摘要
目的 :研究二烯丙基三硫(diallyl trisuli de,DATS)对人白血病细胞HL-60细胞中还原型烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate,NADPH)氧化酶活性的影响及可能的作用机制。方法:DATS(150μmol/L)作用于HL-60细胞不同时间后,采用硝基四氮唑蓝(nitro blue-tetrazolium,NBT)还原实验检测NADPH氧化酶的活性;实时荧光定量PCR检测DATS对微RNA-34a(microRNA-34a,miR-34a)以及Src、Gab1和Shp-2 mRNA表达的影响及miR-34a对Src、Gab1和Shp-2 mRNA表达的影响;蛋白质印迹法检测DATS及miR-34a对Src、Gab1和Shp-2蛋白磷酸化的影响;细胞计数实验检测DATS和miR-34a对HL-60细胞增殖的影响。结果 :DATS作用于HL-60细胞后能明显提高细胞中NADPH氧化酶的活性,且呈时间依赖性,而Src激酶抑制剂PP2可抑制这种作用;DATS能上调HL-60细胞中miR-34a的表达水平,呈时间依赖性;miR-34a过表达可抑制Src、Gab1和Shp-2 mRNA及磷酸化蛋白的表达;DATS能抑制磷酸化Src、Gab1和Shp-2蛋白的表达水平。DATS和miR-34a过表达能抑制HL-60细胞的增殖,而抑制miR-34a的表达则可提高白血病细胞的增殖能力。结论 :DATS可能通过促进HL-60细胞miR-34a-Src-Gab1-Shp途径调控NADPH的氧化酶活性,从而起到抑制白血病细胞增殖的作用。
Objective： To investigate the effect of diallyl trisulfide （DATS） on activity of nicotinamide adenine dinucleotide phosphate （NADPH） oxidase and to explore its possible mechanism. Methods： The activity of NADPH oxidase in leukemia HL-60 cells treated with 1S0 mol/L DATS at different time points was detected by nitro blue-tetrazolium （NBT） test. The mRNA expression levels of microRNA-34a （miR- 34a）, Src, Gab1 and Shp-2 in HL-60 cells treated with DATS and the mRNA expression levels of Src, Gab1 and Shp-2 in HL-60 cells transfected with miR-34a were detected by real-time fluorogenic quantitative- PCR. The expressions of phosphorylated Src, Gab1 and Shp-2 proteins in HL-60 cells after treatment with DATS and transfection with miR-34a were detected by Western blotting. The effect of DATS and miR-34a on the proliferation of HL-60 cells was detected by cell counting assay. Results： DATS could enhance the activity of NADPH oxidase in HL-60 cells in a time-dependent manner, and this effect could be inhibited by Src inhibitor PP2. DATS could increase the expression of miR-34a in HL-60 cells in a time-dependent manner. Overexoression of miR-34a could decrease the mRNA and phosphorylated protein levels of Src, Gabl and Shp-2. DATS could inhibit the protein expressions of phosphorylated Src, Gab1 and shp-2. DATS and overexpression of miR-34a could reduce the proliferation of HL-60 cells, and the inhibition of the expression of miR-34a could enhance the proliferation of HL-60 cells. Conclusion： DATS can regulate the activity of NADPH oxidase through miR-34a-Src-Gab1 -Shp pathway to inhibit the proliferation of leukemia cells.
出处
《肿瘤》
CAS
CSCD
北大核心
2014年第5期437-442,449,共7页
Tumor
