The Mechanismof Long Noncoding RNA-LLEST Promoting Apoptosis In Acute Myeloid Leukemia And Construction Of Lentivirus Vectors

【Objectives】1. To analyse the expression of LLEST in acute myeloid leukemia(AML) and myelodysplastic syndromes(MDS) compared with normal, and ensure the importance of LLEST in AML. And to predict the genes associated with the impact of LLEST.2. To elucidate the mechanism of proapoptosis impact of LLEST.3. To construct Lentivirus Vectors that could overexpress LLEST, and package the Lentivirus and then ensure the efficacy of the Lentivirus Vector. As a consequence, it lays a foundation for following research of the impact of LLEST on primary leukemia cells.【Methods】LLEST diversity expression was got inAMLand MDS throughlarge sample gene microarrayanalysis in NCBI database. The target genes of LLEST were predicted by utilizing gene microarray technology and bioinformatics software. We chose four patients’ bone marrow samples that before and after achieving chemotherapy, K562 cell line which overpressed LLEST(LLEST cell line), K562 cell line which served as control(Vector cell line) and tumor blocks that come from nude mice subcutaneous tumor formation by LLEST cell line and Vector cell line to explore associated genes expression and the proteins based on the BCL2 gene predicted.Then we conducted our research that mechanism of LLEST’s pro-apoptosis through dual-luciferase reporter assay to deepen our understanding.On the same time, we chose the bone marrow samples of 18 patients LLESTcell line and Vector cell line,and explored the expression of LLEST and XIAP based on the prediction of the gene of XIAP. The last,we constructed recombinant plasmid containing the gene of LLEST, achieved the package of Lentivirus, and lay the foundation of the following research.【Results】1. We realized the expression of LLEST in thebone marrow mononuclear cells of AML and MDS through Haferlach’s datum about the gene microarray outcomes of AML and MDS in NCBI database.The expression of LLEST in AML and MDS was obvious decreasedcompaed with normal.2. We attained 1613 genes which expressed differently in LLEST cell line by utilizing gene microarray technology, and got 1518 genes by predicting promising gene by bioinformatics software.At last, there were 11 genes existing in the two databases. And we only observed lower consistent expression of BCL2 gene in samples of AML receiving treatment compared to the sample before receiving therapy, and it was not same for the other genes predicted. The expression difference showed also in LLEST cell line and Vector cell line. LLEST cell line presented lower BCL2 expression level of gene. In addition, the tumor blocks come from nude mice subcutaneous tumor formation were tested by western blot to explore the expression of BCL2 protein. The tumor blocks resulting from LLEST cell line had lower expression protein level of BCL2.The further research carried by dual-luciferase reporter assay showed that LLEST inhibitated BCL2 gene directly rather than indirectly.3. The gene expression of gene of LLEST and XIAP declined simultaneously in 4 out of 18 primary AML patients after receiving chemotherapy.LLEST cell line showed lower expression of XIAP gene compared with Vector cell line.4. We chose LV5 plasmid as carrier to construct recombinant plasmidcontaining the gene of LLEST, LV5-LLEST, achieve the package of Lentivirus in 293 T cell line, and then transfect K562 cell line.LLEST gene presented high expression in K562 cell line by quantitative PCR.【Conclusions】1. LLEST expression is different in AML and MDS.2. LLEST plays a role of pro-apoptosis by inhibitating BCL2 gene directly.2. XIAP is associated with LLEST’s role of pro-apoptosis, the mechanism is needed further research.3. We have conducted successfully recombinant plasmid carring LLEST, LV5-LLEST, acheived package of Lentiviruspreliminarily and lay the theoretical basis and experimental foundation of the following research in primary leukemia cells.