| Objective:To investigate the relationship between serum exosomal circRNA(exo-circRNA)and peripheral neuropathy(PN)in patients with multiple myeloma(MM)of cold coagulation and blood stasis through clinical research,bioinformatics analysis and cell experiments,so as to preliminarily explore the diagnostic and prognostic role and possible pathogenesis of exosomal circRNAs in multiple myeloma peripheral neuropathy(MMPN).Methods:According to the Traditional Chinese Medicine syndrome score table for MM patients of cold coagulation and blood stasis,which was self-drafted by referring to the diagnosis items of Yang deficiency and cold coagulation syndrome and blood Stasis syndrome of bone rheumatism disease in 《The Standard for TCM Disease and Syndromes therapeut ic results》,25 MM patients with cold coagulation and blood stasis syndrome and 5 healthy controls(HC)were included in the research.The serum of6 MM patients and 5 HC was extracted,and aberrant expression of exo-circRNAs was detected by high-throughput sequencing assay.qRT PCR was used to verify the abnormal expression level of circ-GRIN2 B in serum exosomes of 25 MM patients.Bioinformatics analysis was performed on the origin genes and downstream target genes of abnormally expressed circRNA in MM using Gene ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG),miRanda,Targetscan and Metascape.Serum exosomes extracted from MM patients and HC were co-cultured with PC12 cells,and the expression of downstream genes of circ-GRIN2 B and the proliferation rates of PC12 cells were evaluated.The correlation between the clinical characteristics of MMPN and serum exosomal circ-GRIN2 B of MM patients was analyzed.ROC curve,univariate and multivariate Cox regression model were conducted to identify the diagnostic and prognostic potiential of circ-GRIN2 B in MMPN.Results:1.The high-throughput sequencing results indicated that there are1265 significantly upregulated circRNAs and 787 downregulated circRNAs in the serum of MM patients compared to HC group.Aberrant expression level of circ-GRIN2 B in serum exosomes of 25 MM patients was verified by PCR.The correlation analysis indicated that the expression of serum exosomal circ-GRIN2 B was positively correlated with the clinical characteristics of PN including monoclonal protein proportions,clinical grades of PN,FACT/GOG-Ntx questionnaire scores and VAS scores in MM patients of cold coagulation and blood stasis.ROC curve,univariate and multivariate COX regression analysis identified that exosomal circGRIN2 B is a clinical diagnostic and independent prognostic factor in MMPN.2.GO and KEGG analysis on the origin genes demonstrated that upregulated circRNAs tend to promote the development of MM and pathogenesis of PN,which was implied by the terms of enriched functions including “histone methyltransferase activity(H3-K4specific)”,“activation of the PI3K-Akt signaling pathway” and “Ionotropic glutamate receptor signaling pathway”.The results of bioinformatics analysis consisted of target gene prediction and functional clustering analysis showed that the overexpressed circ-GRIN2 B in the exosomes of MM patients with cold coagulation and blood stasis might induce MMPN through ce RNA mechanism by downregulating the level of hsa-miR-6829-3p.3.The expression of hsa-miR-6829-3p was significantly downregulated in the serum of MM patients.The viability of PC12 cells co-cultured with the serum exosome of MM patients was inhibited,w ith elevated expression of circ-GRIN2 B and GRIN2 B.Conclusion:Compare to HC,there are aberrant highly expressed circRNAs in the serum of MM patients of cold coagulation and blood stasis,among which circ-GRIN2 B was significantly upregulated and its expression levels has the potential to be a diagnostic biomarker and independent prognostic factor for MMPN in MM patients with cold coagulation and blood stasis.Besides,circ-GRIN2 B might induce MMPN through ce RNA mechanism,implying that it has a potential to become an effective therapeutic target for MMPN. |
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