The Mechanism Of Long Non-coding RNA-HOTAIR Participate In Drug Resistance In Acute Myeloid Leukemia

Objective:To study the abnormal expression of Long non-coding RNA(LncRNA)in acute myeloid leukemia multidrug resistance,and evaluate whether there is the abnormal expression of LncRNA HOTAIR in the Myelocytic leukemia cells of patients with different disease status;Analyzing whether LncRNA HOTAIR knockdown could reserve the therapy drug resistance of leukemia multi drug resistance cell line and study the mechanism of this procedure;Explore whether HOTAIR could be packaged in exosome and participate in drug resistance in leukemia.Methods:The expression level of four kinds of LncRNA:HOTAIR,PVT1,H19 and GAS5 in leukemia cell line K562 and its parallel resistant cell line K562/A02 were detected by qRT-PCR.qRT-PCR were also used to detected HOTAIR expression in Myelocytic leukemia cells of different disease status patients;Lentiviral transfection technology was performed to knockdown of HOTAIR expression in K562/A02 cells,the efficiency of knockdown was detected by Fluorescence microscopy and qRT-PCR.After transfection,the expression of Notchl and P21 were analyzed by qRT-PCR.Cell apoptosis after transfection was detected by Flow cytometry.Cell proliferation and chemotherapy doxorubicin sensitivity of K562/A02 cell line after transfection was detected by CCK8 kit.The expression of P21,AKT,pAKTs473,Notch1 protein was analysed by Western-blot.Exosomes extraction from K562,K562/A02,HOTAIR knockdown(si-HOTAIR)and control group cell culture supernatant by ExoQuick-TC kit method.qRT-PCR were used to detected HOTAIR expression in exosomes.Results:qRT-PCR detection showed that the expression of HOTAIR and H19 in leukemia resistant cell line K562/A02 was significantly higher than sensitive cell line K562,while GAS5 expression was lower than sensitive cell line,and PVT1 expression showed no statistically different.The results of clinical trials showed that the expression of HOTAIR in Myelocytic leukemia cells from refractory and relapsed patients was higher than that of primary leukemia patients,non-cancer doner own lower expression of HOTAIR compared with primary leukemia patients,two patients showed HOTAIR decrease when they received CR;HOTAIR knockdown could inhibit the proliferation of K562/A02 and increase apoptosis rate as well as the sensitive of K562/A02 to Adriamycin.qRT-PCR test results showed that HOTAIR knockdown directly increase the expression of P21 either in gene and protein level,and Western-blot results showed that pAKTs473 and Notchl protein expression decreased significantly after HOTAIR knockdown.In addition,qRT-PCR detection showed that the expression of HOTAIR in K562 secretory exosomes was higher than that in K562/A02,while after Lentiviral transfection,HOTAIR expression in exosomes from positive group(si-HOTAIR)is higher than negative group(si-NC).Conclusion:A variety of LncRNA may be involved in the formation of leukemia drug resistance,and the expression level of HOTAIR in Myelocytic leukemia cells can be used as a biomarker to detect the different disease status of patients.HOTAIR may promote the occurrence of leukemia resistance by down regulating cell cycle regulation gene P21 expression and affecting AKT and Notch signaling pathway.HOTAIR can be encapsulated into the exosomes and maybe participate in drug resistance of leukemia.