Study On The Mechanism Of CircHNRNPU On Promoting The Malignant Proliferation Of Multiple Myeloma And Chinese Medicine Intervention

Background and ObjectiveMultiple Myeloma（MM）is a malignant tumor originating from B cells in the bone marrow and characterized by abnormal proliferation of bone marrow plasma cells and monoclonal immunoglobulin,ranking second in hematological malignancies.MM is particularly hazardous,whose incidence rate is increasing year by year,seriously threatens the life of patients.As a most severe subtype among all types of MM,immunoglobulin D multiple myeloma（IgD MM）is very rare comprising only 1 to 2%of all MM cases featured by diagnosis in relatively young patients,and often accompanied by multiple adverse prognosis,such as extraosseous lesions,renal failure,extramedullary involvement,amyloidosis.The most significant characteristics of IgD MM are refractory and poor prognosis,with an overall survival of only 13 to 21 months,while the median overall survival of common MM is 3 to 6 years.The advanced research achievement of the microenvironmental interactions between MM cells and the BM niche,and their roles in the progression of disease and acquisition of drug resistance,has promoted the development of novel therapeutic drugs for MM treatment.The intercellular interaction in BM niche through exosomes and circular RNAs（circRNAs）attracts extensive attention.CircRNAs,back-spliced products of exonic or intronic sequence of precursor mRNA（pre-mRNA）,are a fascinating class of conserved single-stranded RNA molecules.It has been reported that circRNAs act as essential players in cancer initiation,progression and drug resistance.In particular,circRNAs may influence tumor microenvironment through intercellular communication due to its abundance in exosomes and human body fluids.Therefore,circRNAs are now being considered as promising biomarkers for cancer.Many studies have suggested that circRNAs are translatable,which are translated into previously unknown protein isoforms.However,the evidences remain insufficient,especially in MM.Therefore,the systematic study of IgD MM is of guiding significance not only for IgD MM but also for other recurrence and refractory MM.In the present study,high expression circRNA circHNRNPU（hsa_circ₀017272）in IgD MM patients were screened by Agilent SBC-ceRNA microarray chips.The present study aims to elucidate the potential role of circHNRNPU acting as a novel therapeutic target,which can be translated into a previously unknown protein affecting the BM microenvironment in MM.Meanwhile,using traditional Chinese medicine monomers to inhibit MM cell proliferation through targeting circHNRNPU biosynthesis would provide therapeutic strategies for clinical research.Methods1.Agilent SBC-ceRNA microarray chips were employed to detect 5 IgD samples,5 IgG patient samples and 3 normal plasma cell samples.To investigate the most abundant and differentially expressed circRNA in IgD MM compared to lgG and NPCs samples,we first used WGCNA（weighted correlation network analysis）.technology to analyze the differentially expressed circRNAs in IgD MM.Then,the Relief algorithm is further used to select the most discriminating genes from the candidate genes.Subsequently,unsupervised cluster analysis is used to check the degree of difference between these selected hot genes and the three groups of samples.On the other hand,we examined the relationship between hotspot genes and other co-expressed genes in the gene co-expression network outputted by WGCNA.Through a series of bioinformatics analyses,circHNRNPU was found to be highly expressed in IgD MM.2.Bioinformatics methods were used to predict the sequence of circHNRNPU,and divergent primers were designed to detect the presence of circHNRNPU in MM cells by RNase R digestion combined with PCR and PCR product sequencing.The circRNA junction probe was designed and BaseScopeTM RNA ISH assay was performed to detect the expression of circHNRNPU in IgD MM,IgG MM patients and healthy control（NP）tissues.PCR was performed to detect the expression of circHNRNPU in blood samples from 48 MM patients and 48 healthy controls.3.The circHNRNPU overexpression plasmid was constructed according to the bioinformatic analysis of the translation mode of circHNRNPU and linked with HA tag.Lentivirus transfection system was used to construct circHNRNPU overexpression MM cell line.QPCR was confirmed that circHNRNPU was overexpressed MM cells.Sanger sequencing following with PCR was conducted to determine the accuracy of the cyclization product.Western blot and MS analysis were used to detect the expression of circHNRNPU₆03aa and prove the specific peptide fragments from circHNRNPU₆03aa.Immunofluorescence was used to determine the subcellular location of circHNRNPU₆03aa.Thiazolyl Blue Tetrazolium Bromide（MTT）assay was used to detect cell viability of circHNRNPU₆03aa.Flow cytometry was employed to detect the effects of knockdown and overexpression of circHNRNPU on the cell cycle.The immune deficiency mouse xenograft tumor model was established by subcutaneous injection of WT and circHNRNPU-OE MM cells to study the effect of circHNRNPU overexpression on MM cell growth in vivo.4.RNA immunocoprecipitation-sequencing（RIP-seq）was performed on MM cells to analyze the effect of circHNRNPU₆03aa on alternative splicing.PCR was used to verify the effect of circHNRNPU₆03aa on SKP2 exon skipping.RIP-PCR method was used to further