Study On The Effect Of Multiple Myeloma(MM) Derived Exosomes On T Cell Immunity And LncRNA H19 Involved In Resistance Of Bortizomib In MM

Objective:To study the effects of multiple myeloma?MM?derived exosomes on the number and function of CD4⁺T,CD8⁺T and regulatory T cells from healthy donors in order to elucidate the role of MM exosomes in T cell immunity.To study the correlation between the expression level of lncRNA H19 in plasma exosomes and the theraputic efficacy in patients with multiple myeloma in order to elucidate the role of lncRNA H19 in the occurrence of bortizomib resistance in multiple myeloma and explore its mechanism.Contents:CD4⁺T,CD8⁺T and CD4⁺CD25⁺CD127^dimTreg cells were isolated from healthy donors and MM patients and then co-cultured with exosomes isolated from human myeloma cell lines?OPM2 and U266?.Apoptosis and proliferation of T cells were detected.The changes of perforin and granzyme B in CD8⁺T cells and IL-10 and TGF-?in Treg cells were measured.The expression of lncRNA H19 in plasma from healthy donors and plasma,exosomes together with CD138⁺myeloma cells from multiple myeloma patients were measured.Culture of bortezomib-resistant myeloma cell lines and to elucidate the role of lncRNA H19 in the development of drug resistance of myeloma.Methods:Part 1.Study on the effect of multiple myeloma derived exosomes on T cell immunity.The extraction of the myeloma derived exosomes was carried out by the method of ultracentrifugation and total exosome isolation kit.The morphology of exosomes was observed by transmission electron microscopy.CD4⁺T cells,CD8⁺T cells,and CD4⁺CD25⁺CD127^dimTreg cells from healthy donors and MM patients were isolated by MACS and co-cultured with MM?OPM2,U266?-derived exosomes for 48h and then detected for apoptosis rate by Annexin?apoptosis detection Kit through flow cytometry and cell vitality by CCK8 kit.The effects of exosomes on the production of the Perforin and GranzymeB in CD8⁺T cells was measured by Flow cytometry.The effects of MM-derived exosomes on the secretion of IL-10 and TGF-?in Treg cells were measured by ELISA.Part 2.Study on the role of lncRNA H19 in Bortezomib resistance in multiple myeloma.The expression of lncRNA H19 in plasma from healthy donors and plasma,exosomes together with CD138⁺myeloma cells from multiple myeloma patients were measured by real-time quantitative PCR.According to the therapeutic efficacy of myeloma,the patients were devided into new diagnosis the expression of lncRNA H19in the plasma of patients with primary,remission and refractory recurrence and healthy donors was compared.The difference of lncRNA H19 expression in plasma of primary diagnosis,remission,refractory recurrent patients and healthy donors was compared.CCK8 and Annexin?methods were used to detect the sensitivity of MM cell lines?OPM2,U266 and LP1?to bortezomib,and real-time quantitative PCR was used to detect the expression level of lncRNA H19.By gradually increasing the concentration of bortezomib,the drug-resistant MM cell lines were successfully cultured,and the expression of lncRNA H19 were compared.LncRNA H19 was transfected with lentivirus and overexpressed in MM cell line?OPM2,U266?,and the sensitivity of MM cell lines to bortezomib was measured after overexpression.The levels of NF-?B pathway and AKT in sensitive and resistant U266 cell lines were detected by Western blot,and the mRNA expression of MDR1 and TRX were detected by real-time quantitative PCR.Results:Part 1 Study of the effect of MM derived exosomes on T cell immunity.The MM-derived exosomes were successfully isolated using total exosome isolation kit and ultracentrifugation method,and there was no obvious difference in morphology by transmission electron microscopy.1.The apoptosis rate of CD4⁺T cells from healthy donors in OPM2 group?6.24±1.24%,P=0.0049?and U266 