Study On Autophagy Induced By Rapamycin In Acute Myeloid Leukemia HL-60Cells And Changes In The Expression Of UCH-L3in Progress Of Autophagy

Objective: Protein degradation mediated by ubiquitin and autophagy are the basicmechanisms involved in cellular self-regulation. The two systems are not independent toeach other, while mutually regulate through the complex mechanism. UbiquitinCarboxyl-terminal Hydrolase L3(UCH-L3) is a member of the family ofdeubiquitinating enzymes. In recent years, it is found that its abnormal expression isassociated with the malignant transformation of cells and tumor genesis. The aim of thisstudy is to investigate anti-proliferation effects of rapamycin on acute myelocyticleukemia cell line HL-60, to compare and analyze the expression differences ofUCH-L3in gene and protein level before and after the HL-60cells were treated byrapamycin, to explore the function mechanism of UCH-L3in autophagic cell death ofleukemia cells, further to develop the novel therapeutic targets and strategies to cureleukemia.Methods:1. Cell proliferation activity of HL-60cells treated with rapamycin(1μM、2μM、3μM、5μM) was measured by CCK-8assay.2. Fluorescence microscopewas used to detect the cells’ modality after the cells were treated with rapamycin indifferent time points (0h、24h、48h).3.After the treatment of rapamycin in different timepoints (0h、24h、48h), the mRNA level of autophagy related gene-LC3-II andgene-UCH-L3was detected by Real Time PCR assay.4.Western blot assay wasemployed to detect the protein expression level of UCH-L3after the cells were treatedwith rapamycin in different time points (0h、24h、48h).Results:1. Different concentrations of rapamycin could inhibit the proliferation ofHL-60cells. All the treatment groups had statistically difference as compared with thecontrol group (P<0.05), and the inhibitory rates were dosage dependent.2. Under the fluorescence microscope, fluorescence intensity in the control group was low, but in thetreatment groups strengthened along with the process time extension, and the cells’crimple and fragment phenomena were observed.3. After treated by2μM rapamycin indifferent time points (24h、48h), compared with the control group, the mRNA level ofLC3-II raised, while that of UCH-L3declined, all the treatment groups had statisticallydifference as compared with the control group (P<0.05).4. The protein expression levelof UCH-L3after the cells were treated with rapamycin in different time points (24h、48h) was declined, and had statistically difference as compared with that of the controlgroup (P<0.05).Conclusions:1.Rapamycin could significantly inhibit the proliferation of the cutemyelocytic leukemia cell line HL-60and induce the autophagic cell death.2. Theexpression level of UCH-L3in gene and protein was declined after the HL-60cellswere induced to autophagy by rapamycin, which means that UCH-L3may play a role inthe regulation of autophagic cell death of leukemia.