The Roles And Mechanisms Of UbcH10 On Bortezomib-resistant Multiple Myeloma Cell Lines

Multiple myeloma(MM)is the second most common hematologic malignancy,with an incidence higher than that of acute leukemia.Although most MM patients are sensitive to initial chemotherapy treatments,relapse often occurs due to an acquired drug resistance.The introduction of bortezomib(BTZ)represented a breakthrough in the treatment of MM.Chemotherapy regimens based on BTZ have become first-line recommendations for MM patients with untreated,relapsed or refractory myeloma,including patients who are eligible for autologous hematopoietic cell transplantation.BTZ resistance has a strong impact on the clinical effectiveness of MM treatments.Therefore,determining the process by which BTZ resistance develops in MM and searching for effective regimens to overcome resistance are critical research areas.Approximately 80-90% of cell proteins are degraded by the ubiquitin-proteasome pathway(UPP);thus,up to 90% of cell proteins may be a target for BTZ.The study of abnormal degradation of UPP pathway proteins may facilitate an understanding of drug resistance in MM.Ubc H10,also known as UBE2 C,is a gene located at 20q13.12 and has a key role in UPP mediated protein degradation.Research indicates Ubc H10 is highly expressed in many cancers,including breast cancer,ovarian cancer,thyroid cancer,oesophageal cancer,lymphoma,MM,and hepatocellular carcinoma.In addition,Ubc H10 overexpression is often associated with a high cancer grade,high proliferation and poor tumour prognosis.There are few studies on the relation between Ubc H10 and chemosensitivity.Although chemotherapy is an important cancer treatment,drug resistance creates significant challenges to eliminate cancer cells and causes poor prognosis.Many studies have shown that mi RNAs are involved in cancer initiation and development cancer by regulating apoptosis,proliferation,differentiation and metastasis as well as influencing drug resistance via their target genes.A study of differentially expressed mi RNAs and the affiliated regulation of drug resistance-associated genes may contribute to understanding the mechanisms of drug resistance.All mutations,misexpression and abnormal processing of mi RNAs impair function and can result in abnormal target gene expression.This study demonstrates,for the first time,that Ubc H10 is highly expressed in the BTZ-resistant myeloma cell lines U-266/BTZ,NCI-H929/BTZ and RPMI-8226/BTZ,which is attributed to the inactivation of post-transcriptional control.The in-depth study reveals that during the development of BTZ resistance in these cells,the hsa-mi R-631 levels decreased,which resulted in the increased expression of the target gene Ubc H10.We also found that the multiple drug-resistant protein MDR1 exhibited a positive correlation with Ubc H10 due to the reduced ubiquitination of MDR1,which was caused by high Ubc H10 expression.By overexpressing mi R-631,both BTZ sensitivity and BTZ-induced apoptosis were enhanced in the resistant cells.Meanwhile,resensitization by mi R-631 overexpression was blocked by exogenous expression of Ubc H10,which was not regulated by intracellular mi R-631.In conclusion,the mi R-631/Ubc H10/MDR1 pathway is closely associated with the development of BTZ resistance in myeloma cells,and the overexpression of mi R-631 can significantly improve BTZ sensitivity in resistant myeloma cells.Part ?: the Study of the Ubiquitin-conjugating Enzyme Gene Ubc H10 Expression in the Myeloma Cell Lines and BTZ-resistant Myeloma Cell LinesObjectives: To detection Ubc H10 m RNA and protein levels in myeloma cells and BTZ-resistant myeloma cell lines,and study its correlation with BTZ resistance in these cells.Methods: The expression of Ubc H10 and protein of m RNA gene in five MM cells(U-1996,U-266,MM.1S,NCI-H929 and RPMI8226)were detected by PCR and Western-Blotting,while the normal plasma cells were used as control.With the method of increasing drug concentration,the bortezomib resistant cell lines were established and identified.The expression of m RNA and protein of Ubc H10 gene in drug resistant cell lines and their parental cell lines were detected by semi quantitative PCR and Western blotting.Results: Compared with normal plasma cells,the content of Ubc H10 m RNA in U-1996,U-266,MM.1S,NCI-H929 and RPMI8226 cells was significantly increased,which was 4.131±0.373(P=0.001),4.950±0.428(P<0.001),6.086±0.606(P<0.001),8.581±0.767(P<0.001),4.278±0.480(P=0.001),respectively.The expression levels of Ubc H10 protein in U-1996,U-266,MM.1S,NCI-H929 and RPMI8226 cells were significantly higher in normal plasma cells,and the difference was statistically significant.We successfully established three drug resistant cell lines U-266/BTZ,NCI-H929/BTZ and RPMI-8226/BTZ.The results of IC50 measurement show that the IC50 values in U-266/BTZ,NCI-H929/BTZ and RPMI-8226/BTZ increased from 11.10±1.24 n M,6.08±0.71 n M and 10.02±1.62 n M in their parental cells to 55.62±4.88 n M,49.12±4.32 n M,and 61.21±5.82 n M,respectively.These figures represented statistically significant differences between the resistant cells and their parental cells(P<0.01).Western blotting results indicated that Ubc10 protein was higher in resistant myeloma cells than in parental cells(P<0.05).While Ubc H10 m RNA increased in the resistant cells,there was no significant difference between the resistant cells and their parental cells(P>0.05).Conclusion: Ubc H10 was highly expressed in