group?5.42±1.07%,P=0.0007?was significantly higher than that in control group?4.37±0.96%?respectively,but there was no significant difference between OPM2 and U266 group?P>0.05?.The apoptosis rate of CD4⁺T cells from MM patients in OPM2 group?14.56±4.65%?and U266 group?16.42±5.08%?was higher than that in control group?13.83±5.69%?,but there was no significant difference.2.The vatality of CD4⁺T cells from healthy donors in OPM2 group?95.34±4.58%,P=0.331?and U266 group?85.45±6.0945%,P=0.0358?was lower than that in the control group.The latter had statistical significance in inhibiting the proliferation of CD4⁺T.The vatality of CD4⁺T cells from MM patients in OPM2 group?98.61±2.80%?and U266 group?100.02±5.48%?was no significant difference compared with control group.3.The apoptosis rate of CD8⁺T cells from healthy donors in OPM2 group?9.97±1.28%,P=0.021?and U266 group?11.00±1.75%,P=0.018?was significantly lower than that in control group?16.12±2.95%?respectively,but there was no significant difference in OPM2 and U266 group?P>0.05?.The apoptosis rate of CD8⁺T cells from MM patients in OPM2 group?14.40±4.86%?and U266 group?14.39±4.36%?was lower than that in control group?17.37±4.05%?,but the decreased apoptosis rate of CD8⁺T was not significant?P>0.05?.4.The vitality of CD8⁺T cells from healthy donors in OPM2 group?112.63±3.88%,P=0.0099?and U266 group?111.70±3.62%,P=0.0322?was significantly higher than that in control group,suggesting that MM-derived exosomes could promote the proliferation of CD8⁺T from healthy donors.The vitality of CD8⁺T cells from MM patients in OPM2 group?101.58±10.82%?and U266 group?100.31±10.80%?was no significant difference compared with control group.5.The level of perforin in CD8⁺T cells from healthy donors in OPM2 group?10.82±2.53%?and U266 group?9.34±2.36%,P=0.044?was lower than that in control group?12.76±3.31%?,and the U266 group had statistical significance compared with control group.There was no significant difference in the perforin between OPM2 and U266 groups?P>0.05?.The level of perforin in CD8⁺T cells from MM patients in OPM2 group?6.48±1.06%,P=0.049?and U266 group?5.63±1.15%,P=0.027?was significantly lower than that in control group?10.55±2.50%?.There was no significant difference in the perforin between OPM2 and U266 groups.6.The level of granzyme B in CD8⁺T cells from healthy donors in OPM2 group?31.87±6.10%?and U266 group?32.99±7.08%?was slightly higher than that in control group?28.74±6.21%?,but there was no statistical significance?P>0.05?.The level of granzyme B in CD8⁺T cell in OPM2 group?34.48%±9.98%?and U266 group?41.49±9.17%?was not significantly different from that in control group?34.91±8.14%??P>0.05?.7.The apoptosis rate of Treg cells from healthy donors in OPM2 group?15.33±3.87%,P=0.041?and U266 group?11.71±2.71%,P=0.008?was significantly lower than that in control group?19.61±3.50%?respectively,but there was no significant difference in OPM2 and U266 group?P>0.05?.The apoptosis rate of Treg cells from MM patients in OPM2 group?30.91±7.25%?and U266 group?37.29±6.54%,P=0.023?was higher than that in control group?29.95±6.68%?,and the U266 group had statistical significance compared with control group.8.The vitality of Treg cells from healthy donors in OPM2 group?139.54±23.24%?and U266 group?173.48±34.99%,P=0.043?was higher than that in control group,and the latter was significantly higher,suggesting that MM-derived exosomes could promote the proliferation of Treg from healthy donors.The vitality of Treg cells from MM patients in OPM2 group?82.29±12.16%,P=0.031?and U266 group?85.09±13.50%?was lower than that in control group,and the OPM2 group had statistical significance compared with control group.9.The level of IL-10 in the supernatant of Treg cells from healthy donor was increased in OPM2 group?18.53±8.08pg/ml,P=0.043?and U266 group?13.51±7.83pg/ml?than