Research On The Mechanism Of Long Non-coding RNA MALAT-1 Mediated Epigenetic Modification In Acute Myeloid Leukemia

Objective:(1)To explore whether MALAT-1signaling pathway is involved in occurrence,progression and prognosis,thus reveal the impact of MALAT-1 mediated epigenetic modification on acute myeloid leukemia,which may provide a new idea for further research on the diagnosis and treatment of acute myeloid leukemia.(2)To further study the impact of MALAT-1 signaling pathway on the function of leukemia cell lines,and also to provide a basis for the future study of targeted therapy of acute leukemia.(3)To further discuss mechanism of MALAT-1 inducing methylation in vitro,which through interacting with DNA and / or histone modification complexes,so as to induce targeted gene promoter CpG islands and / or histone methylation,and finally mediates the occurrence and development of acute myeloid leukemia.And the possible mechanisms of epigenetic regulation mediated by MALAT-1 and its related pathways were elucidated.Methods:(1)Newly diagnosed acute myeloid leukemia(AML)patients and normal bone marrow mononuclear cells were chosen as the cell models,then RT-qPCR(Real-time Quantitative PCR)method was used to detect MALAT-1 and PRC2 complex(EZH2,SUZ12,EED)related protein-coding genes expression,and the acute leukemia patients were included for follow-up,so as to analyze whether MALAT-1 mediated in the occurrence,development and prognosis of acute leukemia.(2)The expression of MALAT-1 was identified in a variety of acute myeloid leukemia cell lines,and acute leukemia cells lines were screened as the research models according to the RT-qPCR results of clinical specimens.Then we constructed the MALAT-1 interference lentiviral vectors,and cells were infected with vetors.CCK-8 Cell growth curve,colony formation assay and flow cytometry were used to evaluate the proliferation of leukemia cell lines.The morphological changes of apoptotic cells were observed by using Hoechst 33258 staining(bisbenzimide,Hoechst 33258).Apoptosis of cells was evaluated using flow cytometry after Annexin V and PI staining.The expression of Caspase-3,Caspase-8 and-9 was assessed by western blot analysis,which helped reveal the changes of cell apoptosis.At the same time western blotting was applied to detect the effect of MALAT-1 on the classic Wnt/ beta-catenin cell signaling pathway.Thus the possible impact of MALAT-1 on the function of leukemia cells was studied.(3)The target genes of MALAT-1 were predicted by literature search,and the expression of target gene was detected by RT-qPCR method after MALAT-1 silencing.Pyrosequencing was used to detect the methylation status of CpG promoter of target genes,and also the methylation rate.At the same time,histone H3K27(histone H3 lysine 27)methylation modification in the promoter region of target genes was detected by chromatin immunoprecipitation(Chromatin Immunoprecitation,ChIP)combined with qPCR method(ChIP-qPCR)detection in leukemia cell lines.RNA immunoprecipitation(RNA Immunoprecipitation,RIP)combined with qPCR(RIP-qPCR)was used to determine whether the chromatin modification complex was formed.And we aim to explore the possible mechanism of epigenetic regulation of target gene mediated by MALAT-1 pathway.(4)Differential expression of target gene was detected by RT-qPCR in acute myeloid leukemia patients and normal people,and pyrosequencing was used to detect the methylation of promoter CpG islands in target gene at the same time,so as to further verify the clinical significance of MALAT-1 and its target gene in patients with acute leukemia.(5)Targeting MALAT-1 microRNAs were screened through bioinformatics methods.Then we constructed the microRNA over-expressed lentiviral vectors and transfected the leukemia cell lines,and MALAT-1 expression was detected by RT-qPCR after transfection.And also the expression of target microRNA was detected in MALAT-1 silenced cells.At the same time,the interaction between MALAT-1 and target microRNA was verified by double luciferase reporter gene assay.Results:(1)Expression of MALAT-1 in patients with AML increased markedly when compared with that of healthy controls,and subgroup analysis showed increased MALAT-1 expression in patients with M5.The results revealed that,compared with patients with low MALAT-1 expression,OS of high expression group significantly decreased in patients with M5.Univariate analysis of OS in M5 patients showed that white blood cell count and MALAT-1 expression level were prognostic indicators while the remaining clinical parameters were not.In multivariate analysis,the Cox proportional hazards model revealed that MALAT-1 overexpression was an independent prognostic factor for OS.(2)The expression of coding genes of EZH2 and EED in PRC2 complex in AML patients were higher than those in the normal control group,however,there was no significant difference observed in SUZ12.Subgroup analysis showed that expression of EZH2,SUZ12,EED in M5 patients were increased,the difference was statistically significant.Correlation analysis revealed that the expression of EZH2 and SUZ12 was positively correlated with the expression of MALAT-1 in patients with M5,and it was speculated that MALAT-1 might be involved in the occurrence of leukemia by histone methylation modification.(3)Based on the results of RT-qPCR in clinical specimens and various AML leukemia cell lines,M5 cell lines,U-937 and THP-1 cells were selected as the cell model for following experiments.