Background: Bone metastasis is the leading cause of mortality and reduced quality of life in patients with metastatic prostate cancer (PCa). Long non-coding RNA activated by DNA damage (NORAD) has been observed to have an abnormal expression in various cancers. This article aimed to explore the molecular mechanism underlying the regulatory role of NORAD in bone metastasis of PCa.
Methods: NORAD expression in clinical PCa tissues and cell lines was detected with the application of qRT-PCR. Cancer cells were then transfected with plasmids expressing NORAD, after which Transwell assay and CCK-8 assay were carried out to detect proliferation, migration, and bone metastasis of PCa. NORAD downstream target molecules were screened through bioinformatics analysis, followed by further verification using dual luciferase assay. Extracellular vesicles (EVs) were labeled with PKH67 and interacted with bone marrow stromal cells. The gain- and loss-function method was applied to determine the internalization and secretion of PCa cells-derived EVs under the intervention of downstream target molecules or NORAD.
Results: PCa tissues and cell lines were observed to have a high expression of NORAD, particularly in tissues with bone metastasis. NORAD knockdown resulted in reduced secretion and internalization of EVs, and suppressed proliferation, migration, and bone metastasis of PCa cells. It was indicated that NORAD interacted with miR-541-3p, leading to the upregulation of PKM2. Forced expression of PKM2 promoted the transfer of PKH67-labeled EVs to bone marrow stromal cells.
Conclusions: NORAD might serve as a ceRNA of miR-541-3p to promote PKM2 expression, thereby enhancing the development of bone metastasis in PCa by promoting internalization and transfer of EVs of cancer cells, providing an insight into a novel treatment for the disorder.
Keywords: Bone metastasis; Extracellular vesicles; NORAD; Prostate cancer; Pyruvate kinase isozymes M2; miR-541-3p.