that in control group?12.76±3.31%?,and the OPM2 group had statistical significance.There was no significant difference in IL-10 between OPM2and U266 groups?P>0.05?.The level of IL-10 in the supernatant of Treg cells from MM patients in OPM2 group?3.29±1.84pg/ml?and U266 group?2.60±1.72pg/ml?was higher than that in control group?1.39±1.02pg/ml?,but the difference was not significant?P>0.05?.10.The level of TGF-?in the supernatant of Treg cell from healthy donor in OPM2 group?417.57±19.33pg/ml?and U266 group?325.96±14.32pg/ml?was not significantly different from that in control group?386.60±28.89pg/ml??P>0.05?.The level of TGF-?in the supernatant of Treg cell from MM patients was significantly lower in OPM2 group?290.29±23.21pg/ml,P=0.0046?and U266 group?230.03±15.96pg/ml,P=0.0068?than in the control group?387.49±21.60pg/ml??P<0.05?.11.The expression level of lncRNA H19 in CD4⁺and CD8⁺T cells was significantly increased in OPM2 group which was co-cultured with OPM2 exosomes(2^1.52±1.99)than that in the control group(2^0.00±2.2).The difference was statistically significant?P=0.012?.Part 2 Study on the role of lncRNA H19 in Bortezomil resistance in multiple myeloma1.The expression levels of lncRNA H19 in plasma were 4.1740?10.07?,3.37849?6.22?,12.8723?38.1?and 1.0867?1.48?in newly diagnosed MM patients,remission patients,refractory and relapsed MM patients and healthy donors respectively and there was significant difference among the four groups?P<0.01?.The expression of lncRNA H19 was significantly higher in the refractory and relapsed patients than in the newly diagnosed MM patients?P=0.005?,remission patients?P<0.001?and healthy donors?P<0.001?,and was higher in the newly diagnosed MM patients than in the healthy donors?P=0.048?.There was no significant difference between the newly diagnosed MM patients and the remission patients?P=0.174?.2.The expression levels of lncRNA H19 in exosomes were 0.7071?1.42?,2.8481?6.80?and 9.5137?23.38?in newly diagnosed MM patients,remission patients and refractory and relapsed MM patients respectively,and there was significant difference among the three groups?P=0.004?.The expression of lncRNA H19 in exosomes was significantly higher in the refractory and relapsed patients than in the newly diagnosed MM patients?P=0.015?and remission patients?P=0.014?.There was no significant difference between the newly diagnosed MM patients and the remission patients?P=0.692?.3.The expression of lncRNA H19 in CD138⁺plasma cells of MM patients was positively correlated with the level of lncRNA H19 in their own plasma?Pearson r=0.745,P<0.001;Spearman r=0.639,P=0.002?.4.The IC50 values of bortezomib in OPM2?22.85nM?,LP1?7.22nM?and U266?4.25nM?were related to their expression level of lncRNA H19 which was 2^0.31±1.85,2^-6.74±0.49 and 2^-9.12±2.00 respectively.5.Cultured for bortezomib resistant cell lines,the vatality of the three bortezomib resistant cell lines was significantly higher than that of the sensitive cells lines after bortezomib,and the apoptosis rate was significantly decreased in U266 and LP1.The IC50 values of OPM2,U266 and LP1 were 82.91nM,370nM and 84.65nM,respectively.Compared with the IC50 values of their sensitive cell lines,the multiples of drug resistance were 3.63 times,87.06 times and 11.72 times respectively.U266 had the highest drug resistance multiple.6.The expression levels of lncRNA H19 in bortezomib resistant OPM2(2^{3.8795±0.5064}),U266(2^{1.7595±0.3154})and LP1(2^{2.6429±0.8559})was increased compared with that in the sensitive cell lines respectively.Among them,bortezomib resistant U266and LP1 cell lines had significantly increased P values of 0.037 and 0.010 respectively,indicating that lncRNA H19 was related to the development of bortezomib resistance in MM cell lines.7.By lentivirus transfection,lncRNA H19 was overexpressed in OPM2 and U266cell lines,and the overexpression times were 1858 and 3,070,410 times respectively.CCK8 assay was used to detect the vatality of OPM2 cell lines overexpressed with lncRNA H19.The concentrations of bortezomib were 10nM,20nM,40nM,80nM and160nM,respectively.After 48h,the vitality?