(4)We successfully constructed the GV248-MALAT-1-RNAi lentiviral vectors and transfected U-937 and THP-1 cells.Then cells were detected by RT-qPCR,the results showed that the expression levels of MALAT-1 in two types of cells were reduced by greater than 50%(P<0.05),indicating the successful construction of cell models.(5)CCK-8 Cell growth curve,colony formation assay and flow cytometry were used to evaluate the proliferation of leukemia cell lines.Silencing MALAT-1 could significantly inhibit the proliferation of U-937 and THP-1 cells and lead to cell cycle arrest in G0/G1 phase.(6)In the MALAT-1-silenced cells,the nuclei were dense and stained,some nuclei disintegrated and fragmented into dense granular particles(apoptotic bodies).Also,the apoptosis rate was detected by flow cytometry.The results showed that the apoptosis rate of U-937 and THP-1 cells were significantly increased.Western blotting showed that the expression of apoptosis related proteins Caspase-3,Caspase-8 and Caspase-9 were up-regulated in U-937 and THP-1 cell lines after transfection.At the same time,western blotting was used to detect the protein involved in classical Wnt/ beta-catenin cell signaling pathway.And the results show that: beta-catenin and c-Myc protein were down-regulated,suggesting that MALAT-1 may affect the proliferation of U-937,THP-1 through regulating the classical Wnt/ beta-catenin signaling pathway.(7)The potential target genes(E-cadherin,PTEN)of MALAT-1 were predicted by literature search.(8)Results of RT-qPCR showed that,after knocking down MALAT-1 in U-937 and THP-1 cell lines,the expression of E-cadherin and PTEN genes were up-regulated;and expression of multiple genes EZH2,SUZ12,DNMT1,DNMT3 A,DNMT3B,C-Myc,beta-catenin were down-regulated,with no significant difference observed in EED.(9)The results of pyrosequencing showed that the methylation rates of CpG islands in the promoter region of PTEN and E-cadherin were decreased in different degrees after knocking down MALAT-1 in U-937 and THP-1 cells.(10)The results of ChIP-qPCR showed that level of histone H3K27me3 in promoter region of target gene E-cadherin and PTEN in U-937 /THP-1 cell lines was significantly decreased after knockdown of MALAT-1.(11)Results of RIP-qPCR in U-937 cells showed that,MALAT-1 was significantly enriched with EZH2 and SUZ12 antibody when compared with the negative control group(IgG).When using EED,DNMT1,DNMT3 A,DNMT3B antibody for RIP-qPCR,compared with the negative control group(IgG),the enrichment of MALAT-1 was not significantly different.A similar result was obtained in THP-1 cells.(12)The results of RT-qPCR showed that the expression of PTEN and E-cadherin gene in patients with M5 were decreased compared with the normal control group.The results of pyrosequencing showed that: when compared with normal control group,the average methylation rates of CpG islands in promoter region of PTEN and E-cadherin genes were significantly higher.(13)Through bioinformatics web,we search for targeting MALAT-1 microRNA,the prediction results show that there are a total of 105 microRNA binding sites in the chain of MALAT-1,and we found that miR-143 and MALAT-1 may have potential binding sites.Then we constructied the microRNA over-expressed lentiviral vectors successfully and transfected the leukemia cell lines,then MALAT-1 expression was detected by RT-qPCR after transfection.The results showed that,compared with control group,the expression of MALAT-1 in U-937 and THP-1 cells was decreased in different degrees.The expression of target microRNA was detected in MALAT-1 silenced cells.There was no significant difference in the expression of microRNA 143 in MALAT-1 silenced cells.We cloned the fragment of MALAT-1 encompassing the miR-143 binding sites into psiCHECK-2 dual-luciferase reporter plasmids and performed luciferase assays in 293-T cells.miR-143 overexpression resulted in a significant decrease in luciferase activity,however,directed mutagenesis of the predicted miR-143 binding sites abolished this effect.The results indicated that MALAT-1 expression may be regulated through direct miR-143 binding.Conclusion:(1)The expression of MALAT-1 was increased in patients with M5,and was closely related to the poor prognosis.Expression of EZH2,SUZ12,EED genes were increased in patients with M5.And expression of EZH2 and SUZ12 was positively correlated with the expression of MALAT-1 in patients with M5,suggesting that MALAT-1 might be involved in the occurrence of AML by inducing DNA and / or histone methylation modification.(2)Silencing MALAT-1 could significantly inhibit the proliferation of U-937 and THP-1 cells and lead to cell cycle arrest in G0/G1 phase.And apoptosis rate of U-937 and THP-1 cells were significantly increased.(3)The potential target genes(E-cadherin,PTEN)of MALAT-1 were predicted by literature search.MALAT-1 may form the MALAT-1-PRC2 complex by combining EZH2 and SUZ12 to induce the histone H3K27 trimethylation of downstream target genes(E-cadherin,PTEN),leading to transcriptional silencing of target genes.MALAT-1 could affect the expression of DNMT1,DNMT3 A,DNMT3B,which may induce methylation of promoter CpG Islands in downstream target genes(E-cadherin,PTEN),so as to regulate the expression of genes.The mechanism remains to be further studied.(4)Bioinformatics analysis revealed a potential binding site of miR-143 in MALAT-1.MALAT-1 expression may be regulated through direct miR-143 binding.