3 ligase RNF147-mediated ubiquitination. HIF-1/DARS-AS1/RBM39 signaling pathways are involved in the tumorigenesis of myeloma.