| Chronic myeloid leukemia(CML)is a kind of myeloproliferative tumor caused by abnormal proliferation of hematopoietic stem cells.The P210 protein encoded by the BCR-ABL1 fusion gene produced by t(9;22)(q34;q11)translocation has high tyrosine kinase activity,which leads to abnormal proliferation and apoptosis of cells.As the first targeted therapy for clinical use,Imatinib Mesylate(IM)opened a new era for CML treatment.However,there are still some patients suffering from primary or secondary resistance,which affects the patient's prognosis and shortens their survival period.MicroRNAs(miRNAs)are a class of non-coding small RNAs of about 18-25 nucleotides that are targeted to the 3'-untranslated region(3'-UTR)of target genes.Horizontal inhibit expression of target gene at the post-transcriptional period includes preventing the translation and accelerating the degradation of the target gene.In the study of leukemia including CML,miRNAs not only participated in the occurrence and development of CML,but also played an important role in IM resistance.Deubiquitinating enzymes(DUBs)can dissociate ubiquitin from ubiquitinated target proteins,prevent proteins from being degraded,and participate in many important life activities,including cell cycle regulation,gene transcription,and kinase activation,protein degradation,DNA repair,etc.Ubiquitin-specific proteases 15(USP15),is a members of the USP family and plays different biological roles in different tumors.However,USP15 has not been studied in CML.This study filtered expression of miR-202-5p in IM CML cell line was screened by high-throughput sequencing,discussed the role and mechanism of miR-202-5p/USP15 axis in CML resistance,thus providing experimental basis for clinical overcoming CML resistance.Part 1 Detection of differential expression of microRNA in CML-resistant cell lines and the expression of miR-202-5p in different CML cell lines and CML patients by high-throughput sequencingObjective:Filter differential expression of microRNA in CML-resistant cell lines and the expression of miR-202-5p in different CML cell lines and CML patientsMethods:1,CCK-8 method was used to detect the IC50 of K562/K562G cells against IM;RT-qPCR was used to detect the expression of drug resistance genes in K562/K562G cells;Western blot was used to detect the expression of P-glycoprotein(P-gp)in K562/K562G cells.2.High-throughput sequencing was used to detect differences in miRNA expression in K562 cells and K562 cell lines.3.Real-time quantitative PCR(RT-qPCR)and in situ hybridization(FISH)methods were used to detect the expression of miR-202-5p in CML cells.4.RT-qPCR and FISH methods were used to detect the expression of miR-202-5p in CML patients.Result:1.The K562G cell line is an IM resistant cell line.CCK-8 method was used to detect the IC50 of K562/K562G cells against IM.The IC of IM in K562G cells was approximately 30 times that of K562 cells;RT-qPCR method demonstrated that the expression level of ABCB1 gene in K562G cells was higher than that of K562 cells;It was confirmed that the expression level of P-gp protein was significantly up-regulated in K562G cells.These demonstrated K562G cells are IM resistant cell lines.2.High-throughput sequencing adopted in expression difference of miRNA in K562 cells and K562G cells.High-throughput sequencing showed that compared with K562 cells,102 kinds of known miRNAs were up-regulated and 92 kinds miRNAs were down-regulated in K562G cells;differentially expressed miRNA target genes were enriched in multiple signaling pathways.3.The expression of miR-202-5p increased in IM-resistant CML cell lines.RT-qPCR and FISH methods showed that the expression of miR-202-5p was highest in drug-resistant cell line K562G and lowest in normal peripheral blood leukocytes.4.The up-regulation of miR-202-5p in ABCB1(+)CML patients.RT-qPCR and FISH showed that the expression of miR-202-5p in ABCB1(+)CML patients was higher than that in ABCB1(-)CML patients,and the expression of miR-202-5p in CML patients was higher than that in healthy donor miR-202.