| Background and Objects:miRNAs are a class of endogenous non-coding regulatory RNAs with more than 20 nucleotides in length.miRNA play a significant role in a variety of important biological processes,such as cell development,proliferation,differentiation and apoptosis,regulating by the cleavage or sequentially specific translational repression of target gene mRNA.Numerous studies have found that there is abnormal regulation of miRNAs in many tumors,thus affecting the evolution,progression,metastasis and invasion of tumor.Diffuse large B-cell lymphoma(DLBCL)is one of the most common types of Non-Hodgkin lymphoma.CHOP(cyclophosphamide,doxorubicine,vincristine,and prednisone)chemotherapy is the most classical therapy for DLBCL that has been proved so far,yet drug resistance or relapse still occur in over 50%of patients.miRNAs are found to be closely related to the drug resistance of DLBCL according to the recent studies.miR-148b is located at human chromosome 12q13.13,which widely shows abnomal expression in colon cancer,gastric cancer,breast cancer,lung cancer,melanoma and other tumor tissues.Evidence indicates that upregulation of miR-148b can reverse cisplatin resistance of non-small cell lung cancer cell lines,while increasing the sensitivity of human Burkitt lymphoma Raji cell lines to radiotherapy.However,the role of miR-148b in the drug resistance of DLBCL has not been studied.This study aims to investigate the potential function and mechanism of miR-148b in the drug resistance of DLBCL,providing new theoretical thinking for the reversal of drug resistance and targeted therapy.Experimental Methods:1.Fresh tumor tissues of DLBCL patients were collected and divided into CHOP sensitive group and CHOP resistant group.The expression of miR-148b in the two groups were compared by real-time fluorescence quantitative PCR.2.The human DLBCL drug-resistant cell lines were established by gradually increasing the drug concentration that started with low concentration,CCK-8 method was used to compare the activity of different concentrations of CHOP on CRL2631 cells and drug-resistant CRL2631/CHOP cells,and Western blot was adopted to detected the expression of MDR-1 protein in CRL2631 cells and CRL2631/CHOP cells.3.Total RNA of CRL2631 cells and CRL2631/CHOP cells were collected,and real-time fluorescence quantitative PCR was performed to compare the expression of miR-148b in the two groups.4.Search for potential target genes of miR-148b according to the bioinformatics prediction software.The wild type/mutant type variant Ezrin 3' UTR luciferase reporter carrier and miR-148b inhibitor/miR-148b mimic were co-transfected into CRL2631 cells,using classical experiment of dual-luciferase reporter gene.According to the dual-luciferase reporter gene detection kits,the signals of luciferase and amplite renilla luciferase was detected in each group and relative luciferase activity was calculated with amplite renilla luciferase as control.5.Real-time fluorescence quantitative PCR and western blot were performed to detect the expression of Ezrin mRNA and protein after respectively transfecting miR-148b inhibitor and miR-148b mimic into CRL2631 cells.6.Ezrin mRNA and protein expression levels of CHOP sensitive group and CHOP resistant group in DLBCL patients were compared by Real-time fluorescence quantitative PCR and western blot,so were the CRL2631 cells and CRL2631/CHOP cells.The correlation between Ezrin mRNA and miR-148b expression in DLBCL patients was analyzed.7.miR-148b inhibitor,NC,si-Ezrin and si-control were tansfected into CRL2631 cells respectively.CCK-8 method was used to compare the effect of different concentrations of CHOP on the acticity of CRL2631 cells in each group.Western blot was adopted to detect the expression of Ezrin and MDR-1 in each group.8.CRL2631/CHOP cells were transfected with miR-148b mimic,pre-NC,pcDNA-Ezrin and pcDNA.CCK-8 method was used to compare the effect of different concentrations of CHOP on the acticity of CRL2631/CHOP cells in each group.Western blot was adopted to detect the expression of Ezrin and MDR-1 in each group.9.agomir-148b and agomir-NC were respectively transfected into CRL2631/CHOP cells,which were then subcutaneously inoculated into tumors in the backs of