The Roles Of Ars2 In Acute Myeloid Leukemia By Modulating The MiR-6734-3p/p27 Axis

Research backgroundAcute myeloid leukemia(AML)is a highly heterogeneous clonal disease caused by malignant proliferation of acquired myeloid cells,which is the most common acute leukemia in adults,accounting for about 70% of the incidence in adult leukemia.During the recent decades,thanks to the development of chemotherapy and supportive therapy,the rate for complete cure and long-term survival of AML patients have been significantly improved.However,the 5-year overall survival rate is still relatively low,only accounting for 40%-50%,and 20%-30% patients have the risk of recurrence.Improving the complete cure rate for initial treatment and overcoming the recurrence have become one of the obstacles in AML treatment.At present,molecular targeted therapy has been greatly focused and become one of the most potential methods for tumour treatment.With the improvement of the knowledge about molecular mechanisms in AML,more and more abnormal genes or signaling pathway have been discovered.These genes and signaling pathways provide various of therapeutic targets and strategies for clinical trails,which are helpful for the development of new drugs or new therapies.Non-coding RNAs(ncRNAs)have been thought to be junk RNA in cells and do not regulate cell proliferation-related processes,but as research progresses,the function of non-coding RNAs is constantly being exploited.Non-coding RNAs are classified into long-non-coding RNAs(lncRNA),short non-coding RNAs(snoRNAs)and mirco non-coding RNAs(miRNAs).Among them,miRNA has received extensive attention in recent years.MiRNA research has become popular in the field of tumors.Some miRNA expression will cause tumorgenesis(onco miRNA),while some miRNA expression will inhibit Tumor Suppressor miRNA.This positive or negative mechanism of action on tumors is largely accomplished by miRNA regulation of the corresponding protein expression.Simultaneous miRNAs are also regulated by translation and shearing by proteins such as RNase.Arsenic resistance protein 2(Ars2)is a highly conserved protein.Previous studies have shown that Ars2 could cooperate with other enzymes to regulate the synthesis,cleavage or translation of RNAs,including miRNA,through binding to the 5' end cap-binding complex(CBC)of the mRNAs.Present studies have revealed that miRNAs play key roles in tumor formation and proliferation,however more and more new miRNAs are still being discovered and proved to be involved in tumor progression.There are clinical evidence showing that Ars2 is highly expressed in samples from AML patients and correlated with the prognosis of patients.However,the mechanisms of Ars2 in regulating AML progression still remain unclear.Some recent studies about miRNA reveal that the dysregulation of some miRNAs,caused by the abnormal expression of Ars2,might lead to the occurrence of AML.Based on these evidence,this study aims to explore the miRNAs regulated by Ars2 during the formation and proliferation of AML and elucidate the mechanism involved,hoping to provide specific targets for clinical therapy.Research purpose:1.Explore the mechanisms involved in Ars2 regulating the proliferation of AML and screen out the relevant target proteins.2.Screen out the miRNA clusters regulated by Ars2 in AML and identify the cell cycle related miRNAs.3.Verify the regulatory sites of miR-6734-3p on P27 and the effects of miR-6734-3p on proliferation of AML cells.Research methodsWe reviewed multiple cancer gene databases to explore the relationship between the expression of Ars2 in the bone marrow of AML patients and the prognosis of patients with AML we reviewed multiple cancer gene databases.Construct the Ars2 overexpression(Ars2 OE)-U937 cell lines and the shArs2-U937 cell lines through plKO.1 system and compare the proliferative differences between control-U937,Ars2 OE-U937 and shArs2-U937 cell lines by cell counting and cell clonal forming experiments.Detect the effects of knocking down Ars2 on the G1 stage in cell cycle process of AML cell lines through flow