The Role Of HOTAIR In The Imatinib Resistance In Chronic Myeloid Leukemia Cells

Chapter I The relationship between HOTAIR expression and multidrug resistance in imatinib in CML patientsBackground:Chronic myeloid leukemia(CML)is a malignant clonal disease of hematopoietic stem cells.Malignant hematopoietic stem cells exhibit uncontrolled proliferation,apoptosis,and differentiation disorders,leading to massive infiltration of leukemia cells in the bone marrow and peripheral blood.CML has been rising year by year in China.In addition,compared with Western countries,the incidence of CML in China is showing a trend of younger.Ph chromosome and BCR-ABL fusion gene appear in blood cells of 90%of CML patients.ABL gene can inhibit cell proliferation and cell cycle transformation.There are three breakpoints of ABL.The BCR-ABL fusion gene encodes different proteins due to different breakpints,including:P210,P190,P230.in CML,P210 is mainly expressed,and P230 is in a small part.The encoded protein has strong tyrosine kinase activity and changes the tyrosine phosphorylation of multiple proteins in cells.The level and function of cellular microfilament mobility proteins disrupt the normal signal transduction pathways in cells,causing cells to lose their reactivity to the surrounding environment and inhibit the occurrence of apoptosis.Allo-HSCT(allo-HSCT)is the only method that is expected to cure CML,but it is often limited by the presence or absence of donors,the patient's age,and other factors that cannot be widely used.Imatinib is the first generation of tyrosine kinase inhibitors(TKIs)used clinically.Its clinical application makes CML patients with a 10-year survival rate of 85%to 90%,making CML a A true chronic disease.Therefore,imatinib is currently gradually replacing allo-HSCT as the first-line option.Imatinib is a targeted therapeutic drug for chronic myelogenous leukemia,and it can inhibit Bcr-Abl tyrosine kinase at the cellular level,and it can selectively inhibit the proliferation of Bcr-Abl positive cells and induce its apoptosis.However,there are still some patients who are not sensitive to imatinib and have primary or secondary resistance to imatinib.There are many mechanisms of imatinib resistance.According to the relationship between drug resistance and BCR-ABL,imatinib resistance can be divided into BCR-ABL-dependent resistance and BCR-ABL-independent resistance.BCR-ABL non-independent drug resistance includes multidrug resistance(MDR)expression and overexpression of Src kinase.Multidrug resistance is the main form of drug resistance in tumor cells and refers to a drug acting on tumors.After the cells make them resistant,the tumor cells gradually have cross-resistance to a variety of antitumor drugs that have not been contacted,have no structure,and have different target and mechanism.Acquired multidrug resistance and relapse of disease are often considered to be the cause of chemotherapy failure in patients with malignant tumors,including leukemia.Overexpression of drug membrane transporters,changes in targets of anticancer drugs,reduction in drug activity,and alterations in apoptotic regulatory pathways all contribute to MDR.The single nucleotide polymorphism of MDR1/ABCB1 gene can cause drug resistance in many diseases and is closely related to the molecular genetic mechanism of imatinib resistance in CML patients.Imatinib can be transported out of cells by multidrug resistance-associated proteins.Overexpression of MDR 1 and its protein P-pg can cause a decrease in the intracellular concentration of imatinib,resulting in resistance.The multidrug resistance-associated protein(MRP)family consists of 9 members(MRP1-9).MRP1 belongs to the ABC superfamily transporter and is also a common efflux transporter.Under physiological conditions,it can protect the cells from foreign active substances.Under pathological conditions,its overexpression can increase the intracellular drug outflow,resulting in a decrease in intracellular drug concentration,a reduction in cell damage,and induction of drug resistance.Recent studies have found that MRP1 is overexpressed in tumor cells of various solid tumors and leukemias.HOTAIR belongs to the family of long-chain non-coding RNA(LncRNA)and is the first lncRNA to be discovered.More and more studies have revealed that HOTAIR is highly expressed in various cancers and is related to tumor metastasis.Reducing HOTAIR can reduce cancer invasion.Although lncRNAs play