| Obiective:(1)Explore the differential long non-coding RNA(Long non-coding RNA,lncRNA)in acute leukemia(Acute leukemia,AL)patients and normal people through the lncRNA(Long non-coding RNA)gene chip to find molecular biomarkers that can guide the prognosis of patients with acute leukemia.(2)The RT-qPCR(Real-time Quantitative PCR)method was used to verify the microarray results in acute leukemia patients and normal human specimens,and the prognosis of patients was analyzed to see if lncRNA ENST00000519507 、 lncRNA ENST00000488259 、 lncRNA ENST00000604266、lncRNA ENST00000511270、lncRNA ENST00000487332、lncRNA ENST00000547417、lncRNA ENSG00000228113、lncRNA ENST00000461818、lncRNA ENST00000545379、lncRNA ENST00000551855 is expected to be a molecular marker for predicting the occurrence,development and prognosis of AL.Methods:(1)Using lncRNA microarray to detect differentially expressed lncRNA in3 patients with acute myelogenous with maturation 2a(Acute myelogenous with maturation 2a,AML-M2a)and 3 normal human bone marrow mononuclear cells.After standardization of the original data,bioinformatics analysis such as GO and ECGG was conducted to find related transcription factors,expressed proteins and signaling pathways,and predict 5 high expression and 5 low expression lncRNA that could guide the diagnosis,treatment and prognosis of acute leukemia.(2)138 cases of acute leukemia and120 normal subjects were collected.Abnormal expression of lncRNA by lncRNA chip was detected by qPCR method.The accuracy of gene chip detection was verified by statistical principle and method.(3)ROC curve analysis for optimal cutoff of differentially expressed lcnRNA in population validation in 110 AL patients who had a feasible clinical prognosis analysis.The differentially expressed genes were divided into high expression group and low expression group using the optimal cut-off value.The prognosis of acute leukemia patients with different expression levels was statistically analyzed,and lncRNA with guiding significance for the prognosis of acute leukemia patients was searched.(4)In 7 patients with allogeneic hematopoietic stem cell transplantation,the prognosis of patients with high expression of lncRNA and lowexpression group was statistically significant.Results:(1)The lncRNA gene chip test showed that a large number of abnormally expressed lncRNA existed in acute leukemia patients and normal people.According to the screening criteria of Fold change ≥ 2.0 and P ≤ 0.05,9199 abnormally expressed lncRNAs were screened,of which 5969 were highly expressed lncRNA and 3230 were low expressed lncRNA.(2)After the raw data was standardized,the lncRNA function analysis was performed.Molecular functional analysis showed that the high expression of lncRNA molecules was mainly enriched in metal ion binding,polyadenylation tail RNA binding,and DNA binding.Low expression of lncRNA molecules is primarily enriched in receptor binding,sugar binding,and transmembrane signaling receptor activity.Biological process analysis revealed that high expression of lncRNA organisms was mainly enriched in gene transcription,translation and regulation.Low expression of lncRNA organisms is primarily enriched in immune responses,inflammatory responses,and cell surface receptor signaling pathways.Cell component analysis showed that high expression of lncRNA was mainly enriched in the nucleus,nucleolus,and ribosomal subunit.Low expression of lncRNA is mainly enriched in the plasma membrane,extracellular bodies,and lysosomes.Pathway analysis showed that Pathway analysis of differential genes using KEGG database,high expression of lncRNA metabolic pathways mainly include ribosome metabolism,RNA transport,and nucleotide metabolism.Low expression of lncRNA metabolic pathways mainly include osteoclast differentiation,cytokine interaction with cytokine receptors,and natural killer cell-mediated cytotoxicity.(3)According to the differential expression FC value,the 5 lncRNAs with significant difference between the high and low expression groups were selected.Finally,lncRNA ENST00000519507,lncRNA ENST00000488259,lncRNA ENST00000604266,lncRNA ENST00000511270,lncRNA ENST00000487332,lncRNA ENST00000547417,lncRNA ENSG00000228113,lncRNA ENST00000461818,lncRNA ENST00000545379,lncRNA ENST00000551855 were used as genes for subsequent population validation and survival analysis.(4)The results of qPCR assay in 138 acute leukemias and 120 normal subjects showed that lncRNA ENST00000545379 and lncRNAENST00000461818 showed high expression in 10 abnormally expressed lncRNAs,and lncRNA ENST00000488259 showed low expression.The coincidence rate between the chip test results and the crowd verification results is 70%.(5)The expression level of lncRNA ENST00000519507 was higher in patients with acute leukemia than in normal humanperipheral blood(P<0.0167),and the level of expression was not significantly different between acute myelogenous leukemia(Acute myelogenous leukemia,AML)and acute lymphocytic leukemia(Acute lymphocytic leukemia,ALL).The expression level of lncRNA ENST00000488259 in AL and normal people was not statistically different,but the normal human bone marrow expression level was higher than that in peripheral blood(P<0.0167).The expression level of lncRNA ENST00000604266 was higher in patients with AL than in normal human bone marrow and peripheral blood(P<0.0167),and the expression level was not significantly different between AML and ALL.The expression level of lncRNA ENST00000487332 in patients with AML was higher than that in normal human peripheral blood(P<0.0083),and there was a statistically significant difference in the expression levels of AML and ALL(P<0.0083).The expression level of lncRNA ENST00000547417 was lower in patients with AML than in normal human peripheral blood(P<0.0083).lncRNA ENST00000545379 normal human bone marrow was higher than peripheral expression,and was statistically significant(P < 0.0167).The expression level of lncRNA ENST00000551855 was lower in patients with AL than in normal human peripheral blood(P<0.0167),and the expression level of bone marrow in normal subjects was lower than that in peripheral blood(P<0.0167).(6)Survival analysis showed that age,white blood cell count,lncRNA ENST00000519507,and lncRNA ENST00000545379 were the influencing factors of overall patient survival(OS).Age,white blood cell count,lncRNA ENST00000519507,lncRNA ENST00000547417,and lncRNA ENST00000545379 were influential factors for progression-free survival(EFS)in patients(p<0.05).lncRNA ENST00000547417 and lncRNAENST00000551855 are factors influencing OS and EFS in patients with hematopoietic stem cell transplantation.Among them,white blood cells,lncRNA ENST00000547417,lncRNA ENST00000519507,lncRNA ENST00000545379 are independent factors influencing patient EFS.Conclusions:(1)The results of lncRNA microarray showed that there are a large number of abnormally expressed lncRNAs in AL patients and normal humans,and different lncRNAs can be associated with various proteins,transcription factors and pathways.(2)The coincidence rate between lncRNA chip detection and qPCR verification was 70%.There were significant differences in the expression levels of lncRNA in AML,ALL,normal human bone marrow and normal human peripheral blood,suggesting that it may participate in the development of leukemia through different mechanisms ofaction.(3)lncRNA ENST00000519507,lncRNA ENST00000547417,lncRNA ENST00000545379,and lncRNAENST00000551855 are related to the prognosis of patients with AL and post-hematopoietic stem cell transplantation.It is expected to be a molecular biomarker for the prognosis of acute leukemia and post-transplant patients,but its specific mechanism remains to be further studied. |
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