Research Of LncRNA HOTAIRM1 Regulates Autophagy In Acute Promyelocytic Leukemia Through The PI3K/Akt/mTOR Pathway And Its Possible Mechanism

Part ⅠConstruction of Lnc RNA HOTAIRM1 low expression and negative control NB4 cell linesObjective:To construct the lentiviral vectors with low expression of lnc RNA HOTAIRM1 gene and construct an acute promyeloctic leukemia NB4 cell line expressing the vector,which will lay a foundation for the subsequent studies about the mechanism of autophagy regulating by lnc RNA HOTAIRM1 in acute promyelocytic leukemia.Methods:We designed the negative control sequence（NC）and lnc RNA HOTAIRM1-sh RNA sequences based on the lnc RNA HOTAIRM1 sequence（NR₀38367）in Genebank.Then the sequences were constructed into the lentiviral vector LV3（H1/GFP&Puro）.The recombinant plasmids were transfected into HEK 293 T cells together with the packaging plasmids ps PAX2 and p MD2.G to obtain lentiviral particles.Then lentiviral particles were transfected into NB4 cells for measuring interferential efficiency.The RNA was extracted and the lnc RNA HOTAIRM1-sh RNA sequence with the highest interference efficiency was detected by real-time quantitative PCR（q RT-PCR）.The lentivirus was used to infect NB4 cells,and the low expression of lnc RNA HOTAIRM1NB4 and negative control cell lines were constructed.Results:The expression of lnc RNA-HOTAIRM1 in NC group was significantly higher than it in normal group（P<0.05）,whereas it was significantly decreased in HOTAIRM1-sh RNA-1 、 HOTAIRM1-sh RNA-2 、 HOTAIRM1-sh RNA-3（p<0.05）about 91.05% 、91.86%、98.46% respectively.Conclusion:The interferential efficiency of three recombinant HOTAIRM1-sh RNAs were higher than 90% at lnc RNA level,of which HOTAIRM1-sh RNA-3 was the highest to 98.46%.At the same time,the low expression and negative control lentivirus vector of lnc RNA HOTAIRM1 was successfully constructed,and the corresponding lentivirus was transfected into NB4 cells.The low expression and negative control NB4 cells of lnc RNA HOTAIRM1 were constructed,which laid a foundation for further study on the autophagy of lnc RNA HOTAIRM1 in acute promyelocytic leukemia and its mechanism.Part IIResearch of lnc RNA HOTAIRM1 regulates autophagy in acute promyelocytic leukemia through the PI3K/Akt/m TOR pathway and its possible mechanismObjective:Acute myeloid leukemia（AML）is a malignant disease caused by abnormal proliferation of hematopoietic stem cells.Acute promyelocytic leukemia（APL）is M3 of AML characterized by the cessation of myeloid differentiation in the promyelocytic phase.In this study,we studied he regulation of lnc RNA HOTAIRM1 in APL on autophagy through PI3K/Akt/m TOR signaling pathway through lentivirus vector transfection,and provided ideas for its early diagnosis and treatment prognosis.Methods:In this study,we constructed low-expressing and negative control NB4 cell lines（hereinafter referred to as NB4-Lv-sh HOTAIRM1,NB4-Lv-NC）by lentivirus vector transfection technology.The cell lines treated by all-trans retinoic acid（ATRA）were tested for PI3K/Akt/m TOR signaling pathway expression level by q RT-PCR and Western Blot.The level of light chain 3（LC3II）of type II autophagy marker protein microtubule-associated protein 1 and p62 were detected in the autologous cell lines.Results:It was found that LC3 II m RNA and protein expression in NC group and sh RNA group increased after ATRA treatment,while p62 m RNA and protein expression decreased.The expression levels of Akt m RNA and protein were slightly increased compared with the untreated group,while the expression levels of m TOR m RNA and protein were slightly decreased.The expression levels of p-Akt and p-m TOR protein were significantly decreased and the differences were statistically significant（p<0.05）.In sh RNA cells,LC3 II m RNA and protein expression levels decreased significantly,while p62 m RNA and protein expression levels increased significantly and the differences were statistically significant（p<0.05）.There were no significant changes in Akt,m TOR m RNA and protein expressions,and the differences were not statistically significant（p>0.05）.The expression levels of p-Akt and p-m TOR proteins were significantly increased,and the differences were statistically significant（p<0.05）.Conclusion:In this study,the low expression of lnc RNA HOTAIRM1 and negative control NB4 cell line constructed in the first part were used to detect autophagic pathway.It was found that the expression of lnc RNA HOTAIRM was reduced,the phosphorylation level of the PI3K/Akt / m TOR pathway increased,and autophagy decreased.This shows that lnc RNA HOTAIRM1 can regulate the autophagy of acute promyeloid leukemia through the PI3K/Akt/m TOR signaling pathway.At the same time,we found that ATRA can promote the autophagy of acute promyeloid leukemia cells.This study will provide ideas for the early diagnosis and treatment of acute promyeloid leukemia.