氨基酸转运蛋白LAT4调控髓系白血病细胞自噬的机制探讨

目的:探究氨基酸转运蛋白LAT4调控髓系白血病细胞自噬的机制。方法:通过RNAi技术敲低HL-60细胞中LAT4的表达,CCK-8检测细胞体外增殖能力的变化,免疫荧光检测自噬标志物LC3Ⅱ的表达变化。Western Blot检测给予LAT4抑制剂BCH梯度浓度抑制后细胞内自噬相关蛋白的表达差异。谷氨酰胺剥夺诱导自噬后加入最适剂量BCH抑制LAT4,采用液滴计数仪检测细胞对3H-Leucine摄取率的变化。结果:干扰LAT4表达能显著降低HL-60细胞体外增殖能力并促进自噬蛋白LC3Ⅱ的表达。BCH梯度抑制LAT4活性后LAT4和pS6K蛋白表达受到显著抑制,LC3Ⅱ蛋白表达量增加,50 μmol/L BCH的LAT4活性抑制效果最佳。3H-Leucine摄取率随BCH抑制LAT4时间增加而降低,BCH作用8 h后3H-Leucine摄取率分别较0 h、4 h降低47.64%和27.05%（P＜0.001）。结论:LAT4通过转运亮氨酸激活HL-60细胞内pS6K表达,抑制自噬发生。

The mechanism of autophagy in acute myeloid leukemia cells regulated by amino acid transporter LAT4

Objective:To investigate the mechanism of autophagy in acute myeloid leukemia cells regulated by amino acid transporter LAT4.Methods:LAT4 was knocked down by RNAi in HL-60 cells.CCK-8 assay was used to detect the changes of cell proliferation in vitro.Immunofluorescence was used to detect the expression of LC3Ⅱ in different cells.The expression levels of autophagy-related proteins were detected by Western Blot after giving BCH gradient concentration suppression.After glutamine deprivation,the optimal dose of BCH was added to inhibit LAT4,and the change of 3H-Leucine uptake rate of was detected by a drop counter.Results:Interfering with LAT4 can significantly reduce the proliferation capability of HL-60 and promote the expression of LC3Ⅱ.The expression of LAT4 and pS6K were significantly inhibited by BCH gradient inhibition while the expression of LC3Ⅱ was increased.Besides,the optimum concentration of BCH was 50 μmol/L.The uptake rate of 3H-Leucine was decreased after treating with BCH and the 3H-Leucine uptake rate was reduced by 47.64% and 27.05% when compared with 0 h and 4 h(P＜0.001).Conclusion:HL-60 can activate intracellular pS6K expression through LAT4 transport of leucine and inhibit autophagy.