Study On RFWD2 In Drug Resistance Of Multiple Myeloma And Drug Intervention

Objective:The research was aimed at analyzing the genes related to the survival rate and drug resistance of MM patients by using gene chip technology,and selecting an important candidate oncogene RPWD2.The relationship between RFWD2 gene expression and MM cell proliferation and drug resistance was investigated in vitro and in vivo.The mechanism of RFWD2 causing MM resistance was explored;Looking for a Chinese medicine that can inhibit the proliferation of MM cells.Methods:1.Bone marrow samples of MM patients after initial diagnosis and high-dose chemotherapy were collected and analyzed using Affymetrix U133 Plus 2.0 gene microarray to obtain the whole genome expression profile and selected a candidate oncogene.Kaplan-Meier survival curve was used to analyze the relationship between the gene expression and the survival rate and drug resistance of MM patients.MM cell lines ARP1 and H929 were infected with RFWD2 targeted cDNA or shRNA virus,and stable expression of RFWD2 was detected by Western blot(WB).MTT colorimetry was used to compare the proliferation of transfected cells and control cells.The expression of RFWD2 in drug-resistant and sensitive MM cell lines was detected by WB.2.The toxicity of doxorubicin,dexamethasone,bortezomib,carfizomib and ixazomib(5 lMM chemotherapy drugs)on ARP1 and H929 cells with normal and high RFWD2 expression was determined by MTT assay,and the drug IC50 value was calculated.Flow cytometry was used to detect the apoptosis of ARPI and H929 cells with normal and high RFWD2 expression induced by the above five chemotherapy drugs.3.MM xenografts in immune-deficient NOD/SCED mice were established by injecting with normal or overexpressed RFWD2 ARP1 cells.Adriamycin or bortezomib were injected into the drug group,and tumor diameter was measured regularly with caliper.The mice were sacrificed until the tumor reached 20 mm in diameter,and the tumors were dissected,photographed,weighed and preserved frozen.4.Ubiquitination assay in vivo verified the effect of RFWD2 expression on P27 ubiquitination.WB verified the relationship between RFWD2 expression and the expression of three E3 ubiquitin ligases(Artemis,KPC2 and RCHY1)of P27,and screened out the E3 related to RFWD2 expression.The interaction between RFWD2 protein and E3 ligase protein was verified through co-immunoprecipitation(Co-IP)experiment.Kaplan-Meier survival analysis was performed by fitting MM patient data with RPWD2 and RCHY1 each expressing 50%.5.MTT assay was used to detect the inhibitory effect of triterpene acids of loquat.leaf on MM cell proliferation in vitro.A model of heterogenous transplanted myeloma mice was established and intervention with triterpene acids of loquat.leaf was given.The tumor diameter was measured regularly with calipers until the tumor diameter reached 20 mm.The mice were sacrificed.Results:1.Gene sequencing results showed that the gene expression of RFWD2 in MM patients was significantly higher than that in monoclonal gammopathy of undetermined significance(MGUS)and normal plasma(NP).Kaplan-Meier survival analysis found that in Total Therapy 2(TT2)patients,overall survival(OS)and event-free survival(EFS)of patients with high RFWD2 expression were significantly lower than those with low expression.In the results of the clinical trials on Assessment of Proteasome Inhibition for Extending Remission(APEX),the OS of the patients highly expressing RFWD2 is significantly lower than that of the patients poorly expressing RFWD2.The WB validation showed that the RFWD2 shRNA and RFWD2 cDNA transfected MM cell models were successfully constructed.MTT assay of cell proliferation showed that,compared with the control group,the down-regulated RFWD2 inhibited the growth of MM cells,while the high expression of RFWD2 promoted the growth of MM cells.Meanwhile,WB results showed that RPWD2 expression was negatively correlated with P27 expressionThe RFWD2 level in relapsed MM patients was significantly higher than the baseline level.In the OS analysis of patients with TT2 test recurrence,the survival rate of patients with high RFWD2 gene expression was significantly lower than that of patients with low RFWD2 gene expression.The RFWD2 gene signal level of the patients who were tolerant to bortezomib was significantly higher than that of the patients who were sensitive to bortezomib.There was no significant difference in RFWD2 gene level between patients with dexamethasone tolerance and those with sensitivity.WB results showed that,compared with normal MM.1 cells,the expression of RFWD2 was lower in MM.1 cells that were resistant to dexamethasone,and there was no difference in the expression of P27.Compared with normal 8226 or ANBL6 cells,RFWD2 expression was significantly increased in bortezomib resistant 8226 or ANBL6 cells,while P27 expression was opposite.2.MTT results showed that there was no significant difference in IC50 of adriamycin and dexamethasone between normal and high RFWD2 expression cells.However,the IC50 values of the three proteasome inhibitors,bortezomib,carfizomib and isazomil,for the cells with high expression of RFWD2 were significantly higher than those of the control group.Cell apoptosis was analyzed by flow cytometry,which was consistent with MTT results.There was no significant difference in apoptosis induced by adriamycin and dexamethasone between MM cells with normal and high expression RFWD2.However,under the stimulation of bortezomib,caffizomib and ishazomib,the apoptosis rate of cells with high expression of RFWD2 was lower than that of the control group.3.The results of animal experiments showed that the tumor weight and tumor volumeof mice overexpressing RFWD2 were remarkably higher than that of the control group.After bortezomib treatment,the tumor weight and tumor volume of mice overexpressing RFWD2 also exhibited an upward trend compared to the control group.However,after treatment with dexamethasone,the tumor weight of mice with high expressing RFWD2 was not significantly different from that of the control group,while the tumor volume was smaller than that of the control group.4.The results of in vivo ubiquitination experiment showed that the level of P27 ubiquitination in cells with high RFWD2 expression was higher than that in the control group.WB results showed that,compared with control cells,only RCHY1 expression was also increased in cells with high RFWD2 expression,while there was no difference in the expression of the other two P27 ubiquitin ligases.Meanwhile,RCHY1 expression was also decreased in RFWD2 down-regulated cells.Co_IP results showed that there is an interaction between RFWD2 and RCHYI.Kaplan-Meier survival analysis showed that in TT2 and APEX patients,the survival rate of patients with both high RFWD2 and RCHYI expression was significantly lower than that of patients with medium and low expression of both genes.5.MTT results showed that triterpene acids of loquat.leaf could not only inhibit the growth of MM wild-type cells,but also inhibit the growth of MM cells with high RFWD2 expression.The results of animal experiments showed that triterpene acids of loquat.leaf could inhibit the growth of tumor,but it was not statistically significant compared with the control group.Conclusions:The high expression of RFWD2 was associated with relapse and drug resistance in MM patients.High expression of RFWD2 induces resistance of MM cells to proteasome inhibitors in vitro.The high expression of RFWD2 induced resistance to bortezomib in MM xenograft mice in vivo.RFWD2 regulates the ubiquitination of P27 by interacting with RCHY1 ubiquitin ligase.The triterpene acids of loquat.leaf can inhibit MM cell proliferation.