verify the effect of circHNRNPU₆03aa on SKP2 alternative splicing.SKP2-NM₀01243120.2 transcript was knocked down by small interference technique,and the proliferation ability of MM cells after knockdown transcript was detected by MTT method.The effects of SKP2-NM₀01243120.2 transcript and SKP2-NM_NM₀05983.4 on c-Myc ubiquitination were detected by immunoprecipitation method after MG132 treatment.5.Exosomes from MM cell supernatant were extracted by ultra-high gradient centrifugation and characterized by transmission electron microscopy and exosome marker proteins.RNase R digestion combined with PCR and PCR product sequencing were used to verify the existence of circHNRNPU in MM cell exosomes.Transwell chamber coculture followed with qPCR,western blot and LC-MS/MS was employed to comprehensively analyze the effects of circHNRNPU on RNA and protein expression of recipient cells.The effects of MM circHNRNPU₆03aa-OE cells on protein expression and cell cycle of recipient cells were detected by immunofluorescence and flow cytometry.6.MTT assay was used to detect the effects of different Chinese medicine monomers on MM cell proliferation.QPCR and Western blot were used to examine the regulation of circHNRNPU biosynthesis of MM cells by Chinese herbal monomers,and to search for the Chinese medicine monomers that regulate circHNRNPU biosynthesis.Results1.Agilent SBC-ceRNA microarray was used to screen out highly expressed circRNAs in IgD MM patients.CircHNRNPU was found to be closely related to disease progression in MM patients.HNRNPU mRNA was significantly increased in MM cells compared with NP cells and monoclonal gammopathy of undetermined significance（MGUS）cells.In addition,high HNRNPU expression was associated with poor outcomes in TT2,GSE136337 and HOVON65 patient cohorts,2.CircHNRNPU RNase R digestion combined with PCR and PCR product sequencing confirmed circHNRNPU expression in MM cells.BaseScopeTM RNA ISH assay showed that circHNRNPU expression in IgD MM patients was significantly higher than that in IgG MM patients and normal controls.PCR results showed that the expression of circHNRNPU in blood samples of MM patients was significantly higher than that of normal control,and the expression of circHNRNPU was positi vely correlated with the poor prognosis of patients.3.The protein circHNRNPU₆03aa encoded by circHNRNPU was successfully identified by western blot and LC-MS/MS.MTT proliferation assay showed that circHNRNPU knockdown inhibited MM cell proliferation,and circHNRNPU overexpression promoted MM cell proliferation.Flow cytometry showed that the G2/M phase decreased after knockdown and increased after overexpression of circHNRNPU.Subcutaneous tumorigenesis experiment in mice showed that the weight and volume of subcutaneous tumor were significantly larger in circHNRNPU overexpression group than those in the control group.4.RIP-seq screened out that circHNRNPU₆03aa regulated SKP2 exon skipping,forming a transcript of SKP2-NM₀01243120.PCR and RIP-PCR further proved that circHNRNPU₆03aa regulated SKP2 exon skipping.After knockdown of SKP2-NM₀0124311.2 transcript,cell proliferation was reduced.Co-IP assay showed that SKP2 bound to its downstream protein c-Myc.Ubiquitination assay showed that SKP2-NM₀01243120.2 transcript competitively inhibited cMyc,thus stabilizing c-Myc and promoting the development of MM.5.Exosomes were extracted from the supernatant of MM cells by ultra-high gradient centrifugation.Transmission electron microscopy showed that exosomes were in typical saucer shape.Western blot confirmed the exosome markers Alix and CD9.CircHNRNPU-OE MM cells were cocultured with other surrounding cells using transwell.CircHNRNPU expression was detected in recipient cells by qPCR,and the circHNRNPU₆03aa protein expression was identified by WB and MS.Moreover,the circHNRNPU₆03aa protein could not be detected under the treatment with exosome inhibitor GW4869,indicating that circHNRNPU was secreted into the bone marrow microenvironment through exosome.Immunofluorescence showed that circHNRNPU₆03aa protein was expressed in the recipient cells in time-dependent manner.MTT assay showed that the cell proliferation of co-cultured cells was significantly increased,and cell cycle assay showed that the G2/M phase of co-cultured cells was significantly increased.6.MTT assay showed that artemisinin,bufalin,norcantharidin,raddeanin A and gambogic acid inhibited the growth of MM cells.QPCR and western blot confirmed the RNA and protein levels of circHNRNPU were significantly decreased under the treatment with artemisinin,bufalin,norcantharidin,raddeanin A and gambogic acid.ConclusionThis study demonstrates that high expression of circHNRNPU in IgD MM patients is closely related to poor prognosis of MM.CircHNRNPU is secreted into the bone marrow microenvironment through exosomal information exchange and translated into a novel protein circHNRNPU₆03aa with biological activity.CircHNRNPU₆03aa regulates SKP2 exon skipping,which competitively inhibits c-Myc ubiquitylation,thereby stabilizes c-Myc and promotes MM cell proliferation.Of great significance,circHNRNPU is expected to be a novel biomarker of MM in diagnosis,treatment and prognosis assessment.In addition,Artemisinin,Bufalin,norcantharidin,raddeanin A and gambogic acid interfere with the biosynthesis of circHNRNPU and inhibit MM cell proliferation,providing a new strategy of application of Chinese medicine in MM.