Mechanism Of SIRT1 In Bortezomib-induced Drug Resistance In Multiple Myeloma

Objective1?To explore the relationship between SIRT1 and bortezomib-induced drug resistance in multiple myeloma.2?To study the mechanism of SIRT1 promoting drug resistance in multiple myeloma through hedgehog signaling pathway.3?To evaluate the effect of targeted SIRT1 on reversing drug resistance and bone destruction in myeloma.MethodsBortezomib-resistant(BR)MM cell lines were established by low-dose continuous induction;Differentially expressed genes were screened by RNA sequencing;Plasma cells were isolated from primary and recurrent MM patients by CD138 + magnetic beads and their SIRT1 expression levels were detected;The correlation between SIRT1 expression level and prognosis of MM patients was analyzed by Pearson correlation analysis;The interaction between SIRT1 and GLI2 protein was detected by western blot,Immunoprecipitation and PLA technology;The expression level of SIRT1 protein in bone tissue samples of MM patients was detected by immunohistochemical staining;The binding of GLI2 to SIRT1 promoter region was detected by chromatin immunoprecipitation technology,and the change of gene transcription activity was detected by luciferase reporter assay;Cell viability or apoptotic rate were detected by CCK8 and flow cytometry;Subcutaneous transplantation tumor model and intramedullary injection of MM cell model were established in NOD/SCID mice to further verify the anti-MM effect of SIRT1 inhibitor EX527 combined with BTZ.Results1?Two BTZ-resistant MM cell lines were established.286 up-regulated genes and 75 down-regulated genes were screened by RNA-seq.SIRT1 was found to be one of the most up-regulated genes.Western blot showed that the expression of SIRT1 protein increased with the increase of BTZ concentration during the induction of BTZ-resistant MM cell lines.IHC staining showed that the expression of SIRT1 in relapsed MM patients was significantly higher.These results suggest that SIRT1 is associated with proteasome inhibitors which can induce resistance in MM cells.2?BTZ combined with epigenetic small molecular inhibitor library(containing 182 compounds)was used to treat MM cell lines to screen for relative cell survival.It was found that SIRT1 specific inhibitors S1541(EX527,Selisistat)and S2804(Sirtinol)had more obvious synergistic effect on anti-MM of BTZ.Western blot results showed that the lower the expression level of SIRT1 protein,the more sensitive the cells are to BTZ.These results together demonstrate that SIRT1 plays a very important role in mediating MM-BTZ resistance.3?RNA-seq data showed that some target genes of SIRT1 and hedgehog signaling pathway were significantly correlated.Luciferase assay showed that the transcriptional activity of GLI was dose-dependent on SIRT1.Immunofluorescence staining showed GLI2 mainly translocated into the nucleus after overexpression of SIRT1,while after knockdown of SIRT1,GLI2 expression concentrated in the cytoplasm.These results suggest that SIRT1 may regulate the activation of Hh signaling pathway to some extent by mediating the key transcription factor GLI2.4 ? The interaction between SIRT1 and GLI2 protein was found by immunoprecipitation assay.The PLA experiment further confirmed the communication between SIRT1 and GLI2.The direct interaction between SIRT1 and GLI2 was confirmed by endogenous immunoprecipitation in MM cell lines.Immunohistochemical staining showed that the expression of SIRT1 and GLI2 in bone marrow sections of the same MM patients had the same characteristics of co-localization and co-expression.5?Immunocoprecipitation assay showed that GLI2 was modified by acetylation.Pull-down results showed that overexpression of SIRT1 significantly weakened the ubiquitination of GLI2,and overexpression of P300 significantly enhanced the ubiquitination of GLI2.Overexpression of GLI2 in cells showed that GLI2 could interact with SIRT1 protein by immunoprecipitation,and SIRT1 could deacetylate GLI2.6?Luciferase assay showed that the transcriptional activity of SIRT1 was gradually activated by GLI2 protein.Through ChIP-qPCR,we found that GLI2 was abundantly enriched in the promoter region of SIRT1.Quantitative PCR and Western blot analysis showed that the level of SIRT1 mRNA and protein increased with the increase of GLI2 expression.The above experiments indicate that SIRT1-GLI2-Hh signaling pathway-SIRT1 positive regulatory ring exists,and the up-regulation of SIRT1 causes the activation of Hh signaling pathway to enhance the transcriptional activity of GLI2.7?Xenograft and intra-bone models were established in NOD/SCID mice.The results showed that the growth of tumors in BTZ-EX527 combination group was obvious inhibited compared with control and the single treatment group,and the overall survival rate was significantly prolonged,while the degree and area of bone destruction were significantly reduced.Conclusions:1?SIRT1 is a key regulatory factor that is independent of mutation but specifically induced by proteasome inhibitors and plays an important role in the study of drug resistance in multiple myeloma.2?SIRT1 stabilizes GLI2 protein and activates Hh signaling pathway by interacting with GLI2.3?SIRT1 is a direct target gene of Hh signaling pathway.It maintains hedgehog activity by forming a positive regulatory pathway of SIRT1/GLI2/Hedgehog pathway/SIRT1.4? In vivo and in vitro experiments,targeted SIRT1 can synergize with bortezomib to improve the sensitivity of multiple myeloma cells to bortezomib.