-5p expression.Summary:miR-202-5p was up-regulated in CML-resistant cell lines and ABCB1(+)CML patients.Part 2 Knocked down miR-202-5p to increase the sensitivity of CML cells to IM.Objective:Verify whether miR-202-5p is directly involved in IM resistance of CML cells.Methods:1,Detect the expression of miRNA-202-5p by RT-qPCR after IM challenge.2.K562 cells over-expressed or knocked down miR-202-5p,detect cells against imatinib against IC50 by CCK-8 method,detect apoptosis after imatinib stimulation with AnnexinV/PI flow cytometry,and detect Cyclin D1 and Caspase-3 expression through Western blot.Result:1.IM treatment down-regulated the expression of miR-202-5p in CML cell line.In K562/K562G cells,IM reduced the expression of miR-202-5p in a dose-dependent manner.2.Over-expression of miR-202-5p decreased sensitivity of K562 cells to IM.Compared with the control group,after overexpression of miR-202-5p in K562 cells,the same concentration of IM significantly increased the IC50 value of K562 cells;Western blot showed that overexpression of miR-202-5p could reverse the decrease of Cyclin D1 and the increase of caspase-3 caused by IM application.3.knockdown of miR-202-5p increased sensitivity of K562G cells to IM.Compared with the control group,after konckdown of miR-202-5p in K562G cells,the same concentration of IM significantly decreased the IC50 value of K562G cells;Western blot showed that knockdown of miR-202-5p could enchance the decrease of Cyclin D1 and the increase of caspase-3 caused by IM application.Summary:miR-202-5p regulates CML resistance to IM.Part 3 miR-202-5p inhibited USP15 regulation of CML resistance to IM.Objective:To confirm the role of miR-202-5p in inhibiting USP15-mediated CML cell line resistance to IM.Methods:1.Bioinformatics prediction of miR-202-5p target gene,RT-qPCR,dual fluorescent reporter gene,Western blot detection of USP15 as miR-202-5p target gene.2.The expression of USP15 in CML patients was detected by RT-qPCR,Western blot and immunofluorescence staining.3.K562 cells overexpress or knock down USP15,or over-express or knock down both of them,then CCK8 detects IC50 on imatinib cells;AnnexinV/PI flow cytometry detects the cell death after imatinib stimulation Western blot was used to detect the expression of Cyclin D1,Caspase-3 and USP15.Result:1.USP15 is the target gene for miR-202-5p in CML cells.Compared with transfected mimic-NEG group,over-expression of miR-202-5p in K562 cells down-regulated the expression of USP15 mRNA.Dual fluorescent reporter gene assays show that miR-202-5p mimic can target the 3' UTR sequence of USP15.Western blot results showed that transfection of miR-202-5p mimics down-regulated the expression of USP15 protein while miR-202-5p inhibitor increased the expression of USP15 protein.These showed USP15 is the target gene of miR-202-5p.2.The decreased expression of USP15 in K562G cells.Compared with K562 cells,the expression level of mRNA and protein of USP15 was significantly decreased in K562G cells,and the same results were obtained by immunofluorescence staining.Moreover,USP15 was mainly located in the cytoplasm.3.USP15 is lowly expressed in CML patients and is less expressed in patients with ABCB1(+)CML.RT-qPCR and Western blot results showed that compared with normal donor PBMCs,the expression of USP15 mRNA and protein in the PBMCs of CML patients was significantly decreased;immunofluorescence staining obtained the same results;and in ABCB1(+)CML patients PBMCs are expressed lower.4.IM treatment up-regulated the expression of USP15 expression in CML cell line.In K562/K562G cells,IM increase the expression of miR-202-5p in a dose-dependent manner.5.Knockdown of USP15 decreased sensitivity of K562 cells to IM.The results of CCK-8 showed that compared with the control group,the IC50 of K562 cells on IM increased after knocking down USP15;After IM treatment of the above cells,Western blot results showed that down-regulation of USP15 up-regulation Caspase-3 protein expression,while down-regulation of Cyclin D1 expression.6.Overexpression of USP15 increased the sensitivity of K562G cells to IM.The results of CCK-8 showed that compared with the control group,the IC50 of K562G cells on IM decreased after overexpression of USP15;Western blot results showed that overexpression of USP15 up-regulate Caspase-3 protein expression,while down-regulation of Cyclin D1 expression.7.The miR-202-5p/USP15 axis regulates IM resistance to CML cells.K562 cells were co-transfected with miR-202-5p mimic and pcDNA3.1-USP15.Western blot results showed that the expression of USP15 protein was increased,overexpression of USP15 abolished miR-202-5p resistance to K562 cells.The miR-202-5p inhibitor and si-USP15 were co-transfected with K562G.Western blot showed that the expression of