Clinical Significance And Mechanism Of MiR-217 In Acute Myeloid Leukemia

Part Ⅰ The clinical significance of serum miR-217 in AcuteMyeloid Leukemia Background:Acute myeloid leukemia(AML)is a malignant clonal disease of a kind of myeloid hematopoietic stem progenitor cells.The abnormal primordial cells and immature cells in the bone marrow proliferate and inhibit normal hematopoiesis,which can infiltrate the liver and spleen,lymph nodes and other organs.The main clinical manifestations of acute myeloid leukemia are anemia,hemorrhage,infection and infiltration.AML is one of the most common hematological malignancies in adults and is among the top ten in mortality caused by malignant tumors.The etiology and pathogenesis of AML is very complicated,so far it is not completely clear.It is clear that it is highly heterogeneous in clinical manifestations,immunophenotyping,cell morphology,molecular biology,cytogenetics,etc.It is generally accepted that karyotypic abnormalities and reproducible gene mutations are the important factors that influence the development of AML currently.Most patients can be stratified according to the degree of risk by classifying patients with karyotypic abnormalities and reproducible gene mutations,and the corresponding treatment plan can be stratified according to the different risk levels of patients,now the five-year survival rate of most AML patients has improved significantly over the previous period.However,a considerable number of patients still have poor prognosis,so it is necessary to further explore the new pathogenesis of AML and improve the existing diagnostic and prognostic evaluation system.In the past 20 years,with the continuous research of epigenetics by scientists,more and more evidence indicates that epigenetics plays an important role in the occurrence and development of AML.As an important member of epigenetics,non-coding RNA has become more and more important in the pathogenesis of AML,the most important of which are micro RNAs(miRNAs).miRNAs are a class of non-coding small RNAs that are 18-25 nucleotides in length and have post-transcriptional regulatory gene expression levels that specifically bind to complementary sequences on the 3’ untranslated region(3’UTR)of the target gene,down-regulate the expression level of the target gene,thereby affecting the occurrence and development of the disease.There are many literatures showing that miRNAs play a key role in the occurrence and development of AML,which deserves further study.OBJECTIVE:This study aimed to explore the clinical significance of serum miR-217 in AML.METHODS:Quantitative reverse transcriptase-polymerase chain reaction(qRT-PCR)was performed to detect miR-217 levels in the blood samples obtained from 89 AML patients and 60 healthy controls.RESULTS:1、The results showed that miR-217 expression was significantly decreased in AML patients compared to controls.2、Serum miR-217 levels were greatly downregulated in the AML patients with poor risk cytogenetic.3、ROC analysis demonstrated that serum miR-217 could effectively differentiate AML patients from normal controls.4、miR-217 expression was markedly increased in patients achieving complete remission after their treatment.5、low miR-217 expression was associated with aggressive clinical features.6、Kaplan-Meier analysis showed that AML patients with low miR-217 expression tended to have shorter overall survival and disease free survival.7、In the multivariate analysis stratified for prognostic parameters,miR-217 was proved to be an independent prognostic indicator.CONCLUSIONS:miR-217 was identified as an independent marker for the diagnosis and prognosis of AML.Part Ⅱ: Antitumor effect and mechanism of miR-217 in acutemyeloid leukemia Background:Acute leukemia is one of the most common diseases in hematological malignancies and is highly heterogeneous.So far its pathogenesis has not been fully elucidated.It is known that karyotype abnormalities,gene mutations,and epigenetic changes are all involved in the development of AML.In recent years,there have been more and more studies about epigenetics on the occurrence,development and prognosis of acute leukemia.Epigenetics refers to changes in gene expression levels based on non-gene sequence changes,including DNA methylation,histone modifications,chromosomal remodeling,and non-coding RNA regulation,primarily through transcription or translation of genes regulation,affecting its function and characteristics.With the widespread use of second-generation sequencing and the growing number of relevant research data,scientists have discovered that long-chain non-coding RNAs(lncRNAs)and micro RNAs(miRNAs)not only play a role in natural life processes such as cell differentiation and somatic development.It plays an important role and can participate in the occurrence and development of many diseases through interaction.Non-coding RNAs do not perform life activities by translating into proteins like RNA encoding.They regulate the occurrence and development of various normal life activities and diseases mainly through transcription,post-transcriptional and epigenetic levels.Among the many non-coding RNAs,miRNAs are the most and in-depth studied so far.Current research has confirmed that miRNAs play an important role in cell proliferation,differentiation,migration,apoptosis,ontogenesis,growth,metabolism,and angiogenesis.Numerous studies have shown that the function of miRNAs is mainly achieved by regulating the expression of target genes,and one miRNA can simultaneously target multiple target genes,and one target gene can also be regulated by multiple miRNAs at the same time.Numerous studies have shown that there is a mutual regulation relationship between lncRNA and miRNA.lncRNA interacts with miRNA as a competitive endogenous RNA(ceRNA)and