The Effect And The Mechanism Of DEPTOR On Angiogenesis In Multiple Myeloma

Objective:To investigate the significance of the difference in DEPTOR protein expression in multiple myeloma(MM)cells.Method:The clinical data and bone marrow samples of 22 newly diagnosed MM patients hospitalized in our hospital from October 1^st,2015 to June 30^th,2017 were collected.Primary MM plasma cells in bone marrow were sorted by CD138 magnetic-activated cell-sorting;CD34 immunohistochemical staining was used to detect bone marrow microvessel density of biopsy(MVD).Western blot analysis was used to detect the expression of DEPTOR and vascular endothelial growth factor(VEGF)protein in plasma cells and MM cell lines.Statistical analysis was performed using Graphpad 7.0 software.Result:The expression of DEPTOR was highest while VEGF was lowest in RPMI8226 cell line.The expression of DEPTOR and VEGF in MM.1S cell line was middle.The expression of DEPTOR was the lowest while VEGF was the highest in U266 cell line.The expression of DEPTOR and VEGF protein in plasma cells are varies in patients;the expression of bone marrow MVD are varies in patients,the number of MVD in 22patients ranged from 16.84/mm² to 412.46/mm²(median MVD was 176.77/mm²);The expression of DEPTOR protein in primary PCs was negatively correlated with the VEGF protein expression(r=-0.8402)and MVD(r=-0.828),p<0.05;VEGF protein expression was positively correlated with MVD(r=0.9583,p<0.05).DEPTOR was divided into high expression and low expression group at the median gray value of 0.132,each group has 11 patients.The clinical characteristics and ISS stage showed no significant difference between the high expression of DEPTOR group and low expression of DEPTOR group,p>0.05.The overall survival(OS)showed no significant difference between the high expression of DEPTOR group and low expression of DEPTOR group,p>0.05.Conclusion:The relative expression of VEGF in bone marrow PCs of newly diagnosed MM patients was positively correlated with MVD.The expression of DEPTOR protein in primary PCs was negatively correlated with VEGF protein expression and MVD.Objective: Construction of a lentiviral vector targeting the DEPTOR gene by CRISPR/Cas technology.Method: CRISPR-related protein 9(Cas9)and single-directed RNA(sg RNAs)were used to silence the expression of DEPTOR gene in MM cell lines.According to the design principle of target,three targets targeting DEPTOR gene were designed.The corresponding sg RNAs were synthesized in vitro,sg RNAs were ligated to the Cas9 lentiviral vector,and the positive clones were selected for sequencing.The successfully constructed vector was co-transfected with packaging plasmid in 293 FT cells,the supernatant was collected and the lentiviral particles were concentrated and titrated.Lentiviral GFP,DEP1,DEP2 and DEP3 were infected with RPMI 8226 cells for 72 h(MOI=40).Total RNA was extracted and PCR primer sequences were designed according to the target.PCR amplification was carried out.The amplified PCR product was digested with CruiserTM Enzyme and subjected to agarose gel electrophoresis;the PCR product fragment was also sequenced.Western blot was used to verify DEPTOR protein silencing with the targeted inhibition of DEPTOR gene.Result: Sequencing of positive clones suggested successful construction of DEPTORsg RNA1,DEPTOR-sg RNA2 and DEPTOR-sg RNA3 in Cas9 lentiviral vectors DEP1,DEP2 and DEP3.Lentiviral vectors DEP1,DEP2 and DEP3 were packaged in lentivirus and concentrated to a concentration of 2.36×10⁸ TU/ m L,2.0×10⁹ TU/ m L and 5×10⁸ TU/ m L,respectively.RPMI 8226 cells esposed to lentivirus for 72 h,more than 80% cells express the GFP fluorescence.The PCR product was digest by Cruiser^TM Enzyme.The results of agarose gel electrophoresis showed that bands of enzyme digestion,which confirmed the DEPTOR gene disruption in mixed pool.Base sequencing of DEPTOR fragment amplification,sequencing results showed that compare to negative control group LV-Cas9-GFP,CGG base deletions in DEP1 and DEP2 group,T base deletions in DEP3 group.Western blot confirmed that the DEPTOR protein in the DEP3 group had the best inhibitory effect.Conclusion: The Cas9 lentiviral vector of DEPTOR-sg RNAs were successfully constructed,the most efficient sg RNA and Cas9 lentiviral vector DEP3 was screened.Objective: To evaluate the effects and possible mechanisms of DEPTOR-targeted inhibition on angiogenesis in MM cell lines.Method: The MM cell lines RPMI8226(MOI=40)and MM.1S(MOI=30)were esposed to lentivirus GFP and DEP3,cell proliferation rate was detected by CCK8;72 h after infection,the medium