Exosome-transmitted PSMA3 And PSMA3-AS1 Promotes Proteasome Inhibitors Resistance In Multiple Myeloma

ObiectiveMultiple myeloma(MM)is a hematological malignancy characterized by the abnormal expansion of clonal plasma cells in the bone marrow(BM).The proteasome inhibitor(PIs)has shown promise in the treatment of MM,but the molecular mechanisms underlying MM resistance to PIs remains elusive.Exosomes are 40–150 nm extracellular vesicles(EVs)released by all cell types.Upon release from the cell surface,exosomes possess the capacity to fuse with the plasma membranes of recipient cells to deliver their contents(m RNA,lnc RNA or proteins)into the cytoplasm.Alternatively,proteins present on the surface of the exosomes can engage cell surface receptors on recipient cells to induce intracellular signaling.Long non-coding RNAs(lnc RNAs)are functionally defined as transcripts >200 nucleotides in length with no protein coding potential.Lnc RNAs and exosomes isolated from tumor microenvironment have been reported to play widespread roles in cellular processes,such as tumorigenesis,development and drug resistance.In this study,we investigated the contributions of mesenchymal stem cells(MSCs)derived exosomes to PIs drug-resistance of MM cells and explored which exosomic lnc RNAs are involved and through which molecular mechanisms they elicit this function.Methods1.We isolated exosomes using standard methods,and confirmed their identity by transmission electron microscope(TEM).Exosome size distribution and number are quantified by Nano Sight analysis.The exosome expressions of Flotillin1 and HSP70,surface antigens commonly used as exosome markers were subsequently confirmed using western blot.2.MM.1S or U266 were cultured with PKH67 fluorescently labeled exosomes isolated from MSCs,respectively.The ability of myeloma cells to uptake MSCs–derived exosomes was confirmed using confocal microscope and flow cytometric analysis.3.U266 or MM.1S cells were planted in 96-well plates with or without different amounts of exosomes and bortezomib or carfilzomib for 72 h.The cell vitality was measured using a cell proliferation assay kit(CCK-8).4.Gene set enrichment analysis(GSEA)was carried out to determine whether defined sets of genes were differentially expressed between bortezomib-sensitive and-resistant patients-derived CD138+ cells.The results were validated by clinical data and PIs resistant myeloma cells.5.Exosomes isolated from PSMA3 or PSMA3-AS1-knockdown r-MSCs,or exosomes isolated from PSMA3 or PSMA3-AS1-overexpression s-MSCs were incubated with MM cell lines;CCK8 was used to detect the viability of MM cells.6.CCK8 was used to detect the sensitivity of MM cells to PIs after using specific si RNA against PSMA3 or PSMA3-AS1 in MM cells.The cell vitality was measured using CCK8 after overexpression of PSMA3 to abolish the decreased level of PSMA3 by PSMA3-AS1 si RNA.7.We used northern blot to detect whether PSMA3-AS1 expressed in MM cells,5'RACE and 3'RACE analyses to determine the transcriptional initiation and termination sites of PSMA3-AS1.The coding potential was predicted by ORF-finder?CPC and CPAT.Cell nucleus/cytoplasm fraction isolation experiment and RNA-FISH were conducted to analyze the location of PSMA3-AS1.8.RNase protection assay(RPA)and ?-amanitin experiment were used to examine the possibility of RNA duplex formation between lnc PSMA3-AS1 and PSMA3.9.To evaluate the therapeutic potential of PSMA3-AS1 in MM in vivo,bioluminescent MM models(U266-luc)were established.10.Exosomes were isolated from plasma of BTZ resistant or sensitive MM patient,q RT-PCR was used to detect circulating exosomal gene expression.The univariate and multivariate Cox survival regressions were carried out to validate whether exosmic PSMA3 and PSMA3-AS1 were related to prognostic.Results1.PIs resistant or sensitive MSCs–derived exosomes can be transferred into MM cells with same ability.2.R-MSCs-derived exosomes significantly reduced the sensitivity of MM cells to PIs.3.PSMA3 and PSMA3-AS1 were significantly upregulated in BTZ resistant samples compared to BTZ sensitive samples.Kaplan-Meier analysis showed that high PSMA3 levels in CD138+ MM cells were correlated with reduced progression-free survival(PFS)and poor overall survival(OS).Our clinical data showed that the m RNA levels of PSMA3 and PSMA3-AS1 were upregulated in CD138+ MM cells derived from BTZ resistant patients relative to those from BTZ sensitive patients,as well as in MM cell lines with resistance to PIs.4.U266 or MM.1S incubated directly with exosomes from r-MSCs displayed decreased cell proliferation,which could be abrogated by si RNA against PSMA3 or PSMA3-AS1 in r-MSCs.Consistent with the si RNA experiments,when PSMA3 or PSMA3-AS1 expression was upregulated in s-MSCs,levels of PSMA3 or PSMA3-AS1 were increased in MM cells incubated with exosomes from PSMA3 or PSMA3-AS1-overexpression s-MSCs compared to vector control cells.In concordance with upregulation of PSMA3 and PSMA3-AS1,cell proliferation was enhanced.5.Knockdown of PSMA3 and PSMA3-AS1 in MM cells could increase the sensitivity of MM cells to PIs.Overexpression of PSMA3 recovered the sensitivity of MM cells to PIs which was decreased by downregulation of PSMA3-AS1.6.CPC and CPAT analysis displayed that PSMA3-AS1 has no coding potentiality.The presence of full length PSMA3-AS1 was further confirmed by northern blotting in MM.1S.RNA FISH and q PCR of nuclear and cytoplasmic fractions suggested that PSMA3-AS1 was located both in the nucleus and in the cytoplasm.7.PSMA3-AS1 formed an RNA duplex with PSMA3-AS1 pre-m RNA and increased its stability.8.Targeting PSMA3-AS1 increased proteasome inhibitors sensitivity in vivo.9.Exosmic PSMA3 and PSMA3-AS1 were highly expressed in BTZ resistant MM patients and they were significantly associated with both PFS and OS in the univariate and multivariate analyses.ConclusionBioactive PSMA3-AS1 in MSCs could be incorporated into exosomes and transmitted to myeloma cells,thus promoting PIs resistance.High PSMA3 levels were correlated with reduced PFS and poor OS.Our study confirmed that PSMA3-AS1 and pre-PSMA3 can form RNA duplex to enhance stability of PSMA3,thus contributing to PIs resistance.Exosmic PSMA3 and PSMA3-AS1 were significantly associated with both PFS and OS in the univariate and multivariate analyses.Therefore,1nc PSMA3-AS1 may serve as a predictor and a potential therapeutic target for PIs resistance.