Study On The Mechanism Of Nuclear Transcription Factor Snail1 Silencing To Improve The Chemotherapeutic Sensitivity Of Bortezomib Resistant Multiple Myeloma Cells

BackgroundMultiple myeloma is a malignant tumor originated from cell line B.Its typical feature is abnormal proliferation of plasma cells,resulting in increased blood calcium,renal damage,anemia,bone diseases and other clinical manifestations.The median age of MM diagnosis is 69 years,and the average overall survival time is 6-7 years.Chemotherapy is the main treatment for this disease.In recent years,with the in-depth exploration of the pathogenesis of MM,researchers have screened targeted therapeutic drugs such as proteasome inhibitors and immunomodulators from the molecular level.The emergence of new drugs has made breakthroughs in the treatment of MM.As proteasome inhibitor,the clinical use of bortezomib is an important event in MM chemotherapy.Bortezomib can not only directly inhibit the proliferation of myeloma cells and induce apoptosis,but also improve the chemosensitivity of dexamethasone and other drugs.Although bortezomib-based chemotherapy has significantly improved the symptoms of MM patients and delayed tumor progression in the short term,almost all MM patients who use bortezomib will show drug resistance characteristics after taking the drug for a period of time,which greatly limits the long-term efficacy of the drug for MM.Up to now,there is little research on the mechanism of MM’s resistance to bortezomib,the mechanism of which is still unclear,and there is no effective clinical solution.Snail1 is the member of the Snail superfamily of zinc-finger transcription factors.It usually acts as a transcriptional repressor.Snail1 transcriptional factor plays a key role in the control of epithelial to mesenchymal transition and fibroblast activation.Snail1 expression and function is regulated at multiple levels from gene transcription to protein modifications.Snail1 is widely expressed in various cancers,and overexpression is frequently associated with migration,invasion and metastasis.At present,researches on Snail1 are mainly focused on hepatocellular carcinoma,gastric cancer,bladder cancer and other diseases,but no report has been found in multiple myeloma.Part Ⅰ The Up-regulation of Expression of Snail1 Gene During the Generation of Bortezomib Resistance in Multiple MyelomaObjective: Clarify the abnormal expression of Snail1 gene in bortezomib resistance mechanism of Multiple myeloma(MM).Methods: Multiple myeloma cells(MMCs)samples were collected before and after the formation of drug resistance mechanism in clinical bortemozib.Total RNA and total cell protein were extracted,respectively,and quantitative fluorescence PCR(realtime-PCR)was used to detect the m RNA content of Snail1 gene.The protein expression of Snail1 was detected by Western blotting,and the transcription and protein expression differences between the two groups of tumor samples were analyzed.Meanwhile,m RNA content and protein expression of MDR1 and P53 were detected.The expression of the two functional genes in the progress of bortezomib resistance mechanism of MMCs was also analyzed.Results: The m RNA and protein expressions of Snail1 gene in MMCs cells in bortezomib were significantly enhanced compared with MMCs before chemotherapy(P<0.01,vs MMCs before chemotherapy).MRNA and protein expression of MDR1 gene were significantly enhanced(P<0.01,vs MMCs before chemotherapy).P53 protein expression was significantly decreased(P<0.01,vs MMCs before chemotherapy),while m RNA content was not significantly different(P>0.05,vs MMCs before chemotherapy).The relative content of hsa-mir-22-3p was significantly enhanced(P< 0.01,vs MMCs before chemotherapy).Conclusion: The Snail1 gene has significantly enhanced m RNA and protein expression during the generation of the mechanism of MMCs’ resistance to bortezomib,which was consistent with the change trend of MDR1 gene transcription and protein expression,contrary to the change trend of P53 protein expression,and consistent with the change trend of hsa-mi R-22-3p relative content.Part Ⅱ Analysis of the Mechanism of Snail1 Silencing to Improve MMCs Resistance to BortezomibObjective: Clarify Snail1 gene silencing improves chemotherapy sensitivity of bortezomib-resistant MMCs and elucidate the mechanism.Methods: Snail and P53 were silenced in bortezomib-resistant MMCs cell line XG-7/Bor and RPMI-8226 /Bor by lentivirus pathway.At the same time,MDR1 was overexpressed.Total RNA and cellular protein were extracted respectively.Realtime-PCR and Western blotting methods were used to detect the changes of m RNA and protein in three groups respectively.Bioinformatics predicted the binding sites of hsa-mi R-22-3p and P53 gene 3 ’non-coding region(UTR),which was confirmed by luciferase reporter gene experiment.Bioinformatics predicted nuclear Transcription factor Snail1 with hsa-mi R22-3p and MDR1 promoter Transcription factor binding sites(TFBS)and confirmed.Snail1 was silenced in XG-7 /Bor and RPMI-8226 /Bor cells.The changes of drug resistance and apoptosis were observed.Total cellular proteins were collected after gene intervention,and changes in MDR1 and P53 protein expressions in each group were detected by Western blotting.In the experiment,MDR1 and P53 were set as the control group to evaluate the dominance of Snail1/MDR1 and Snail1/ hsa-mi R-22-3p /P53 action pathways.Results:The lentivirus pathway could effectively silence Snail1 and P53 in XG-7 /Bor cells and RPMI-8226 /Bor cells,and effectively overexpress MDR1 gene(P<0.01,vs cell control group or NC control group).Hsa-mi R-22-3p inhibited the expression of P53 protein by binding to the P53 gene’s 3 ’UTR seed region " 5’-GGCAGCU-3’".Snail1 had TFBS binding sites in both MDR1 and has-mi R-22-3p promoter regions,and the transcription of both was positively regulated.Snail1 silencing can effectively reduce the IC50 value of XG-7 /Bor and RPMI-8226 /Bor.In XG-7/Bor cells,Snail1 gene silencing can effectively up-regulate the apoptosis rate of cells(P<0.01,vs cell control group or NC control group).It can inhibit MDR1 protein expression and up-regulate P53 protein expression(P<0.01,vs cell control group or NC control group).The overexpression of exogenous MDR1 gene or the silencing of P53 gene could significantly inhibit the influence of Snail1 gene silencing on its expression(P<0.01,vs Snail1 gene silencing group).Conclusion: Lentivirus pathway is an effective method to improve the efficiency of gene transduction in MMCs cells.Snail1 gene silencing effectively improved the drug resistance of XG-7 /Bor and RPMI-8226 /Bor drug-resistant cells through the Snail1/MDR1 action pathway,and induced apoptosis through the Snail1/hsa-mi R-22-3p /P53 action pathway.