LncRNA MEG3过表达对胰腺癌细胞PANC1自噬、凋亡及mTOR通路的影响  

Effects of overexpression of LncRNA MEG3 on autophagy,apoptosis and mTOR pathway in pancreatic cancer cells PANC1

作　　者：张丽萍 吴卫春[2] 陈丽娟[3] Zhang Liping;Wu Weichun;Chen Lijuan(Department of Clinical Laboratory,First People’s Hospital of Yuhang,Hangzhou 311100,China;Department of Thoracic Surgery,First People’s Hospital of Yuhang,Hangzhou 311100,China;Traditional Chinese Medicine,First People’s Hospital of Yuhang,Hangzhou 311100,China)

机构地区：[1]杭州市余杭区第一人民医院(浙江大学医学院附属第二医院余杭院区)检验科,311100 [2]杭州市余杭区第一人民医院(浙江大学医学院附属第二医院余杭院区)胸外科,311100 [3]杭州市余杭区第一人民医院(浙江大学医学院附属第二医院余杭院区)中医科,311100

出　　处：《中华内分泌外科杂志》2021年第4期413-418,共6页Chinese Journal of Endocrine Surgery

基　　金：浙江省医药卫生科技计划项目(2017KY565)。

摘　　要：目的本研究旨在探讨长链非编码RNA母系表达基因3(LncRNA MEG3)过表达对胰腺癌细胞(PANC1)自噬、凋亡及哺乳动物雷帕霉素靶蛋白(mTOR)通路的影响。方法构建pCMV-N-Flag-MEG3表达质粒并转染入PANC1细胞。实时荧光定量聚合酶链式反应(qRT-PCR)法检测HPDE6C7(正常胰腺细胞)组、PANC1(空白对照)组、Vector(PANC1细胞转染空载体)组和MEG3(PANC1细胞转染pCMV-N-Flag-MEG3重组质粒)组中LncRNA MEG3表达量;四甲基偶氮唑蓝比色(MTT)法、流式细胞术和单丹磺酰尸胺(MDC)染色法分别检测LncRNA MEG3过表达对胰腺癌细胞PANC1增殖、凋亡和自噬的影响;蛋白免疫印迹(Western blot)法检测LncRNA MEG3过表达对胰腺癌细胞PANC1中抑凋亡因子(Bcl-2)、促凋亡因子(Bax)和自噬因子(Beclin 1)蛋白表达水平及mTOR通路中mTOR、核糖体p70S6激酶蛋白(SK61)和核起始因子4E结合蛋白1(4E-BP1)磷酸化水平的影响。结果与PANC1组和Vector组相比,MEG3组LncRNA MEG3表达水平(0.36±0.08 vs 0.35±0.11 vs 0.69±0.09)、胰腺癌细胞PANC1增殖抑制率(3.35%±0.12%vs 3.23%±0.09%vs 36.77%±0.13%)、自噬率(29.32%±1.03%vs 26.73%±1.32%vs 57.76%±1.09%)、凋亡率(9.85%±1.58%vs 9.73%±1.12%vs 35.89%±1.05%)、Bax(0.26±0.08 vs 0.29±0.05 vs 0.83±0.08)和Beclin 1(0.15±0.06 vs 0.17±0.02 vs 0.61±0.03)表达水平显著升高(均P<0.05),Bcl-2表达水平(0.79±0.12 vs 0.81±0.09 vs 0.30±0.03)及mTOR通路中mTOR(1.08±0.05 vs 1.06±0.08 vs 0.37±0.10)、SK61(1.12±0.06 vs 1.11±0.09 vs 0.41±0.03)和4E-BP1(0.97±0.07 vs 0.95±0.03 vs 0.39±0.05)磷酸化水平显著降低(均P<0.05)。结论LncRNA MEG3过表达能抑制胰腺癌细胞PANC1增殖促进细胞凋亡,促进细胞自噬囊泡的形成,可能与阻滞mTOR通路有关。Objective To investigate the effects of overexpression of long non-coding RNA maternally expressed gene 3(LncRNA MEG3)on autophagy,apoptosis and mammalian rapamycin target protein(mTOR)pathway in pancreatic cancer cells(PANC1).Methods The pCMV-N-Flag-MEG3 expression plasmid was constructed and transfected into PANC1 cells.The expression of LncRNA MEG3 in hpde6c7(normal pancreatic cells)group,PANC1(blank control),Vector(PANC1 cell transfected empty vector)group and MEG3(PANC1 cell transfected with pCMV-N-Flag-MEG3 recombinant plasmid)group was detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR);methyl thiazolyl tetrazolium(MTT),flow cytometry and monodansylcadaverin(MDC)staining were used to detect the effects of overexpression of LncRNA MEG3 on the proliferation,apoptosis and autophagy of PANC1 cells;Western blot was used to detect the effects of overexpression of LncRNA MEG3 on the expression levels of Bcl-2,Bax and Beclin-1 in PANC1 cells,and the phosphorylation levels of mTOR,ribosomal p70S6 kinase protein(SK61)and uclear initiation factor 4E binding protein 1(4E-BP1)in mTOR pathway.Results Compared with those in PANC1 group and Vector group,the expression level of LncRNA MEG3(0.36±0.08 vs 0.35±0.11 vs 0.69±0.09),proliferation inhibition rate(3.35%±0.12 vs 3.23%±0.09 vs 36.77%±0.13),autophagy rate(29.32%±1.03 vs 26.73%±1.32 vs 57.76%±1.09),apoptosis rate(9.85%±1.58 vs 9.73%±1.12 vs 35.89%±1.05),expression levels of Bax(0.26±0.08 vs 0.29±0.05 vs 0.83±0.08)and Beclin 1(0.15±0.06 vs 0.17±0.02 vs 0.61±0.03)of PANC1 cells in MEG3 group were significantly higher(all P<0.05),and the expression level of Bcl-2(0.79±0.12 vs 0.81±0.09 vs 0.30±0.03)and phosphorylation levels of mTOR(1.08±0.05 and 1.06±0.08 vs 0.37±0.10),SK61(1.12±0.06 and 1.11±0.09 vs 0.41±0.03)and 4E-BP1(0.97±0.07 and 0.95±0.03 vs 0.39±0.05)in mTOR pathway were significantly lower(all P<0.05).Conclusion Overexpression of LncRNA MEG3 can inhibit the proliferation of PANC1 cells,promote apoptosis

关 键 词：长链非编码RNA母系表达基因3 胰腺癌细胞 自噬 凋亡 哺乳动物雷帕霉素靶蛋白 

分 类 号：R735.9[医药卫生—肿瘤]