an important role in the development of myeloid leukemia,little is known about the role of CML in acquiring acquired resistance to imatinib.The role of HOTAIR in chronic myelogenous leukemia has not been studied.Objective:Imatinib resistance is a major cause of clinical failure in CML patients.This topic will analyze the relationship between HOTAIR expression and multidrug resistance in CML patients through analysis of clinical data to further explore the mechanism of drug resistance in CML patients and provide theoretical guidance for clinical treatment of imatinib-resistant patients.Methods:Thirty-four CML patients(including 16 males and 18 females,ranging in age from 22 to 75.years with a median age of 41.7 years)and four volunteers from October 2011 to December 2013 were randomly selected clinically.Age from 18 to 45 years,median age 35.8 years).The bone marrow fluid of the subject was collected by posterior superior iliac spine bone marrow puncture.According to real-time PCR detection of MRP 1 expression levels,according to the median expression of MRP 1,34 cases of CML patients were divided into low expression group and high expression group of multidrug resistance associated protein 1(MRP1),and then by real-time quantitative PCR(qPCR)The expression of IncRNA HOTAIR in different expression groups of MRP 1 was detected.Result:1.The expression of IncRNA HOTAIR was significantly.up-regulated in all CML patients compared with healthy controls(p=0.0095),with HOTAIR expression higher in the MRP1 high expression group than in the low MRP1 expression group(p=0.0185).Tip:HOTAIR may play an important role in the prognostic stratification of CML.2.The expression of IncRNA HOTAIR was positively correlated with MRP1 level,y=0.5382x+0.0003 R2=0.7495.Conclusion:High expression of HOTAIR may be one of the causes of imatinib resistance in CML patients.Chapter ? The Mechanism of HOTAIR in Inducing Imatinib Resistance in CML CellsBackground:Imatinib is the earliest first-generation TKI in clinical application,it can inhibit Bcr-Abl tyrosine kinase,inhibit the proliferation of Bcr-Abl positive CML cells and induce its apoptosis.It has replaced allogeneic stem cell transplantation and has become the first-line treatment option for patients with CML.Although imatinib has achieved milestone achievements in the treatment of CML,imatinib resistance is still a problem that plagues clinical CML therapy.There are many reasons for the resistance to imatinib.BCR-ABL-independent resistance means that the resistance is not related to the BCR-ABL fusion protein.The study found that this may be related to the metabolism and transport process of imatinib in vivo and BCR-ABL.It is related to the abnormal activation of kinase downstream signaling pathways.At present,more and more attention has been paid to the study of non-coding RNA and drug resistance in tumor cells.Non-coding RNA(ncRNA)refers to an RNA that does not encode a protein and is not translated into a protein after being transcribed from the genome and directly performs biological functions at the RNA level.Long-chain non-coding RNA(LncRNA)is a recently discovered class of ncRNAs with regulatory activity.Abnormal expression of LncRNA in tumors can inhibit the apoptosis of tumor cells and promote the infiltration,metastasis and drug resistance of tumor cells.HOTAIR belongs to the lncRNA family and is highly expressed in a variety of tumors and promotes the development of tumors.Some scholars have found that HOTAIR is a marker of poor prognosis in acute myeloid leukemia.Our previous study also revealed that HOTAIR mRNA expression is elevated in patients with CML and is positively correlated with the expression of multidrug resistance-associated protein MRP1.It showed that HOTAIR high expression may be one of the reasons for drug resistance in CML patients.But what is the specific mechanism of HOTAIR-induced imatinib resistance?Previous research reported:The autophosphorylation of tyrosine at position 177 of the BCR-ABL 1 gene resulted in the formation of a complex between GRB2,GAB2 and SOS,activating the PI3K/Akt signaling pathway.Therefore,activation of a series of downstream signal molecules,leading to increased protein translation,increased cell proliferation and other biological behavior,leading to the occurrence of CML,then we speculated that HOTAIR high expression has effect on PI3K/Akt signaling pathway.The phosphatidylinositol 3-kinase(PI3K)family is a