泛素结合酶10表达对三阴性乳腺癌自噬的影响及其机制

Effect of ubiquitin conjugating enzyme10 on autophagy in triple-negative breast cancer and the possible mechanism

目的探究泛素结合酶10(UbcH10)对三阴性乳腺癌自噬的调节作用及其作用机制。方法在人三阴性乳腺癌细胞系MDA-MB-231中,利用小干扰RNA(siRNA)进行瞬时转染低表达UBCH10基因,转染阴性对照siRNA的细胞作为阴性对照组,未经处理的细胞作为空白对照组。通过实时荧光定量反转录聚合酶链反应(RT-qPCR)和蛋白质印迹法(Western blot)检测转染后UbcH10的表达。MDA-MB-231细胞的增殖情况及单磷酸腺苷活化蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(mTOR)通路相关蛋白的表达情况分别用细胞增殖试验(CCK-8)法及Western blot法检测。组间差异采用单因素方差分析和t检验。结果RT-qPCR和Western blot实验结果显示,转染后siUbcH10-1组和siUbcH10-2组UbcH10的mRNA和蛋白的表达水平低于阴性对照组,差异有统计学意义(0.182±0.138比0.917±0.031、0.127±0.136比0.917±0.031,t=9.191、9.876,P<0.001)、(0.430±0.245比0.430±0.245、0.434±0.093比0.434±0.093,t=3.685、3.651,P<0.05),证明低表达细胞株构建成功。CCK-8结果显示,siUbcH10-1组和siUbcH10-2组细胞12、24、48 h的吸光度值均低于阴性对照组(0.160±0.004比0.211±0.004、0.155±0.003比0.211±0.004、0.246±0.006比0.286±0.012、0.202±0.004比0.286±0.012、0.308±0.007比0.432±0.004、0.308±0.013比0.432±0.004,t=14.965、16.423、4.649、9.629、13.737、13.756,P<0.01),差异有统计学意义。Western blot实验结果显示siUbcH10-1组和siUbcH10-2组微管相关蛋白1轻链3BⅡ(LC3BⅡ)/微管相关蛋白1轻链3BⅠ(LC3BⅠ)的比值及AMPK的磷酸化水平高于阴性对照组(4.294±0.812比2.552±0.002、4.554±0.216比2.552±0.002,t=4.033、4.633,P<0.05)、(1.533±0.177比0.564±0.185、1.519±0.263比0.564±0.185,t=4.859、4.792,P<0.05),差异有统计学意义,mTOR的磷酸化水平低于阴性对照组(0.482±0.028比1.153±0.069、0.504±0.072比1.153±0.069,t=8.708、8.433,P<0.05),差异有统计学意义。结论低表达UbcH10可通过抑制AMPK/mTOR信号通路来诱导三阴性乳腺癌细胞发生自噬。

Objective To explore the regulation and mechanism of ubiquitin conjugating enzyme10(UBCH10)on autophagy in triple-negative breast cancer(TNBC).Methods Human TNBC cell line MDA-MB-231 was transfected by small interfering RNA(siRNA)-UBCH10 to knockdown UBCH10,cells transfected with negative control siRNA served as negative control group,and untreated cells served as blank control group.The expression level of UBCH10 was assessed by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)and Western blotting.The proliferation of TNBC cells and the expression of adenosine 5′-monophosphate activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)pathway related proteins were detected by cell counting kit-8(CCK-8)and Western blotting,respectively.One-way ANOVA and t-test were used to compare difference between groups.Results The result of RT-qPCR and Western blotting showed that mRNA and protein expression of UbcH10 in siUbcH10-1 and siUbcH10-2 groups were significantly lower than negative control group(0.430±0.245 vs.0.929±0.053,0.434±0.093 vs.0.929±0.053,t=9.191,9.876,P<0.01),(0.430±0.245 vs.0.430±0.245,0.434±0.093 vs.0.434±0.093,t=3.685,3.651,P<0.05),which proved the successful construction of low-expressing cell lines.CCK-8 results showed that the absorbance(A)at 12,24 and 48 h in siUbcH10-1 and siUbcH10-2 groups was significantly lower than negative control group(0.160±0.004 vs.0.211±0.004,0.155±0.003 vs.0.211±0.004,0.246±0.006 vs.0.286±0.012,0.202±0.004 vs.0.286±0.012,0.308±0.007 vs.0.432±0.004,0.308±0.013 vs.0.432±0.004,t=14.965,16.423,4.649,9.629,13.737,13.756,P<0.01).Western blotting showed that the ratio of microtubule-associated protein 1 light chain 3BⅡ(LC3BⅡ)/microtubule-associated protein 1 light chain 3BⅠ(LC3BⅠ)and the expression level of p-AMPK in siUbcH10-1 and siUbcH10-2 groups were significantly higher than negative control group(4.294±0.812 vs.2.552±0.002,4.554±0.216 vs.2.552±0.002,t=4.033,4.633,P<0.05),(1.533±0.177 vs.0.564±0.185,1.519±0.263 vs.0.564±0.185,t=4.859,4.792,P<0.05),and the p-mTOR expression was significantly lower than negative control group(0.482±0.028 vs.1.153±0.069,0.504±0.072 vs.1.153±0.069,t=8.708,8.433,P<0.05).Conclusion Low-expression of UBCH10 may induce autophagy in TNBC cells by inhibiting the AMPK/mTOR signaling pathway.