USP15 protein decreased.The IC50 of IM transfected cells and single transfected miR-202-5p inhibitor cells against IM were detected by CCK8 method.IC50 of co-transfection group was decreased.These results indicate that USP15 mediates the chemotherapy resistance of miR-202-5p to CML cell lines.Summary:miR-202-5 targeting inhibition of USP15 expression regulates CML cell line resistance to IM.Part 4 Morin induces apoptosis of CML cells by regulating miR-188-5p/PTEN axis and increases the sensitivity of CML cells to IMObjective:To determine whether morin induces apoptosis of CML cells through the miR-188-5p/PTEN axis and increases the sensitivity of CML cells to IM.Methods:1.CCK-8 method was used to detect the proliferation of the cells treated with lignin or combined with IM,and AnnexinV/PI flow cytometry was used to detect the apoptosis rate.2.The cells were treated with Morin.The expression of PTEN gene was detected by RT-qPCR.Western blot was used to detect the expression of PTEN protein and downstream pathway proteins.3.Bioinformatics prediction of miRNAs targeted to PTEN gene,RT-qPCR detection of miR-188-5p expression after treatment with mulcinomycin,double fluorescent reporter gene,Western blot detection of PTEN as a target gene of miR-188-5p.Result:1.Morin can inhibit the proliferation of CML cells and induce the apoptosis of CML cells.Morin inhibited the proliferation of CML cell lines and induced the apoptosis of CML cells.With the increase of the concentration of morin,the apoptosis of CML cells increased significantly.2.Morin can increase the sensitivity of CML cells to IM.The results of CCK-8 showed that compared with the control group,the combination of morin and IM can reduce the IC50 of IM,and the combined use of drugs can significantly increase the cell apoptosis rate.3.Morin inhibits PI3K/AKT pathway activation and promotes apoptosis by increasing PTEN protein expression.The results of RT-qPCR showed that the expression of PTEN mRNA was increased in CML cells treated with morin;the expression of PTEN protein was increased,the protein expression of p-AKT was decreased,the protein expression of Caspase-3 was increased,and the protein level and mRNA of BAX increased in Western blot.,BCL-2 protein levels and mRNA levels decreased significantly.4.Morin can reduce the expression of miR-188-5p in CML cells and up-regulate the expression of PTEN.Bioinformatics predicts that miR-188-5p can target the PTEN gene;RT-qPCR results show that the expression of miR-188-5p decreased significantly after treatment with morin;the fluorescent reporter gene showed transfection of miR-188-5p mimic.Afterwards,the fluorescence activity of the PTEN wild-type 3'UTR group was reduced by 33%;Western blot showed that the expression level of PTEN protein was decreased in the transfected miR-188-5p mimic,while the miR-188-5p inhibitor was treated.In the group,the expression of PTEN protein increased.The treatment of K562 cells with morin was transfected with the miR-188-5p mimic.Simultaneously,it was found that transfection with miR-188-5p mimic significantly reversed the increased expression of PTEN.These results suggest that miR-188-5p directly targets PTEN 3'UTR in K562 cells and inhibits its protein expression.5.Morin induces apoptosis of CML cells by inhibiting the expression of miR-188-5p and increases the sensitivity of CML cells to IM.CCK-8 results showed that knockdown of miR-188-5p group,IM of IC50 decreased;transfected miR-188-5p inhibitor group compared with the control group,the rate of apoptosis was significantly higher;CCK-8 method to detect cell proliferation The rate and IC50 of imatinib show that,by transfection of miR-188-5p mimic,the synergistic effect of morin and IM can be reversed.The above results show that the enzymatic enhancement of CML cells by lutein is achieved by inhibiting the expression of miR-188-5p.Summary:Morin induces apoptosis of CML cells through the regulation of miR-188-5p/PTEN axis and increases the sensitivity of CML cells to IM.Conclusion1.miR-202-5p was up-regulated in CML-resistant cell lines and ABCB1(+)CML patients.2.miR-202-5p regulates CML resistance to IM.3.miR-202-5 targeting inhibition of USP15 expression regulates CML cell line resistance to IM.4.Morin induces apoptosis of CML cells by regulating miR-188-5p/PTEN axis and increases the sensitivity of CML cells to IM. |
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