Different effects of long noncoding RNA NDRG1-OT1 fragments on NDRG1 transcription in breast cancer cells under hypoxia

Hypoxia plays a crucial role in the aggressiveness of solid tumors by driving multiple signaling pathways. Recently, long non-coding RNA (lncRNA) has been reported to promote or inhibit tumor aggressiveness by regulating gene expression. Previous studies in our laboratory found that the lncRNA NDRG1-OT1 is significantly up-regulated under hypoxia and inhibits its target gene NDRG1 at both the mRNA and protein levels. At the protein level, NDRG1-OT1 increases NDRG1 degradation via ubiquitin-mediated proteolysis. However, the repressive mechanism of NDRG1 at the RNA level is still unknown. Therefore, the purpose of this study was to study how NDRG1-OT1 transcriptionally regulates its target gene NDRG1. Luciferase reporter assays showed that NDRG1-OT1 decreased NDRG1 promoter activities. Mass spectrometry, bioinformatics tools, genetic manipulation, and immunoblotting were used to identify the interacting proteins. Surprisingly, different fragments of NDRG1-OT1 had opposite effects on NDRG1. The first quarter fragment (1-149 nt) of NDRG1-OT1 had no effect on the NDRG1 promoter; the second quarter fragment (150-263 nt) repressed NDRG1 by increasing the binding affinity of HNRNPA1; the third quarter fragment (264-392 nt) improved NDRG1 promoter activity by recruiting HIF-1 alpha; the fourth quarter fragment (393-508 nt) down-regulated NDRG1 promoter activity via down-regulation of KHSRP under hypoxia. In summary, we have found a novel mechanism by which different fragments of the same lncRNA can cause opposite effects within the same target gene.