| Objective This research was aimed to investigate the expression level of miR-21、Let-7b、miR-18a、miR-146 a and PD1,PD-L1,c-SRC,CD138 derived from serum exosomes and the prognostic clinical value of all above exosomes contents in patients with multiple myeloma(MM).Methods1.Serum exosomes were precipitated from 57 MM patients and 20 normal control using Exoquick exosome isolation reagent,transmission electron microscope was used observe exosomes the morphology and size,and Western blot was used detecte exosomes marker relate protein CD63,CD9,CD81 and HSP70.2.The relative expression level of miR-21、Let-7b、miR-18a、miR-146 a derived from serum exosomes was measured by q RT-PCR.3.The relative expression levels of protein molecules PD-1,PD-L1,c-SRC and CD138 derived from serum exosomes were detected by Western blot.4.Correlations between the expression level of miR-21、miR-18a、miR-146a、Let-7b and different clinicopathological parameters in patients with MM were analyzed by Spearman correlation,and different clinical features expression level were analyzed by Kruskal-Wallis H test.The expression level of all above miRNAs betwween 20 relapsed and refractory myeloma(RRMM)and 20 remission myeloma(Remit-MM),and the differences in expression levels between 44 patients treated with bortezomib and13 patients without bortezomib were analyzed by Mann-Whitney U test.5.Kaplan-Meier survival curve,COX proportional hazard model and area AUC under ROC curve were used to analyze the role of miR-21,miR-18 a,miR-146 a and Let-7b expression in predicting the overall survival of patients with myeloma.6.The expression level of PD-1,PD-L1,c-SRC and CD138 derived from myeloma serum circulating exosomes were analyzed by Kruskal-Wallis H test in different ISS stages.Results1.Eelectron microscope result display that the exosome was round or oval in shape,with a clear bilayer membrane structure,and CD63,CD9,CD81 and HSP70 marker proteins was detected in exosomes.2.The expression levels of derived from serum exosomes miR-21(0.537 ±0.260vs1.304 ±0.177,P < 0.001),Let-7b(0.844 ±0.414vs1.319 ±0.242,P < 0.001)and miR-18a(0.912 ±0.429vs1.415 ±0.218,P < 0.001)in patients with myeloma were lower than those in normal controls group,while the expression level of miR-146 a between the two groups was not significantly different(P>0.05).3.The study on the correlation between miRNA in serum circulating exosomes and different clinical features of myeloma showed that:3.1 According ISS to grop,expression level of miR-21,Let-7b and miR18 a in circulating exosomes of derived with stage Ⅱ and Ⅲ myeloma were lower than those of derived with stage I,and we result show that miRNA level of miR-21 was decrease in serum circulating exosomes derived from stage Ⅲ myeloma than stage II myeloma.3.2 According DS to grop,the result show that miR-21 was especially reduce in stageⅢ myeloma than derived from stage Ⅰ and stage Ⅱ(P < 0.05).3.3 In our study,compared with patients with renal function favourable,the miR-21 with renal dysfunction was especially lower.3.4 Compared with the patients who did not secrete M protein(negative),our data show that miRNA of miR-21 was especially reduce with upregulated level M protein.3.5 The miRNA of miR-146 a was insignificace change derived from myeloma with different clinical characteristics.4.The study showed that miR-21,Let-7b and miR-18 a were positively correlated with myeloma clinicopathological parameters hemoglobin(r=0.582,r=0.309,r=0.370 respectively,P<0.05)and show negatively correlated with clinicopathological parameters serumβ2-microglobulin concentration(r=-0.83,r=-0.362,r=-0.542)and cystatin C(r=-0.627,r=-0.461,r=-0.434).In our data,miR-146 a was insignificace change in myeloma clinicopathological parameters P>0.05.5.When miR-21,Let-7b,miR-18 a and miR-146 in serum circulating exosomes were used as prognostic indicators for patients with myeloma:5.1 The miR-21,Let-7b,miR-146 a and miR-18 a in serum circulating exosomes with RRMM were lower than by treatment remission,and the expression was also lower in no choice bortezomib treatment.5.2 Kaplan-Meier analysis showed that the overall survival time was especially declined of myeloma when miR-21,Let7 b and miR-18 a was decreased in serum exosomes,and pairwise or three combinations result showed that the miRNA is reflects batter overall survival time of myeloma(P < 0.05).5.3 The results of COX proportional hazard model and area under ROC curve AUC showed that miR-21,Let-7b,miR-146 a and miR-18 a in serum circulating exosomes combined with ISS increased the accuracy of prognosis prediction.6.In ISS stage,the results of Western blot detection showed that CD138,c-SRC,PD-1and PD-L1 in serum circulating exosomes were especially upregulated in three stage,respectively(P<0.001).Conclusions1.The expression of miR-21,Let-7b and miR-18 a in serum circulating exosomes was closely related to the deterioration of clinical symptoms and the progression of the disease in myeloma.2.The serum circulating exosome miR-21,Let-7b and miR-18 a could be used as a value in the therapeutic effect evaluation and monitoring prognosis.3.The miR-21,miR-18 a,miR-146 a and Let-7b in serum circulating exosomes can monitor the therapeutic effect of myeloma patients and increase the accuracy of prognosis prediction.4.The protein CD138,c-SRC,PD-1 and PD-L1 in serum circulating exosomes could be used as a prognostic factor for patients with myeloma. |
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