the BTZ-resistant myeloma cell lines U-266/BTZ,NCI-H929/BTZ and RPMI-8226/BTZ,which was attributed to the inactivation of post-transcriptional control.Part ?: Knockdown of Ubc H10 Expression by RNA Interference Inhibits Myeloma Cell Proliferation and Enhances Cell ApoptosisObjective: To investigate the effects of Ubc H10 gene silencing on proliferation and apoptosis of myeloma cell line U-1996.Methods: According to the Ubc H10 genetic information,the si RNA sequence targeting Ubc H10 was designed.It was transfected into U-1996 cells via lipofectamine2000.Ubc H10 m RNA and protein were examined 48 h after transfection,respectively.Cells without transfection were served as blank controls and those transfected with negative sequence as negative controls.The cell proliferation was detected by CCK-8 assay 24 h,48 h and 72 h after transfection,and the cell apoptosis rate was detected by FCM 48 h after transfection.Results: In this study,the datas showed that the expression level of Ubc H10 m RNA and protein was knocked down by Ubc H10 si RNA as indicated by RT-PCR and western blotting analysis.Proliferation of U-1996 cells was significantly reduced compared with that of control at 24,48 and 72 h after transfection.Ubc H10 gene silencing can significantly inhibit the proliferative activity of U-1996 cells and increase the cells apoptosis by the flow cytometric analysis.Conclusion: Ubc H10 gene silencing can significantly inhibit the proliferative activity of U-1996 cells and increase the cells apoptosis.Part ?: Hsa-mi R-631 Resensitizes Bortezomib-resistant Multiple Myeloma Cell Lines by Inhibiting Ubc H10Objective: To study the role of hsa-mi R-631 in bortezomib resistant cell lines,and to analyze the regulation mechanism of drug resistance.Methods: Three selected MM cell lines,U-266,NCI-H929,and RPMI-8226 were cultured with BTZ in a gradient of increased concentrations.Cells were cultured in a medium containing 51.2 n M BTZ for two additional passages.These cell lines were named U-266/BTZ,NCI-H929/BTZ and RPMI-8226/BTZ.CCK-8 assay was employed to determine cell inhibition by BTZ and to calculate the drug dose causing 50% growth inhibition(IC50)values.Meanwhile,the resistant and parental cells were harvested for total RNA and protein extraction,followed by real-time PCR and western blotting to measure the Ubc H10 m RNA,hsa-mi R-631 and Ubc H10 proteins.The hsa-mi R-631 binding region of Ubc H10 gene 3 'UTR region was predicted by bioinformatics method and the binding site was verified by luciferase reporter gene assay.By lentivirus experiment system,over expression of has-mi R-631 in three strains of resistant cell lines,and then use different concentrations of bortezomib treatment after 48 hours.Cell viability was determined with CCK-8 method,and obtained the cells with bortezomib IC50 values.Results: In this study,we established three BTZ-resistant cell lines,including U-266/BTZ,NCI-H929/BTZ and RPMI-8226/BTZ,by using gradual induction.We examined the m RNA and protein expression levels of Ubc H10 in the resistant cells and their parental cells.The data showed that Ubc H10 protein was significantly increased in the resistant cell lines while m RNA was slightly increased.We searched for mi RNAs with a binding site on the 3' UTR of Ubc H10.We then quantitatively measured the mi RNAs in the resistant and parental cells.The results indicated hsa-mi R-631 was negatively correlated with the Ubc H10 protein.A luciferase reporter assay verified that hsa-mi R-631 was able to bind to Ubc H10-3'UTR and inhibit protein expression through the seed site.The results suggest that the inactivation of Ubc H10 regulation by hsa-mi R-631 may be a molecular mechanism for resistance in MM cell lines.Genetic intervention was conducted in the three resistant cell lines using a lentiviral approach.The gene delivery efficiency was close to 100%,according to the GFP levels.Lv-mi RNA631 infection significantly increased mature mi R-631 levels in both resistant cell lines and parental cells(P < 0.01).Western blotting results demonstrated that LV-mi R631 significantly decreased Ubc H10 protein(P<0.01 vs.control).Lv-Ubc H10 infection significantly increased Ubc H10 protein(P < 0.01 vs.control),as did the combination of Lv-mi R631 and Lv-Ubc H10.No obvious difference was found in these groups compared to the Lv-Ubc H10 infection group(P > 0.05).The changes in the MDR1 proteins were in similar to those found in Ubc H10.The overexpression of mi R-631 in three BTZ-resistant cell lines reduced BTZ IC50 values.In this study,we also verified the specificity of the pathway,as mi R-631 overexpression reversed the resistance to BTZ by inhibiting Ubc H10 expression while promoting the ubiquitination of MDR1 and reducing the MDR1 protein levels.Moreover,the overexpression of exogenous Ubc H10 under the control of a CMV promoter while free of the mi R-631 binding site inhibited the reversion of BTZ resistance through mi R-631 overexpression.Conclusion: We identified the mi R-631/Ubc H10/MDR1 pathway was involved in the development of BTZ resistance in myeloma cells.In addition,the study increased the sensitivity of myeloma cells to BTZ by expressing mi R-631 using genetic engineering.These results may help resolve the issue of BTZ resistance.The study also indicated mi R-631 may be used as a genetic marker for the selection of therapy regimes in MM.However,for patients with low mi R-631 levels,therapies other than those based on BTZ may be more effective.