DOCK1通过AMPK/mTOR通路对多发性骨髓瘤细胞增殖、凋亡和自噬的调控机制研究  

Regulation Mechanism of DOCK1 on Proliferation,Apoptosis and Autophagy of Multiple Myeloma Cells Through AMPK/mTOR Pathway

作　　者：李丹[1] 张忠山[1] 李洵婷 曹苏娟[1] LI Dan;ZHANG Zhong-shan;LI Xun-ting;CAO Su-juan(Department of Oncology,the Affiliated Hospital of Xiangnan College,Hunan Chenzhou 423000,China)

机构地区：[1]湘南学院附属医院肿瘤科,湖南郴州423000

出　　处：《现代检验医学杂志》2022年第3期62-68,共7页Journal of Modern Laboratory Medicine

基　　金：湖南省科技计划一般项目(2019SK3151)。

摘　　要：目的探讨胞质分裂作用因子1(dedicator of cytokinesis 1,DOCK1)在多发性骨髓瘤(multiple myeloma,MM)中的表达及其对癌细胞增殖、凋亡和自噬的影响及相关机制。方法选取MM患者骨髓组织及人MM细胞株U266和RPMI 8266进行研究,采用实时荧光定量PCR(real time-quantitative PCR,RT-qPCR)法检测MM骨髓组织及细胞中DOCK1相对表达;构建敲低DOCK1表达细胞系,采用CCK-8法和流式细胞术检测DOCK1对MM细胞增殖与凋亡的影响;Western blot实验检测细胞增殖相关蛋白(c-Myc,cyclin D1)、凋亡相关蛋白(Bax,Bcl-2)、自噬相关蛋白(LC3-II/LC3-I,P62)及AMPK/mTOR通路相关蛋白的表达水平。结果LV-DOCK1-shRNA2组的U266和RPMI 8266细胞增殖率在24,48和72h均显著低于空白对照组和阴性对照组,差异均有统计学意义(F_(U266)=21.130~100.108,F_(RPMI8266)=35.067~95.677,均P<0.001)。LV-DOCK1-shRNA2组的U266和RPMI 8266细胞增殖相关蛋白c-Myc,cyclin D1表达水平均显著低于阴性对照组和空白对照组(F_(U266)=56.061,71.584;F_(RPMI8266)=93.248,62.146,均P<0.001)。LV-DOCK1-shRNA2组的U266,RPMI 8266细胞24h,48h凋亡率显著高于阴性对照组和空白对照组(F_(U266)=77.051,61.533;F_(RPMI8266)=60.868,68.306,均P<0.001)。与空白对照组和阴性对照组比较,LV-DOCK1-shRNA2组U266和RPMI 8266细胞凋亡相关蛋白Bax表达水平明显升高,Bcl-2表达水平明显降低(F_(U266)=92.588,21.940;F_(RPMI8266)=82.736,14.511,均P<0.05);自噬相关蛋白LC3-II/LC3-I表达水平明显升高,P62表达水平明显降低(F_(U266)=147.1,26.342;F_(RPMI8266)=173.300,31.477,均P<0.05)。LV-DOCK1-shRNA2组U266,RPMI 8266细胞AMPK/mTOR通路相关蛋白p-AMPK表达水平明显高于阴性对照组和空白对照组(F_(U266)=40.542,F_(RPMI8266)=40.892,均P<0.05);p-mTOR表达水平明显低于阴性对照组和空白对照组(F_(U266)=38.707,F_(RPMI8266)=43.002,均P<0.05)。在LV-DOCK1-shRNA2组再次转染DOCK1过表达质粒使其表达恢复后,p-AMPK和p-mTOR表达水平也被逆转得到恢复�Objective To investigate the expression of dedicator of cytokinesis 1(DOCK1)in multiple myeloma(MM)and its effects on proliferation,apoptosis and autophagy of cancer cells and related mechanisms.Methods Bone marrow tissue of MM patients and human MM cell line U266 and RPMI 8266 were selected for the study.Real time-quantitative PCR(RT-qPCR)was used to detect the relative expression of DOCK1 in MM bone marrow tissues and cells.Knockdown DOCK1 expression cell lines were constructed,and the effects of DOCK1 on MM cell proliferation and apoptosis were detected by CCK-8 method and flow cytometry.Western blot assay was used to detect the expression levels of proliferation-related proteins(C-MYC,cyclin D1),apoptosis-related proteins(Bax,Bcl-2),autophagy related proteins(LC3-II/LC3-I,P62)and AMPK/mTOR pathway related proteins.Results The proliferation rates of U266 and RPMI8266 cells in LV-DOCK1-shRNA2 group were significantly lower than those in blank control group and negative control group at 24,48 and 72h,the differences were statistically significant(F_(U266)=21.130~100.108,F_(RPMI8266)=35.067~95.677,all P<0.001).The expression levels of proliferation-related proteins C-myc and Cyclin D1 in LV-DOCK1-shRNA2 group were significantly lower than those in negative control group and blank control group(F_(U266)=56.061,71.584,F_(RPMI8266)=93.248,62.146,all P<0.001).Apoptosis rates of U266 and RPMI8266 cells in LV-DOCK1-shRNA2 group were significantly higher than those in negative control group and blank control group at 24h and 48h(F_(U266)=77.051,61.533,F_(RPMI8266)=60.868,68.306,all P<0.001).Compared with blank control group and negative control group,the expression level of apoptosis-related protein Bax in U266 and RPMI 8266 cells in LV-DOCK1-shRNA2 group was significantly increased,and the expression level of Bcl-2 was significantly decreased(F_(U266)=92.588,21.940,respectively.F_(RPMI8266)=82.736,14.511,all P<0.05).The expression level of autophagy related protein LC3-II/LC3-I was significantly increased,and the expre

关 键 词：多发性骨髓瘤 胞质分裂作用因子 AMPK/mTOR通路 增殖 凋亡 自噬 

分 类 号：R733.3[医药卫生—肿瘤] R730.43[医药卫生—临床医学]