| Background and Purpose: Multiple myeloma(MM)is the second most common tumor of the hematological system,characterized by malignant proliferation of monoclonal plasma cells and secretion of large amounts of monoclonal immunoglobulins,with the main clinical manifestations being hypercalcemia,renal failure,anemia,and bone disease.MM occurs mainly in the elderly,and its incidence is increasing year by year.In recent years,treatments such as proteasome inhibitors,immunomodulators,daratumumab,and autologous hematopoietic stem cell transplantation have significantly improved the prognosis of MM patients,and the median survival of MM is now more than 6 years.However,MM remains an incurable tumor.The pathogenesis of MM is very complex,and the specific mechanisms of action of its development are not fully understood.Therefore,there is a need to further investigate the molecular mechanisms of MM pathogenesis to provide targets for treatment.The role of non-coding RNAs(nc RNAs)in physiological and pathological processes has been widely explored.Nc RNAs mainly include micro RNAs(mi RNAs),long non-coding RNAs(lnc RNAs),and circular RNAs.Many of the tumor-specific mutations detected in tumors are located in the sequences of nc RNAs.Recent studies have shown that a small number of small open reading frames in nc RNAs actually have the potential to encode peptides or proteins.Nc RNAs play an important role in the progression of several cancers through their regulatory roles in glucose metabolism,methylation pathways and ubiquitination pathways.Lnc RNAs are more than 200 base pairs in length and are transcribed from a separate promoter by RNA Pol II.Noncoding RNA activated by DNA damage(NORAD)was first identified in2016 and was mainly involved in DNA damage repair and maintenance of genomic stability.NORAD plays different roles in different tumors,and it exhibits pro-cancer effects in a variety of tumors,mainly related to mechanisms such as chemotherapeutic drug resistance,promotion of cell proliferation,migration and angiogenesis.However,it showed cancer-inhibiting effects in lung and breast cancers.So far,there is no report on the role and mechanism of action of NORAD in MM at home and abroad.In this study,we will investigate the regulation of apoptosis,cell cycle and cell proliferation by NORAD and its mechanism,as well as its role in vivo,to demonstrate that NORAD is a good therapeutic target for MM.Methods: The GEO database was used to analyze the differential expression of NORAD in healthy donors(HD)and MM.The bone marrow of 60 patients with newly diagnosed MM(NDMM),30 patients with MM(PTMM)who achieved CR after 4 courses of treatment and 17 patients with iron deficiency anemia as HD group from February 2021 to June 2022 were collected and the expression of NORAD gene was detected using q RT-PCR method.The clinical prognostic value of NORAD was also analyzed with the clinical data.A stable transgenic MM cell line with silenced NORAD expression was constructed to study the effects of NORAD gene on apoptosis,proliferation,cell cycle and tumorigenesis of myeloma cells in vivo.The downstream signaling pathway of NORAD action on MM cells was explored using m RNA-seq and protein blotting,and bone morphogenetic protein 6(BMP6)was found to be the most obvious differential gene elevated after silencing NORAD.By further silencing the expression of BMP6 in a stable transgenic strain of MM with silenced NORAD expression,a reversion assay was performed to observe its effects on apoptosis,proliferation,cycle and tumorigenesis of myeloma cells in vivo.Results: The GEO database indicates that NORAD is highly expressed in MM.Our data also found high expression of NORAD gene in NDMM patients compared to HD,and low expression of NORAD gene in PTMM patients compared to NDMM.Silencing NORAD expression in MM cell lines increased the apoptosis rate of MM cells,induced MM cell cycle arrest in G0/G1 phase,and reduced the proliferation rate of MM cells.m RNA sequencing revealed that BMP6 was the most significantly elevated differential gene after silencing NORAD,and silencing BMP6 in a stable transient strain of MM with silenced NORAD expression reverted the effect of NORAD gene on MM cell apoptosis,and cell cycle effects.Further investigation of downstream mechanisms revealed that NORAD exerts these effects through the BMP6/P-ERK1/2 axis.Animal experiments also found that the volume and weight of subcutaneous tumors decreased and the expression of BMP6 and P-ERK1/2 increased after silencing NORAD gene.Conclusion: In this study,NORAD was found to be highly expressed in MM and correlated with its poor prognosis.NORAD promotes myeloma cell proliferation and inhibits its biological behavior such as apoptosis through the BMP6/P-ERK1/2 axis.NORAD is a potential therapeutic target for MM. |
