Arsenic Trioxide Affect The Warburg Effect In Acute Promyelocytic Leukemia Cell Line Of NB4

Part Ⅰ Study of warburg effect in leukemic cell linesObjective:To detect whether the warburg effect exists in leukemic cell lines.Methods:The inhibition rate of cell growth was detected by MTT after glycolysis inhibitor (2-DG) or oxidative phosphorylation inhibitor (oligomycin A:OA) added and incubated with THP-1, K562, HL-60and NB4cell line respectively for48h. Using Lactic acid kit and glucose assay kit to detect the consumption of glucose and production of lactic acid in all leukemic cell lines which were cultured in RPMI1640with low FCS, and calculation the ratio of glucose and lactic acid.Results:The growth inhibition of leukemia cell lines in a concentration dependent manner by2-DG. Cell growth inhibition of NB4and HL-60was obviously after48h intervention by2-DG, but K562is not sensitive to2-DG The growth inhibition of leukemia cell lines was not in a concentration dependent manner by OA for48h. THP-1was found to exhibit a higher sensitivity to OA, but K562and NB4was found to exhibit a low sensitivity instead. The glucose uptake of NB4were much more significant than other leukemia cell lines, and the ratio of consumption of glucose and production of lactic acid is about1/2.Conclusion:In four kinds of leukemic cells lines mentioned as before, NB4is the most sensitive one to the inhibitors of glycolysis, and the ratio of consumption of glucose and production of lactic acid is about1/2. These result suggest that the Warburg effect of NB4was significant. Part Ⅱ Regulating effect of arsenic trioxide on miRNA-122in NB4Objective:To detect the expression of miRNA-122after treaded with arsenic trioxide or all-trans retinoic acid in NB4wich is acute promyelocytic leukemia cell line.Methods:The expression of miRNA-122was detected by real-time quantitative PCR.Results:After treaded with1μmol/L ATO and1μmol/L ATRA for NB448h, the results of real-time quantitative PCR detection showed that miRNA-122expression increased nearly4times after ATO, but1.2times after ATRA.Conclusion:ATO can promote the expression of miRNA-122under the same concentration. Part Ⅲ PKM23’UTR is a direct target of miR-122.Objective:To verify the PKM23’UTR is a direct target of miR-122. Inhibition of miR-122can relieve the negative regulation of PKM2and leads to increased expression of PKM2Methods:Using TargetScan Human5.1software to predicted binding sites of PKM2by miRNA-122, designing primer sequences of PKM23’UTR and wild mutation PKM23’ UTR, connecting the amplified sequence and the psiCHECK-2vector, then transformated into plasmid. miR-122mimics, miR-122inhibitor and negative control were co-transfected into NB4for48hours with plasmid using cationic liposomes method. Cell were harvested and assayed by Dual Luciferase Assay System to detect the activity of luciferase. Constructing lentivirus carrying miR-122inhibition and transfected into NB4cells, screening of NB4-mir-122inhibitor and NB4-NC inhititor cell lines stably by G418, and detect the expression of miR-122by real-time quantitative PCR. Using MTS and Western Blot to detect the expression of PKM2and cell proliferation after inhibition of miR-122Results:After co-transfection the PKM23’UTR plasmid and miR-122, significant decreased of luciferase activity were obesrved, there was no significant difference in luciferase activity in mutPKM23’UTR-1plasmid which was cotransfected with miR-122, the luciferase activity of mutPKM23’UTR-2and miR-122dropped significantly, but the luciferase activity of mutPKM23’UTR-3which was mutation of two predicted binding sites at the same time has no significant difference. Constructed and selected successfully of the NB4-mir-122inhibitor and NB4-NC inhititor cell lines. Compared with NB4-NC inhititor, NB4-mir-122inhibitor exhibited elevated expression of PKM2and enhanced cell proliferation.Conclusion:The rescults demonstrated that PKM2is a major target of miRNA-122not only by Dual Luciferase Assay System but also by protein level. As for the two binding sites of PKM2by miRNA-122, the first binding site plays a key role, second binding sites plays a supportive role. The low expression of miR-122in NB4can promote the proliferation of tumor cells through the enhancement of the Warburg effect.