The Biological Function And Mechanism Of CircELK4 And The Protein It Encoded In Multiple Myeloma

BackgroundMultiple myeloma(MM)is considered as a malignant disease caused by the proliferation of monoclonal plasma cells,which lead to clinical symptoms.As the second most prevalent malignant disease in hematology,the incidence of MM is approximately4.5-6 of each hundred thousand people.MM mostly occurs in flat bones,such as the skull and pelvis,and causes clinical symptoms including hypercalcemia,anemia,bone destruction,and renal insufficiency.In recent years,the prognosis of MM patients has improved significantly due to the new treatment regimens.However,MM is still incurable,and patients’ disease gets relapsed and refractory even after multiple lines of treatment.Patients eventually facing the situation of multidrug resistance and incurability.Therefore,it is important to investigate the underlying mechanisms of MM progression,develop and identify novel diagnostic and prognostic markers and therapeutic targets,and improve the prognosis of patients.Previous studies have shown that 98% of genomic DNA transcription produces nc RNAs,and several nc RNAs play important roles in physiological and pathological processes’ regulation.Circ RNA is a special type of nc RNA,which is structurally distinct from m RNA due to its 5’ and 3’ ends are covalently linked into a loop.Subsequent studies have elucidated the regulatory role of circ RNAs in tumor relapse and progression.Recently,more and more studies have suggested that specific structures within circ RNAs,such as internal ribosome entry site(IRES),can facilitate their post-transcriptional recruitment of ribosomes and translation of polypeptides.This process has greatly broadened the field of nc RNA research,and a variety of previously overlooked polypeptides have been reported to have important regulatory functions in tumor cells.Exploring the biological functions and regulatory mechanisms of these peptides in tumors will undoubtedly provide new directions for future research in predicting tumor prognosis,improving therapeutic response,and finding therapeutic targets.Therefore,this project aims to investigate the existence of circ RNAs that are specifically expressed in MM cells and have the ability to encode functional peptides,and further investigate their functions and potential mechanisms to provide a new basis and reference for understanding of MM pathogenesis and the formulation of nc RNA/peptide molecule-based MM therapeutic strategies.MethodsTumor cells were using magnetic beads sorted from the bone marrow samples of 5patients with primary diagnosis of MM.Normal plasma cells from bone marrow samples of 3 normal volunteers were sorted as control.In addition,we analyzed and integrated the data from the GEO database(GSE133058)in order to verify the differential circ RNAs.Combining the m RNA data,circ RNA data,sample type,and tumor subtype data in SBC microarray,we performed weighted correlation network analysis(WGCNA,)to screen out circ RNAs with potentially important functions in MM cells--hsa＿circ＿0000175(hereinafter referred to as circ ELK4).Using PCR of specific divergent primers,sanger sequencing,q RT-PCR,and fluorescence in situ hybridization,we verified the loop structure and cellular localization of circ ELK4 in MM cells.Then,stable knockdown and overexpression of circ ELK4 MM cell line was constructed by transfection with lentivirus to test the regulatory role of circ ELK4 in MM cells in vitro through cell function experiments with CCK8,Ed U staining,cell cycle flow cytometry test,and soft agarose clone formation.By querying the Circ DB and Tran Circ databases for integrated predictions of previous research data,we hypothesized that circ ELK4 possesses IRES structure and has the ability to encode functional peptide.To verify the encoding ability of circ ELK4 and the biological functions of its encoded products,we then constructed an overexpressing circ ELK4 cell line fused with Flag protein within ORF and a cell line overexpressing circ ELK4 translated peptide,and performed the cell functional experiments,Western blot,and IP experiments.Finally,considering the similarity of circ ELK4 translated peptide sequence with the sequence of parental gene ELK4,we hypothesized that the downstream molecule of circ ELK encoded polypeptide regulation should be consistent with ELK4 gene,and this conclusion was verified by western blot in the subsequent part of the study.ResultsWe normalized the SBC microarray data and screened a total of 6686 differentially expressed circ RNAs in MM,including 3341 up-regulated differential circ RNAs and 3345down-regulated circ RNAs.Combined with the results of differentially expressed circ RNAs in GSE133058 data,we screened 28 up-regulated differentially expressed circ RNAs.Combined the SBC microarray m RNA data and differentially expressed circ RNA data,we constructed the WGCNA network to integrate 11 co-expressed gene modules.Among them,the purple module was most associated with myeloma and Ig D subtype.The hub gene within the purple module was screened and we obtained circ ELK4 as a target for subsequent experimental exploration.The results of PCR with specific divergent primers,q RT-PCR and sanger sequencing all verified the loop structure of circ ELK4;the results of FISH experiments revealed the cytoplasm-dominated cellular localization of circ ELK4.Functional experiments showed that proliferation capacity of MM cells increased after overexpression of circ ELK4,and the result of knockdown is opposite.After predicting the open reading frame structure of 439 aa and IRES within circ ELK4,we examined the endogenous and stable translation ability of circ ELK4 using Western blot and IP assays.Functional experiments verified that circ ELK4 encoded439 aa peptide is consistent with the pro-oncogenic effect of circ ELK4 and has a significant role in promoting tumor cell proliferation.In the final mechanistic study,we found that circ ELK4 encoded 439 aa could increase the level of c-FOS protein in MM cells,the function of which is similarly to its parental gene ELK4 without affecting the protein level of ELK4,and promote the development of myeloma disease by increasing cell proliferation.ConclusionWe found that MM cells specifically overexpressed circ ELK4 compared to normal plasma cells.Circ ELK4 could promote the proliferation ability of MM cells with upregulating c-FOS levels through encoding 439 aa polypeptide.This result suggests that circ ELK4,as a highly expressed cancer-promoting circ RNA in MM cells,has the potential to be a therapeutic target molecule for MM.