cytometry.Detect the cell cycle related proteins and screen out the differentially expressed proteins between Ars2 OE-U937 and shArs2-U937 cell lines.Construct the shArs2-U937 cell lines with lentiviral infection system and perform the Immunoprecipitation of CBP80 to compare the expression level of CBP80 binding Ars2 when Ars2 being knocked down.Then carry out immunoprecipitation for Ars2 on shArs2-U937 cell lines to detect the changes of Ars2 binding CBP80.Detect the levels of RNase Drosha/Pasha and Dicer1 binding to Ars2 by immunoprecipitation to confirm the regulation of Ars2 on the process of pri-miR-6734-3p being cleaved to become miR-6734-3p.Sent the control-U937 and shArs2-U937 cell lines out to the Ribo Bio.(Guangzhou,China)for high-throughout miRNA microarray analysis and identify the miRNA clusters positively regulated by Ars2.Identify the miRNA clusters that could bind to the 3' end of p27/Kip1 through exploring the public miRNA databases.Overexpress the specific miRNA in U937 cell lines by miRNA mimic and determine whether the miRNA could inhibit p27/Kip1 on mRNA and protein levels by western-Blot and qRT-PCR.Calculate the specific binding site of the miRNA to the 3' end of p27/Kip1 based on the RNA22 database(cm.jefferson.edu/rna22/Interactive/).Then construct the dual luciferase reporter system of the sequence and confirm the efficiency.Transfect miR-6734-3p mimic and miR-6734-3p inhibitor into U937 cell lines and confirm the effects of miR-6734-3p on G1 stage of AML cell cycle by flow cytometry.Compare the expression level of miR-6734-3p in healthy human samples and AML patients by qRT-PCR.Construct the AML mouse model by injecting saline,U937 or shArs2-U937 into mice tail veins and record the changes in body-weight and the survival time of the mice.At 40 days after the injection,take the liver and kidney from the mice models and examine them by immunochemistry.Meanwhile,detect the infiltration of AML into bone marrows of the mice through flow cytometry.Result1.The public database showed that the expression level of Ars2 was related to the prognosis of patients.The higher the expression of Ars2,the worse the prognosis.Public database statistical results and clinical sample PCR results showed that Ars2 was highly expressed in AML patient samples.2.Ars2 regulates the proliferation and colony formation ability of AML cells.The higher the expression of Ars2,the faster the proliferation rate and colony formation ability of AML.Silenced Ars2 expression causes AML cell proliferation was slower,and the ability of colony formation became weaker.Ars2 affects the proliferation rate of AML cells by regulating cell cycle progression.3.In AML,Ars2 binds to CBC,and the miRNA cleavage enzyme Drosha is recruited to perform cleavage function on pri-miRNA,thereby maintaining the normal synthesis of miR-6734-3p.MiR-6734-3p inhibits the expression of p27.Ars2 knockout causes the expression of miR-6734-3p decreased,and the expression of p27 increased,which eventually leads to the G1 phase arrest of AML cell cycle.MiR-6734-3p was highly expressed in AML patient samples.4.The expression of Ars2 in mouse leukemia model was positively correlated with the proliferation rate of AML.Ars2 knockout could slow the infiltration rate of AML,delay the timing of mouse lesions,and prolong the survival time of AML mice.Conclusions1.High expression of Ars2 may cause poor prognosis in patients with AML.Compared with normal samples,Ars2 has a high expression trend in AML.2.Ars2 can regulate the AML cell cycle and thus positively regulate the reproductive rate of AML cell lines.3.In AML,Ars2 binds to CBC,and recruits RNase DROSHA to maintain miRNA cleavage.When Ars2 expression is deleted,the expression of miR-6734-3p is decreased,and its inhibition of p27 is weakened,which causes cell cycle G1 arrest in AML cells.MiR-6734-3p is highly expressed in AML patient samples.4.In the AML NOD/SCID model,Ars2 regulates AML proliferation and other processes,and presents a dose-effect trend.5.Ars2 and miR-6734-3p have potential and value in the treatment of cancer in clinical treatment.