Page 180 - Haematologica - Vol. 105 n. 6 - June 2020

J. Tong et al.
AS1 could not influence the HIF-1α mRNA level in RPMI 8226 cells (Online Supplementary Figure S7C). We speculat- ed that the DARS-AS1 silencing inhibited the translation of HIF-1α by suppression of the mTOR pathway.17,18 Altogether, HIF-1 enhanced the transcription activity of DARS-AS1; in turn, DARS-AS1 might accelerate the translation rate of HIF-1α expression via the mTOR path- way.
Discussion
Hypoxia-responsive genes are involved in the malignant progression and poor prognosis of human cancers.19 Numerous lncRNA have been reported to show abnormal expression in cancers.20 In the present work, we found that hypoxic-regulated DARS-AS1 was required for the sur- vival and tumorigenicity of myeloma cells in vitro and in
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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 pos- itive control. The data represent the average and standard deviation of three independent experiments. (C) Chromatin immunoprecipitation assay certified the bind- ing of HIF-1 with the predicted two HIF response element (HRE) regions; LDHA was used as a positive control. (D) RNA immunoprecipitation experiments were per- formed 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 dimethyl- sulfoxide (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.
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haematologica | 2020; 105(6)