Impact Of Long Noncoding RNA-LLEST In Acute Myeloid Leukemia

【Objectives】1. To analyze the different expression of long noncoding RNA-LLEST in bonemarrow mononuclear cells of AML patients compared with non malignancies blooddisease and healthy volunteers. And analyzed the correlation of clinical prognosis andrelapse between LLEST and AML patients.2. To study the effects of the biological function in human leukemia cell lines (K562cell line), which forced high expressed LLEST, and we explored the possible mechanismof these phenomenon.3. The function of long noncoding RNA-LLEST in vivo.【Methods】Mononuclear cells in bone marrow samples were collected from more than100patients of untreated AML, myelodysplastic syndrome (MDS) and aplastic anemia (AA)according to MICM criterion. The healthy volunteers and non malignancies blood diseasepatients (e.g. anemia, thrombocytopenia) as control group. The different expression ofLLEST between MDS and AA was analysised by gene chip technology. At same time, weuse real-time RT-PCR to detect the LLEST expression in AML patients. We also analyzethe expression of LLEST in bone marrow mononuclear cells of5AML patients betweennewly diagnosised and release after chemotherapy. We confirmed that the correlationamong LLEST expression quantity, patient’s condition, the proportion of blast cells in bonemarrow, AML-ETO fusion gene quantitative values of AML-M2patients. The clinical andlab data of19AML-M2cases were collected, calculate the median of the LLEST after acourse of chemotherapy, and their clinical characteristics therapies and relapse were thenfollowed-up. We study the effects of the biological function in human leukemia cell lines(K562cell line) by molecular biology technology, which forced high expressed LLEST,and explored the possible mechanism of these phenomenon. Finally, we use nude mouse as in vivo to further study the function of LLEST.【Results】1. Different expression of LLEST and its clinical significance.Gene chip results show that the expression of LLEST of bone marrow CD34+cells inchronic aplastic anemia (CAA) patients was higher than healthy volunteers, but its lowexpression in MDS, a relative malignant hematopoietic diseases. In the process of thebenign hematopoietic diseases transformate to malignancies, the expression of LLEST wasreduced gradually, until the CD34+cells in AML patients do not expression. Wedemonstrated that the expression quantity of LLEST was very low in AML patients (M1,M2, M3, M5) compared with control group through the real time RT-PCR. For the selected5AML-M2patients, the LLEST expression was very high when untreated. After inducedchemotherapy to the state of release, the expression of LLEST were higer than before. Thisresults showed that there is a relevance between LLEST expression quantity and the stateof disease. Enlarging the sample, further study found that there exist a inverse ratiobetween LLEST expression and tumor load, that is to say, the more tumor load result inmore blast cells proportion in bone marrow, the higher AML-ETO fusion gene values, theworse condition of disease, but the LLEST expression is low. The results have statisticallysignificant (p <0.05). For a part of patients (n=19) followed-up, we found that a relevancebetween LLEST expression level and disease relapse after a course of chemotherapy, thehigher LLEST expression quantity, the less rate of relapse, but the sample size were toosmall, we need enlarge sample size in further.2. The effects of biological behavior on leukemia cell lines which forced highexpression of LLEST, and explorate its possible mechanism.Through the molecular cloning and cell culture technology, using liposome2000carry plasmid which contains LLEST gene transfected human leukemia K562cell linesforced high expressed LLEST. Detected the biological behavior in transfected cells, wefind that LLEST can inhibit the growth of tumor cells, the cell cycle arrest in G2/M phase,promoted cell apoptosis and differentiation. Western Blot results confirm that the increasedof cleaved caspase9protein and decreased of the BCL-2were the main mechanism tomediate cell apoptosis. In addition, we also studied the ATP6v1c1gene. We demonstratethat there is a significant down regulation of ATP6v1c1, so we think over-expressedLLEST might weaken a growth advantage through these effects. 3. In vivo results.We further confirmed that high expression of LLEST in leukemia cell line canreduced the incidence of tumor and inhibit its growth through the experiment of nudemouse subcutaneous tumor.【Conclusions】We conclude that LLEST is a long noncoding RNA by bioinformatics analysis, andLLEST was decreased in malignant hematopoietic diseases. The high expression ofLLEST in AML-M2patient, the less proportion of blast cells and relapse rate, and have agood prognosis. Cell line experiments confirm that forced high expression of LLEST genein leukemia cell line can negative regulate the malignant biological behavior, it can inhibitthe proliferation of tumor cells, making the cell cycle arrest in G2/M phase, promote cellapoptosis and differentiation. in this process, numerous intracellular proteins (e.g. caspase9and BCL-2) and gene (e.g. ATP6v1c1) abnormal expression and participated in thisregulation together. In vivo, the experiment of nude mouse subcutaneous tumor also provedthat the LLEST can inhibit the growth of tumor cell.