nude mice.The growth of subcutaneously transplanted tumors in the two groups was observed and compared by CHOP protocol.Tumor tissue was removed and the expression of miR-148b in the two groups was detected by Real-time fluorescence quantitative PCR.Real-time fluorescence quantitative PCR and western blot were used to detect the mRNA and protein expression of Ezrin in the two groups.Results:1.Human DLBCL resistant cell line CRL2631/CHOP was induced.Compared to the CRL2631 cells,the sensitivity of CRL2631/CHOP cells to CHOP was decreased,and the IC50 value was significantly increased,as well as the expression level of MDR-1 protein.2.The expression level of miR-148b in tumor tissues of DLBCL patients in the CHOP sensitive group was significantly higher than that in the CHOP resistant group,while tmiR-148b expression level of CRL2631/CHOP cells was greatly lower than that of CRL2631 cells.3.Ezrin was found to be the direct target gene of miR-148b by microrna.org.Compared with the control group,luciferase activity was remarkably reduced in miR-148b mimic+wild-type Ezrin 3'UTR carrier(WT)group,while luciferase activity was not significantly changed in miR-148b mimic+mutant-type Ezrin 3'UTR carrier(MUT)group.On the contrary,in the miR-148b inhibitor+WT group,luciferase activity evidently increased compared with controls,but the luciferase activity change in the miR-148b inhibitor+MUT group shows no significant difference compared with controls.4.After the up-regulation of miR-148b,the expression levels of Ezrin mRNA and Ezrin protein in CRL2631 cells were both greatly decreased compared with the control group.In addition,Ezrin mRNA and protein expression levels of CRL 2631 cells were increased after the down-regulation of miR-148b compared with the control group.5.Ezrin mRNA and protein expression levels in the CHOP sensitive group of DLBCL patients were significantly lower than those in the CHOP resistant group,while Ezrin mRNA and protein expression levels in CRL2631/CHOP cells were evidently higher than those in CRL2631 cells.Futhermore,a linear negative correlation was found between miR-148b and Ezrin mRNA expression in patients with DLBCL.6.After miR-148b inhibitor transfected into CRL2631 cells,Ezrin protein expression was up-regulated,sensitivity of cells to CHOP was decreased,IC50 value was significantly increased and MDR-1 protein expression was also significantly increased.After co-transfected with miR-148b inhibitor and si-Ezrin,Ezrin expression of CR2631 cells in the miR-148b inhibitor+si-Ezrin group was evidently declined,the sensitivity of cells to CHOP was remarkably increased and MDR-1 protein expression was decreased,compared with the miR-148b inhibitor group.7.After transfection with miR-148bmimic into CRL2631/CHOP cells,Ezrin protein expression was down-regulated,sensitivity of cells to CHOP was enhanced,IC50 value was significantly declined and MDR-1 protein expression was also significantly declined.However,after co-transfection with miR-148b mimic and PcDNA-Ezrin,Ezrin expression in miR-148b mimic+PcDNA-Ezrin was greatly up-regulated,sensitivity of cells to CHOP was remarkably reduced and MDR-1 protein expression was evidently increased,compared with the miR-148b inhibitor group.8.After nude mice were treated with CHOP,the volume of subcutaneously transplanted tumor in the agomir-148b group was greatly reduced compared with that in the agomir-NC group.Besides,the expression level of miR-148b in the agomir-148b group was evidently higher than that in the agomir-NC group while the expression level of Ezrin mRNA and protein was remarkably lower than that in the agomir-NC group.Conclusion:1.miR-148b showed low expression in DLBCL patients with drug resistance and CRL2631/CHOP cells.2.Ezrin was highly expressed in DLBCL patients with drug resistance and CRL2631/CHOP cells.3.Ezrin was the target gene of miR-148b.4.miR-148b regulates the resistance of DLBCL cells to CHOP by targeting to Ezrin.5.The results of this study are helpful to explore the molecules and the regulatory mechanisms that play a key role in the drug resistance of DLBCL cells,providing novel theoretical ideas for the reversal of drug resistance and targeted therapy. |
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