The Expression And Mechanism Of LncRNA-ANRIL In Myelodysplastic Syndromes

Context:Myelodysplastic syndromes(MDS)is a group of heterogeneous diseases.Due to the hematopoietic stem cell differentiation and development disorder,the formation of blood cells is abnormal in quality and quantity.It is characterized by dysplasia of bone marrow,progressive cytopenia and easy transformation into acute myeloid leukemia(AML).The clinical manifestations include infection,bleeding and anemia to varying degrees.MDS can occur at any age,in the majority with middle-aged and old.In recent years,with the advent of an aging society,the incidence of the disease has increased significantly.The incidence of the disease is about 3-4 per 100,000 people over the age of 60,while it can be as high as 50 per 100,000 people over the age of 70.MDS has an atypical clinical presentation with complex and nonspecific hematological changes,making it more complex than leukemia in the diagnostic process,especially when the morphological changes are not significant.The clinical prognosis of MDS varies widely,with a minimum median survival of less than 6 months and a maximum of more than 5 years.The specific pathogenesis of MDS is still unclear.But with the development of evidence-based medicine and continuous scientific research,epigenetic mechanisms have been identified as playing an important role in the development of MDS.Hypermethylation of the promoter regions of tumor suppressor genes(TSGs),such as P15 and P16 genes,leads to inactivation of tumor suppressor genes and imbalance of cell proliferation and differentiation,which leads to tumorigenesis.The hypermethylation status of P15 and P16 genes was prevalent in MDS patients,accompanied by decreased or absent expression of p15 and p16 genes.However,DNA methylation levels and P15 and P16 gene expression levels were not strictly correlated,and some patients responded poorly to hypomethylating agents,suggesting that there may be other factors leading to inactivation of TSGs.As a research hotspot,long non-coding RNA(lncRNA)has not only been used in tumor diagnosis and prognosis evaluation,but also become a new method of targeted therapy.ANRIL is a long non-coding RNA transcribed from the CDKN2A/B gene cluster on Chr9p21.More and more evidences have shown that ANRIL is involved in tumorigenesis by affecting cell proliferation,apoptosis and metastasis.Current studies suggest that ANRIL is associated with many malignant tumors and metabolic diseases,including lung cancer,stomach cancer,breast cancer,liver cancer,bladder cancer,ovarian cancer,type 2 diabetes,obesity,coronary artery disease and so on.Ras-induced senescence requires the inhibition of ANRIL.High level of ANRIL expression is associated with chemoresistance,and knockdown of ANRIL promotes chemotherapy sensitivity.It has also been studied in hematological diseases.It has been reported that ANRIL is highly expressed in AML patients and is associated with AML pathogenesis.It has also been shown that,ANRIL acts synergistically with EZH2 to promote the proliferation of adult T-cell leukemia cells.To date,there are no studies on the expression of ANRIL in MDS and its mechanism.Objective:The expression of ANRIL in bone marrow(BM)specimens from MDS patients and healthy controls was measured to evaluate its efficacy for clinical molecular diagnosis and to explore its potential as a diagnostic tool for MDS.By interfering with ANRIL in SKM-1 cell line and observing its effect on P15 and P16 gene expression,we can analyze the potential molecular mechanism and provide new ideas for the diagnosis and treatment of MDS.Method:A total of 50 BM samples were collected from newly diagnosed MDS patients hospitalized at the First Affiliated Hospital of Zhengzhou University between January 2017 and December 2019.Clinical data including cytomorphology,immunophenotypic analysis,cytogenetics,relevant genetic testing and biochemical assays were complete.BM samples from 15 healthy bone marrow donors were collected as controls.Real-time fluorescence quantification polymerase chain reaction(qRT-PCR)was applied to determine the expression levels of ANRIL.In SKM-1 cell line,after ANRIL interference,cell cycle changes and apoptosis were observed by flow cytometry,and cell proliferation was measured by cell counting kit-8(CCK8).The expressions of P15 and P16 mRNA and related proteins were detected by Western-blot.Chromatin immunoprecipitation(ChIP)was used to detect the binding ability of CBX7,EZH2 and H3K27me to P15 and P16.The data were statistically analyzed by Graphpad 8.0 and SPSS 23.0.Student’s t-test test was used to analyze the differences between the two groups.The independent sample Kruskal-Wallis test was used for comparison between multiple groups.Receiver operating characteristic(ROC)curve analysis and area under curve(AUC)of ROC were established to evaluate their diagnostic value.Survival analysis was plotted using Kaplan-Meier curves,and statistical significance was determined using the log-rank test.Multivariate analysis was performed using Cox proportional risk model,Hazard Ratio(HR)and 95%Confidence interval(CI)were calculated.The statistical significance level was set asp<0.05.Results:1.The expression of ANRIL is significantly higher in MDS patients than in controls.2.ANRIL expression is significantly higher in the higher risk group of International Prognosis Scoring System-Revised(IPSS-R)than in the lower risk group.3.ANRIL expression level is an independent prognostic factor affecting the overall survival(OS)of MDS patients.4.After ANRIL interfered with SKM-1 cell line,cell proliferation was inhibited and apoptosis rate increased.5.The relative expression of P15mRNA and p15 protein was decreased and the expression of P16mRNA and p16 protein was increased after ANRIL interference with SKM-1 cell line.6.ChIP showed that ANRIL interference affected the binding of CBX7,EZH2,and H3K27me to P15 and P16 promoter sites.ConclusionANRIL may inhibit the transcription and expression of P16 gene by mediating the aggregation of polycomb repressive complex in the promoter region of P16 gene,and promote the proliferation of SKM-1 cell line cells.Combined with the clinical results,ANRIL plays a role of oncogene in the development of MDS.It may be an important molecular biomarker in the diagnosis and prognosis of MDS.