proto-oncogene that is an important kinase for phosphatidylinositol and inositol and plays a regulatory role in cell proliferation,differentiation,apoptosis,and glucose transport.According to its structure and different substrates,PI3K includes three types.Currently the most studied type is PI3K,which includes a regulatory subunit and.a catalytic subunit.The regulatory subunit,also known as p85,can interact with the target protein at the corresponding binding site;the catalytic subunits include the p110?,?,?,and ?classes;and Akt,also known as protein kinase B(PKB),is A serine/threonine kinase is one of the major downstream effector molecules of PI3K.After activated,AKT can act on its corresponding substrates such as Bad factor,Forkhead(Foxo or FH)gene,NF-?B and GSK-3 glycogen synthase kinase-3(GSK-3).Causes phosphorylation of target genes and inhibits apoptosis.The PI3K/Akt signaling network plays a crucial role in many physiological processes.The role of drug resistance in tumor cells is increasingly receiving attention.Once this signaling pathway is activated,it will make tumor cells Accelerated growth,faster metastasis,and the ability to induce blood vessel growth,making tumor cells resistant to chemotherapy-resistant drugs.Further studies have confirmed that this pathway may induce tumor cell resistance to chemotherapeutic drugs by inducing increased expression of multidrug resistance proteins,anti-apoptosis and other pathways,and blocking the signal pathway reversible transformation therapy drug resistance,restoring tumor cells to chemotherapy Drug sensitivity.Recent studies have shown that abnormal activation of the PI3K/Akt pathway can lead to the development of resistance in a variety of leukemias.However,the role of this pathway in imatinib resistance in CML patients,especially its role in the involvement of HOTAIR in imatinib resistance,has not been studied.Objectives:To investigate the specific mechanisms of HOTAIR-induced imatinib resistance in CML cells and to provide theoretical guidance for the treatment of imatinib-resistant CML patients.Methods:The imatinib-resistant K562 cell line(K562-R)was established by increasing concentration gradients.siRNA was used to interfere with the expression of HOTAIR in the transfected cells.MTT assay and flow cytometry were used to detect cell survival before and after inhibition of HOTAIR expression.The rate and apoptotic rate were detected by western blot for the expression of related proteins and ABL1 protein in the PI3K/Akt signaling pathway.Results:1.Expression of MRP 1 and HOTAIR in K562 and K562-R Cell Lines1.1 Compared with K562 cells,the expression of MRP1 in K562-R cells was significantly up-regulated,and the resistance of cells was increased(the IC50 in K562 cells was 250nM,but it was increased to 10p.M in K562-R cells);1.2 The expression level of IncRNA HOTAIR in K562-R cells was significantly higher than that in K562 group.Proved;High expression of HOTAIR is associated with imatinib resistance in K562 cells.2.The effect of HOTAIR knockdown on CML cells2.1 with IncRNA HOTAIR knockdown,K562-R cells showed increased drug sensitivity to imatinib(IC50 reversed to 500 nM,while in control cells,10 ?M),and cell apoptosis increased.2.2 BCR-ABL1 mutations are often associated with CML resistance,and we also examined whether inhibition of HOTAIR expression is achieved by altering the expression of ABL1.The results show that K562-R cells transfected with siRNA-1 did not significantly alter ABL1 expression. Showed:1)with IncRNAHOTAIR knockdown,apoptosis of K562-R cells increased,drug resistance to imatinib was reversed;2)The reversal of this resistance is not achieved by changing BCR-ABL1.3.Effect of HOTAIR on PI3K/Akt signaling pathway3.1 The level of PI3 kinase(PI3K)p110? catalytic subunit and phosphorylated Akt is significantly up-regulated in imatinib-resistant cells(K562-R).3.2 With IncRNAHOTAIR knockdown,the protein expression of PI3 kinase p110?and phosphorylated Akt was significantly decreased.Showed:lncRNA HOTAIR mediates imatinib resistance through the PI3K/Akt dependent pathway.Conclusions1.High expression of HOTAIR is one of the causes of imatinib resistance in CML patients2.HOTAIR may be an upstream regulator of the PI3K/Akt signaling pathway.lncRNA HOTAIR mediates imatinib resistance through a PI3K/Akt dependent pathway.3.HOTAIR-induced imatinib resistance does not change BCR/ABL occurrence and is a BCR/ABL-independent drug resistance.