then by regulating the expression of target genes.Conversely,miRNAs can also play a biological role by regulating lncRNA through RISC(RNA-induced silencing complex),both of them involved in the development of many diseases.lncRNA and miRNA can play a role through multiple sites and multiple targets,and participate in various physiological and pathological processes,such as cell proliferation,differentiation,migration,apoptosis and the development of various diseases.The first part of the study showed that miR-217 plays a role in tumor suppressor gene in AML.In order to further study its specific mechanism of anti-cancer effect in AML,we predicted its upstream regulated lncRNA and downstream through bioinformatics software.A large number of literatures were examined to identify lncRNA and target genes associated with AML and validated by a series of experiments.Objective:To explore the regulation mechanism of miR-217 expression down-regulation in AML,determine its downstream target gene and upstream-regulated lncRNA,verify the interaction of lncRNA MALAT-1-miR-217-KRAS,and participate in the regulation of AML occurrence and development together.Method:1.The binding between miR-217 and lncRNA MALAT1 and the binding between miR-217 and KRAS were verified by dual luciferase reporter assay.The Rescue assay verified the interaction between lncRNA MALAT1,miR-217 and KRAS.2.The results of qRT-PCR showed that the relative expression of lncRNA MALAT1 and KRAS was significantly decreased in miR-217 overexpressing AML cells.The relative expression of lncRNA MALAT1 and KRAS was significantly increased in miR-217-inhibited AML cells.In AML cells down-regulated by lncRNA MALAT1,the relative expression of miR-217 was significantly increased,and the relative expression of KRAS was significantly reduced.3.Overexpression and inhibition of miR-217 in human AML cells,WB assay to detect expression of KRAS protein.Inhibition of lncRNA MALAT1-1 in human AML cells,WB assay to detect expression of KRAS protein.Inhibition of lncRNA MALAT1-1 and miR-217 in human AML cells,WB assay to detect expression of KRAS protein.4.Inhibition of lncRNA MALAT1-1 and miR-217 in human AML cells,CCK8 assay to evaluate cell proliferation.5.Inhibition of lncRNA MALAT1-1 and miR-217 in human AML cells,to perform balloon formation experiment.6.Inhibition of lncRNA MALAT1-1 and miR-217 in human AML cells,cell cycle was detected by flow cytometry to verify the effect of lncRNA MALAT1 and miR-217 on AML cell cycle.7.Inhibition of lncRNA MALAT1-1 and miR-217 in human AML cells,apoptosis was detected by flow cytometry to verify the effects of lncRNA MALAT1 and miR-217 on apoptosis of AML cells.Results:1.The results of the dual luciferase reporter assay indicated that hsa-miR-217-5p can bind to both lncRNA MALAT1 and KRAS 3’UTR,The targeted regulation of ceRNA may exist between lncRNA MALAT1,hsa-miR-217-5p,and KRAS.2.The results of qRT-PCR showed that the expression of hsa-miR-217-5p was negatively correlated with lncRNA MALAT1 and KRAS,while the expression of lncRNA MALAT1 was positively correlated with KRAS expression and negative with hsa-miR-217-5p.The targeted regulation of ceRNA may exist between lncRNA MALAT1,hsa-miR-217-5p,and KRAS.3.WB results showed that overexpression of miR-217 significantly inhibited the expression of KRAS protein;while the expression of KRAS protein was up-regulated after inhibition of miR-217.It is indicated that the expression level of KRAS protein is negatively correlated with the expression of miR-217,and miR-217 may target the regulation of KRAS protein expression.Compared with NC,the protein level of KRAS was significantly decreased after knocking down MALAT1-1,but the expression level of KRAS protein was significantly increased after knocking down MALAT1-1 and inhibiting miR-217.This indicates that MALAT1-1 may affect the protein expression level of KRAS through miR-217.4.The results of CCK8 assay showed that knockdown of MALAT1 could effectively reduce the cell viability of THP-1,and the addition of hsa-miR-217-5p inhibitor could offset the effect of knockdown of MALAT1 on the decrease of THP-1 cell viability.This indicates that MALAT1 may indirectly affect the cell proliferation of AML cells by affecting miR-217-5p.5.The results of balloon formation experiments showed that knockdown of MALAT1 significantly inhibited balloon formation in AML cells,but inhibition of hsa-miR-217-5p restored the balloon formation ability of AML cells.This indicates that MALAT1 may indirectly affect the balloon formation ability of AML cells by affecting hsa-miR-217-5p.6.The results of cell cycle experiments showed that there was no significant change in the G1/S+G2 phase of THP-1 cells after knocking down MALAT1 and overexpressing hsa-miR-217-5p,indicating that MALAT1 may have no significant effect on AML cell cycle.7.The results of apoptosis assay showed that Knockdown of MLALT1 increased the percentage of apoptosis in AML cells,and knockdown of MALAT1 followed by inhibition of miR-217 down-regulated the proportion of apoptotic cells,suggesting that inhibition of miR-217 can rescue the effect of MALAT1 on AML cells.Therefore,MALAT1 may indirectly affect apoptosis of AML cells by affecting miR-217-5p.Conclusion:miR-217 acts as a tumor suppressor in AML,lncRNA MALAT1 is an upstream targeting regulator of miR-217,KRAS is a downstream target gene of miR-217,and lncRNA MALAT1 may adsorb miR-217 through a sponge,thereby Indirectly regulate the expression of downstream target gene KRAS,which in turn affects the biological functions such as apoptosis and proliferation of AML cells.lncRNA MALAT1,miR-217,KRAS exist in the mutual regulation of ceRNA,lncRNA MALAT1-miR-217-KRAS through the ceRNA mechanism to participate in the regulation of AML occurrence and development.