Study On The Roles Of LncRNA CRNDE And Its Molecular Mechanisms In The Progression Of Drug Resistance In Acute Myeloid Leukemia

Acute myeloid leukemia(AML)is an aggressive clonal disorder of hematopoietic stem cells(HSCs)and primitive progenitors.AML is the most common acute leukemia in adults and the second most common acute leukemia in children.Acute myeloid leukemia is a disease characterized by heterogeneous biology,leading to different clinical outcomes,including recurrence.AML still remains challenging to treat owing to patient factors(age and coexisting diseases)and intrinsic biologic factors.Although AML patient survival rates have improved with the use of targeted drugs such as midotolin and enacinib,relapse rates remain high and a large number of patients continue to die from AML.Currently,drug resistance is a major barrier to AML chemotherapy.Most patients with AML who respond to initial chemotherapy still develop multidrug resistance(MDR)to chemotherapy agents and recurrence of the disease has an adverse effect on patient survival.Therefore,there is a great medical need to study the molecular mechanisms involved in MDR to improve the prognosis of AML patients.In recent years,the mechanism of non-coding RNA involvement in multidrug resistance in acute myeloid leukemia has been confirmed.Long noncoding RNAs(LncRNAs)have been shown to play an important role in the development of physiology and pathology.Dregulation of LncRNAs has been reported to be involved in a variety of functional cellular processes in many cancers,including cell proliferation,apoptosis,invasion,migration,and metastasis.More and more evidence shows that long non-coding RNAs are related to drug resistance or drug sensitivity,and a large number of studies also focus on revealing the exact molecular mechanism of LncRNA regulated drug resistance.LncRNA CRNDE(CRNDE)is a gene locus hCG 1815491 on chromosome 16 located on the strand opposite to the adjacent IRX5 gene and first confirmed to be elevated in colorectal adenomas.The up-regulated CRNDE has been confirmed as a biomarker for AML and contributes to the malignant progression in AML cells.However,the specific roles and mechanism of CRNDE in MDR in AML is still unclear.In this study,the expression of CRNDE in peripheral blood mononuclear cells(PBMCs)is investigated.The relationship between CRNDE expression in patients with acute myeloid leukemia and patients after adriamycin(ADR)-based chemotherapy was analyzed.The expression of multidrug resistance related protein in cells was detected.Furthermore,drug-resistant cell lines(KASUMI-1/ADR,HL60/ADR)were constructed to study the cell functions and molecular mechanism in vitro.The effects of CRNDE knockdown on cell proliferation,apoptosis,cell cycle and function-related proteins were studied.CRNDE knockdown on drug-resistant proteins in cells and its effect on drug sensitivity of ADR.And the signal pathway and mechanism involved are analyzed and verified.Finally,animal experiments were conducted to verify the effects of CRNDE knockdown on the growth and drug sensitivity of AML cells in vivo and the possible mechanisms.This study aims to explore the roles of CRNDE in AML MDR and its potential molecular mechanisms.The results of this study may help provide a new therapeutic strategy for reversing multidrug resistance in AML patients.This topic includes the following three parts:1.Expression of LncRNA CRNDE in AML patients before and after chemotherapy and correlation study of chemotherapy resistance;2.Study on the effect of knockdown of LncRNA CRNDE expression on biological function and drug resistance of AML cells and its mechanism;3.Study on the effect of knockdown of LncRNA CRNDE expression on the growth and drug sensitivity of AML cells in animals and its possible mechanism.The first chapter:Expression of LncRNA CRNDE in AML patients before and after chemotherapy and correlation study of chemotherapy resistanceMethods1.qRT-PCR was used to detect CRNDE and MDR1 expression in PBMC cells of AML patients(19 before and 19 after chemotherapy)and healthy volunteers.2.Western blot was used to detect P-gp expression levels.3.Gel electrophoresis was used to semi-quantify the expression of CRNDE in PBMC cells from 5 AML patients and healthy volunteers.4.The expression of CRNDE in PBMC cells of AML patients before and after chemotherapy was statistically analyzed by SPSS software.5.The expression of MDR1 and CRNDE after chemotherapy was statistically analyzed by SPSS software.Results1.qRT-PCR results showed that compared with healthy controls,in patients with AML PBMC cells,CRNDE levels were significantly increased.2.Nucleic acid gel electrophoresis and half quantitative analysis showed that compared with healthy controls,in the random selection of 5 cases of AML patients,CRNDE expression was also significantly increased.And p-gp expression was also significantly increased.3.In addition,qRT-PCR analysis showed that CRNDE levels were significantly increased in PBMCs AML patients after chemotherapy.4.Statistical analysis showed that MDR1 expression level and CRNDE level was significantly positively related.The second chapter:Study on the effect of knockdown of LncRNA CRNDE expression on biological function and drug resistance of AML cells and its mechanismMethods1.KASUMI-1/ADR cell lines were obtained by induction with varing ADR doses in this laboratory.2.MTT assay was used to detect the growth curve of cell activity in each group,and