%?of OPM2 cell lines overexpressed with lncRNA H19 was100.50±14.85,83.04±11.24,77.51±15.75,73.00±3.47,and 70.88±10.71,respectively.The vatality?%?of blank transfected OPM2 cells was 88.00±8.20,80.22±6.08,72.89±8.50,59.74±13.89,and 51.83±8.71,respectively.The vitality of the control group was 83.01±2.84,54.66±7.61,18.41±3.79,11.69±2.16and 7.87±0.93,respectively.After OPM2 overexpression of lncRNA H19,the vatality of OPM2-H19 transfected group was significantly higher than that of the control group at bortezomib 20nM?P=0.007?,40nM?P<0.001?and 160nM?P<0.001?,respectively,indicating that the sensitivity of OPM2-H19 transfected cells to bortezomib was decreased.8.WB showed that AKT,pAKT and NF-?B were significantly inhibited in bortezomil group compared with control group of U266 sensitive cell lines.But the inhibition of AKT and pAKT in U266 resistant cell was not obvious in the control group compared with the control group.NF-?B in U266 resistant cell lines was almost unexpressed,which suggested that NF-?B may not play a major role in the mechanism of bortezomib resistance.9.qPCR showed that there was a positive correlation between the level of MDR1and lncRNA H19 in MM cell lines?r=0.546,P=0.035?.There may be a correlation between the level of TRX and lncRNA H19 in MM cell lines?r=0.513,P=0.051?.Conclusions:1.MM cell lines derived exosomes can promote apoptosis,inhibit the proliferation of normal CD4⁺T cell and inhibit the apoptosis and promotes the proliferation of normal CD8⁺T,but inhibit the secretion of perforin.MM cell lines derived exosomes inhibit the apoptosis and promote the proliferation of normal Treg cells,and promote the secretion of IL-10 in Treg cells.It suggests that the exosomes derived from myeloma can be used as an antigen to stimulate the proliferation of CD8⁺T cells and Treg cells,but inhibit the immune function of normal T cells.The expression of lncRNA H19 in T cells is increased by the exosomes derived from myeloma,suggesting that the changes of immunal function of T lymphocytes may be associated with lncRNA H19.2.The expression of lncRNA H19 in plasma was significantly increased in multiple myeloma patients than that in healthy donors,and was significantly increased in refractory and relapsed patients than in the newly diagnosed and the remission patients of MM.The results showed that lncRNA H19 was closely related to the occurrence and progression of multiple myeloma.The expression level of lncRNA H19 in plasma was positively correlated with the level of lncRNA H19 in plasma exosomes and CD138⁺plasma cells in patients with multiple myeloma.It is suggested that lncRNA H19 in plasma is derived from the malignant plasma cells in bone marrow and can be released into plasma through exosomes.3.The expression level of lncRNA H19 in multiple myeloma cell lines?OPM2,U266and LP1?was correlated with the drug sensitivity of bortezomib.The higher expression of lncRNA H19,the higher IC50 value of bortezomib,suggesting that lncRNA H19 is related to bortezomib resistance.By increasing the concentration of bortezomib,drug-resistant cell line?U266?was established.It was showed that lncRNA H19 was significantly increased in drug resistant cell line compared with sensitive one.After treated with bortezomib,the apoptotis of drug-resistant cell decreased significantly and the cell proliferation activity increased significantly.So it was suggested that lncRNA H19 was associated with bortezomib resistance in multiple myeloma and the mechanism of drug resistance might be related to MDR1,NF-?B and PI3K/Akt pathway.