supernatant was collected,human umbilical vein endothelial cell(HUVEC)cell line angiogenesis ability was evaluated by matrigel tube formation assay;72 h after infection,MM cells staining with mitochondrial superoxide red fluorescent probe,mitochondrial ROS was detected by flow cytometry and fluorescence microscope;the fluorescence enzyme expression of NF-?B reporter gene plasmid p NF?B-TA-luc was detected by microplate reader;the expression of DEPTOR protein,autophagy-related proteins Beclin 1 and LC3II/I,VEGF protein,Cyt C protein and NF-?B signaling pathway-related proteins I?B-?,p-p65 and p-p50 were detected by western blot.The expression of IL-6 and VEGF in the culture supernatant were detected by ELISA.Result: DEP3 lentivirus successfully inhibited the expression of DEPTOR protein and autophagy-related proteins Beclin 1 and LC3II/I in MM cell lines RPMI8226 and MM.1S compare to the GFP group.Targeted inhibition of DEPTOR could inhibit cell proliferation of MM cell lines RPMI 8226 and MM.1S.Targeted inhibition of DEPTOR in MM cell lines RPMI8226 and MM.1S promotes HUVEC tube-like structure formation.Targeted inhibition of DEPTOR in RPMI 8226 cell line,the number of tube-like structures increased from(6.5±2.3)to(11.33±1.98);targeted inhibition of DEPTOR in MM.1S cells,the number of tube-like structures was increased from(7.3±1.3)to(12.05±1.87),the difference between the groups was statistically significant,p<0.05;Cyt C entered the cytoplasm from mitochondria for targeted inhibition of DEPTOR in MM cell lines.Mito-SOX fluorescence of DEPTOR-targeted inhibition RPMI 8226 cells was increased from(36154±3021)to(42138±4340)compared to GFP group,the difference between the groups was statistically significant,p<0.05;the Mito-SOX fluorescence of DEPTOR-targeted inhibition MM.1S cells was increased from(37564±2135)to(51321 ±4166)compared to GFP group,the difference between the groups was statistically significant,p<0.05.The expression of NF-?B reporter gene p NF?B-TA-luc luciferase was increased,and NF-?B pathway related protein p-p65 and p-p50 of the nucleus was increased in DEP3 group compared to GFP group.The expression of IL-6 and VEGF protein in the culture medium were increased,the difference between the two groups was statistically significant,p<0.05.Conclusion: Targeted inhibition of DEPTOR in MM cell lines RPMI8226 and MM.1S inhibited the autophagy.At the same time,it promotes the formation of HUVEC tubelike structure through mitochondrial ROS-activated NF-?B signaling pathway in MM cell lines.Objective: To investigate the role and mechanism of autophagy in angiogenesis of DEPTOR-targeted inhibition MM cell lines RPMI 8226 and MM.1S.Method: MM cell lines RPMI 8226 and MM.1S were exposed to lentivirus GFP and DEP3 for 72 h;then treated with autophagy enhancer rapamycin(100 n M)or mitochondrial ROS scavenger Mito-TEMPO(10 ?M)for 24 h;the medium supernatant was collected,HUVEC cell line angiogenesis ability was evaluated by matrigel tube formation assay;mitochondrial superoxide red fluorescent probe staining was used for mitochondrial ROS was detected by flow cytometry;Western blot was used to detect the cell DEPTOR protein,autophagy related proteins Beclin 1 and LC3II/I,VEGF protein,Cyt C protein,NF-?B signaling pathway-related proteins I?B-?,p-p65 and pp50 expression;the expression of IL-6 and VEGF in the supernatant were detected by ELISA.Result: Both rapamycin and Mito-TEMPO reversed the pro-angiogenic effect of targeting ibhibition of DEPTOR expression in RPMI 8226 and MM.1S cell lines;rapamycin successfully activated targeted inhibition of DEPTOR-mediated autophagy inhibition in MM cell lines.Both rapamycin and Mito-TEMPO reversed the Cyt C entry into cytoplasm;the fluorescence intensity of Mito-SOX in RPMI 8226 cells exposed to rapamycin or Mito-TEMPO decreased to(28172±3765)or(25320±4132),respectively,which were significantly lower than the control group(41320±4003),p<0.05;the fluorescence intensity of Mito-SOX in MM.1S group exposed to rapamycin or MitoTEMPO decreased to(30214±2922)or(33159±3207),which were higher than the control group(51321 ±2166),p<0.05;both rapamycin and Mito-TEMPO reversed the NF-?B pathway-related proteins p-p65 and p-p50 in the nucleus and the downstream IL-6 and VEGF expression induced by targeted inhibition of DEPTOR expression in RPMI 8226 cells and MM.1S cells,the difference between the groups were statistically significant,p <0.05.Conclusion: Autophagy regulate angiogenesis induced by targeted inhibition of DEPTOR through mitochondrial ROS regulating NF-?B signaling pathway and its downstream IL-6 and VEGF.