Study Of The Function And Mechanism Of A Novel Long Non-coding RNA CUST37778 In The Progression Of Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia(Acute lymphoblastic,leukemia,ALL)is a clonal malignant disorder characterized by an uncontrolled proliferation of immature B or T lymphocytes,accounting for 20-25%of acute leukemia,the incidence of ALL mainly occoured around 5 and 50 years old;in the current treatment conditions,children with ALL have good curative effect,but for adult ALL,the curative effect and prognosis is poor,the complete remission rate is less than 80%and the survival rate of 3 year is only 30-40%.Although the treatment effect of adult ALL has been continuously improved,10%of adult ALL has turned to refractory and relapse ALL after induction therapy,and 50%of the patients who achieved complete remission will eventrually turned to relapse.There are many genetic and molecular biological abnormalities in the development of ALL.Long non-coding RNAs(lncRNAs)are a heterogeneous class of RNAs that are generally defined as non-protein-coding transcripts longer than 200 nucleotides.lncRNAs regulate the expression of genes in the form of RNA on epigenetics,transcriptional and post transcriptional levels.Some studies have shown that lncRNAs has a very important biological function in hematological malignancies,and it can be used as an indicator of stratified therapy and prognosis in leukemia in the future.However,up to now,there is only few comprehensive and systematic reports on the correlation between the unknown LncRNAs and the development and prognosis of acute lymphoblastic leukemia.ObjectiveIdentify the lncRNA that is differentially expressed in primary and relapse patients with acute B lymphoblastic leukemiaMethodsA new gene expression profiling chip was used to identify lncRNAs that are differentially expressed in primary and relapse patients with acute B lymphoblastic leukemia compared with those in acute B lymphocytic leukemia patients who has achived complete remission and healthy controls.Identify the selected differentially expressed lncRNA with the bone marrow samples of acute B lymphocytic leukemia patients from XX hospital.We classify primary and relapse ALL patients to two groups according to ROC analyse.And collect the clinical data including age,gender,initial peripheral blood WBC counts,platelets,hemoglobin,BM blasts,cytogenetic and molecular abnormalities.Chi square test or a two-sided Fisher exact test were used to compare clinical variables across groups.Survival data were analyzed by Kaplan-Meier method,and the differences between survival curves was compared by log-rank test.Univariate and multivariate analysis of OS and DFS by COX proportional hazards model.Interference siRNA and overexpression plasmid are used in achiving knockdown and overexpression lncRNA357in B-ALL cell line,the effect on cell proliferation,cell apoptosis and cell cycle were tested by flowcytometry and CCK-8.Q-PCR and Western blot were used to analyse the signal pathway which is important to cell biological activity.Results1.We found that lncRNA CUST37778 are differentially expressed in primary and relapse patients with acute B lymphoblastic leukemia compared with those acute B lymphocytic leukemia patients who has already achived complete remission,and healthy controls.There is significant correlation between primary and relapse patients vs.patients who has achived complete remission(P=0.0006),and significant correlation between primary and relapse patients vs.healthy controls(P=0.0001),while there is no significant difference between patients who has achived complete remission vs.healthy controls(P=0.9348).2.Through analysis of ROC curve,the cut off value of the expression level of lncRNA CUST37778 in 27 primary and relapse patients is 0.2135,and divide the 27patients into 2 groups:high expression group and low expression group.Peripheral blood platelets<100×10~9(p=0.0370*)、BM blasts≥42%(p=0.0130*)is correlated with high expression of lncRNA CUST37778;while there is no significant difference between the gourps when it comes to gender(p=0.125),age(p=0.8947),initial peripheral blood WBC counts(p=0.2059),HB(p=0.8857),Ph chromosome is positive or not(p=0.3261).The CR rate of the 27 patients is 51.85%,whereas there is no significant correlation between the two groups(P=0.7448).Survival analysis reveals that high expression of lncRNA CUST37778 in ALL patients trun to have significantly shorter OS(P=0.0436)and also shorter DFS(P=0.0471)between the two groups.3.LncRNA CUST37778 is highly expressed in BALL-1 cell line and Nalm-6 cell line.Downregulate the expression of lncRNA CUST37778 in the two cell lines can promote cell apoptosis,downregulate cell proliferation,prelong G1 phrase and shorten G2phrase.Downregulate the expression of lncRNA CUST37778 can also promote the expression of Bax,P21and P27,downregulate Bcl-2 and PCNA.P-ERK1/2,p-AKT and p-NF-kB can also be downregualted and the inhibitor of them be used to prove that the influence can be reversed.The apply of interference siRNA and overexpression plasmid transfection achieved satisfied lncRNA CUST37778expression knockdown and overexpression effects in PCa cell lines in vitro,finding out that lncRNA357 could enhance proliferation.ConlusionLncRNA CUST37778 are differentially expressed in primary and relapse patients with acute B lymphoblastic leukemia compared with those acute B lymphocytic leukemia patients who has achived complete remission and healthy controls.Peripheral blood platelets<100×10~9 and BM blasts≥42%(p=0.0130*)is correlated with high expression of lncRNA CUST37778 while there is no significant correlation between other clinical parameters and high expression level of lncRNA CUST37778.High expression level of lncRNA CUST37778 is a poor prognostic factor for DFS and OS but is not enough for being an independent prognostic factor.Downregulate the expression of lncRNA CUST37778 in the B-ALL cell lines can promote cell apoptosis,deregulate cell proliferation,prelong G1 phrase and shorten G2phrase.Downregulate the expression of lncRNA CUST37778 can also promote the expression of Bax,P21and P27,deregulate Bcl-2 and PCNA.P-ERK1/2,p-AKT and p-NF-kB can also be deregualted and the inhibitor of them be used to prove that the influence can be reversed.The apply of interference siRNA and overexpression plasmid transfection achieved satisfied lncRNA CUST37778expression knockdown and overexpression effects in PCa cell lines in vitro,finding out that lncRNA357 could enhance proliferation.