the IC50 value of cells in each group was calculated..3.CRNDE expression in HL60,KASUMI-1,KASUMI-1/ADR,HL60/ADR and human bone marrow stromal cells HS-5 was detected by qRT-PCR.4.The expression of ABC membrane transporter MDR1/P-gp in HL60,KASUMI-1,KASUMI-1/ADR and HL60/ADR cells was detected by qRT-PCR and Western blot assays.5.Lentivirus-mediated gene expression knocked down of CRNDE in HL60,KASUMI-1,KASUMI-1/ADR and HL60/ADR cells was performed,and qRT-PCR was used to detect the knockdown efficiency.6.The proliferation of in each group was detected by MTT and EDU assays,and the expression of Ki67 in each group was detected by Western blot assay.7.Apoptosis and cell cycle distribution were detected by flow cytometry assay,and the levels of apoptosis related proteins and cyclin-related proteins were detected by Western blot assay.8.The levels of MDRl/P-gp in resistant cell lines HL60/ADR and KASUMI-1/ADR with CRNDE knockdown were detected by qRT-PCR and Western blot assays.9.MTT assay was used to detect the effect of CRNDE knockdown on ADR drug sensitivity of HL60/ADR and KASUMI-1/ADR cells,and IC50 was analyzed.10.Western blot assay was used to detect the protein levels in the Wnt signaling pathway in HL60/ADR and KASUMI-1/ADR cells.11.Reversal assays by overexpressing Wnt5a in cells was performed.MTT and western blot assays were used to detect the effects of CRNDE knockdown on ADR drug sensitivity in HL60/ADR and KASUMI-1/ADR cells.Results1.qRT-PCR analysis showed that compared with HS-5 cells,CRNDE expression was significantly increased in HL60,Kasumi-1,Kasumi-1/ADR,and HL60/ADR cells,and the level of MDR1 in KASUMI-1/ADR and HL60/ADR cells was increased.2.Lentivirus-mediated gene interference significantly knocked down the expression of CRNDE in HL60,Kasumi-1,Kasumi-1/ADR,and HL60/ADR cells.3.Both MTT and EDU assay results showed that CRNDE knockdown inhibited the proliferation of HL60,Kasumi-1,Kasumi-1/ADR,and HL60/ADR cells,and western blot results showed that the expression level of proliferation marker protein Ki67 was consistent with the results of cell proliferation.4.Flow cytometry results showed that CRNDE knockdown increased the apoptosis rates of HL60,Kasumi-1,Kasumi-1/ADR,and HL60/ADR,and inhibited the normal process of cell cycle.The results of apoptosis and cell cycle-associated protein levels further showed that CRNDE knockdown increased the apoptosis of HL60,Kasumi-1,Kasumi-1/ADR,and HL60/ADR,and inhibited the normal process of cell cycle.5.qRT-PCR and Western blot analyses showed that CRNDE knockdown reduced the levels of MDRl/P-gp in drug-resistant HL60/ADR and KASUMI-1/ADR cells.6.MTT results showed that CRNDE knockdown promoted the drug sensitivity of HL60/ADR and KASUMI-1/ADR cells and significantly reduced the IC50 value of ADR in cells.7.At the molecular levels of Wnt signaling pathway proteins,the expression ofβ-catenin and its downstream target proteins(c-myc and cyclinD1)in HL60/ADR and KASUMI-1/ADR cells were significantly inhibited by CRNDE knockdown.8.Overexpression of Wnt5a reversed the inhibitory effect of CRNDE knockdown on P-gp level in HL60/ADR and KASUMI-1/ADR cells and then promoted the activation of the Wnt signaling pathway.9.Wnt5a overexpression reversed the inhibition of CRNDE knockdown on drug sensitivity of HL60/ADR and KASUMI-1/ADR cells.The third chapter:Study on the effect of knockdown of LncRNA CRNDE expression on the growth and drug sensitivity of AML cells in animals and its possible mechanism.Methods1.AML tumor model was obtained by subcutaneous injection of cells in mice.The mice were treated with ADR(experimental group)or normal saline(control group)periodically.2.The growth of transplanted tumor and survival rate of mice were observed and recorded.3.The Wnt signaling pathway and drug-resistance related proteins were detected by Western blot assay.Results1.Combination of ADR and CRNDE knockdown treatment significantly inhibited tumor growth and increased the survival rate of mice,compared with normal saline group and ADR+sh-Ctrl-HL60/ADR group.2.The Wnt signaling pathway was inhibited in the transplanted tumor tissues with combination of ADR and CRNDE knockdown treatment,compared with normal saline group and ADR+sh-Ctrl-HL60/ADR group,and the level of drug-resistant related protein P-gp was significantly inhibited.Conclusion1.CRNDE level in PBMC cells of AML patients was significantly increased compared with healthy control group.In addition,CRNDE level in PBMC cells of AML patients after chemotherapy was significantly increased,and the expression level of drug-resistant related gene MDR1 was positively correlated with CRNDE level.These results suggested that CRNDE might be related to the ocurrence and development of AML multidrug resistance.2.CRNDE knockdown inhibited the proliferation and cell cycle progression of AML cells and promoted apoptosis.CRNDE knockdown inhibited the expression level of MDRl/P-gp in drug-resistant AML cells and increased the drug sensitivity of drug-resistant AML cells by inhibiting the expression of P-gp by inhibiting the Wnt signaling pathway.3.In vivo tumor transplantation experiments showed that ADR and CRNDE knockdown inhibited the growth of AML xenograft and improved the survival rate of mice.Combination of ADR and CRNDE knockdown inhibited the Wnt signaling pathway and P-gp levels in the xenograft tissues.It was speculated that CRNDE knockdown could reverse the drug resistance of AML cells to some extent.