沉默表皮生长因子受体的表达对肺腺癌A549细胞自噬的影响

doi:10.3969/j.issn.1004-616x.2019.05.002

癌变·畸变·突变 ›› 2019, Vol. 31 ›› Issue (5): 347-351,358.doi: 10.3969/j.issn.1004-616x.2019.05.002

沉默表皮生长因子受体的表达对肺腺癌A549细胞自噬的影响

马阳, 吴娟, 纪晓坤, 王蕊, 杜芸, 王珩, 郭晓, 张艳

河北医科大学第四医院癌检中心, 河北石家庄 050011

收稿日期:2019-01-09 修回日期:2019-07-30 出版日期:2019-09-30 发布日期:2019-10-09
通讯作者: 杜芸,E-mail:yydd40@126.com E-mail:yydd40@126.com
作者简介:马阳,E-mail:18931166383@163.com。
基金资助:
河北省医学科学研究重点课题（20110118）

Silencing EGFR expression influenced autophagy activity in A549 cells

MA Yang, WU Juan, JI Xiaokun, WANG Rui, DU Yun, WANG Heng, GUO Xiao, ZHANG Yan

Department of Cancer Detcetion, the Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, Hebei, China

Received:2019-01-09 Revised:2019-07-30 Online:2019-09-30 Published:2019-10-09

摘要/Abstract

摘要： 目的：研究降低表皮生长因子受体（EGFR）的表达对肺腺癌A549细胞自噬活性的影响。方法：A549细胞中加入EGF处理，分为4组，即未处理组和10、25、50 ng/mL EGF处理组，用Western blot方法检测EGFR的蛋白表达；采用转染siRNA降低肺腺癌细胞A549细胞EGFR的表达，将A549细胞分为4组，即未处理组、EGFR siRNA-1组、EGFR siRNA-2组和EGFR siRNA-3组，分别采用Real-time RT-PCR和Western blot方法检测EGFR mRNA和蛋白表达水平来评价3个siRNA的沉默效果；将A549细胞分为转染EGFR siRNA组、转染空载体组、转染试剂组（只加入等量转染试剂）及未处理组，且4组均加入等量的50 ng/mL EGF，用Western blot检测LC3Ⅱ/LC3Ⅰ蛋白表达，观察沉默EGFR表达对自噬活性变化；A549细胞分为转染EGFR siRNA组和转染空载体组，用透射电子显微镜观察自噬小体。结果：转染siRNA后A549细胞EGF蛋白的表达降低，LC3Ⅰ向LC3Ⅱ的转化水平升高（P < 0.05），细胞内出现较多自噬小体，自噬活性明显增加。结论：降低EGFR的表达可以提高肺腺癌A549细胞自噬活性。

关键词: 表皮生长因子受体, 肺腺癌, 自噬, siRNA, LC3-Ⅱ/LC3-Ⅰ

Abstract: OBJECTIVE:The role of EGFR in autophagy activity in lung adenocarcinoma A549 cells was investigated using small interference RNA to silence EGFR expression. METHODS:A549 cells were divided into four groups:untreated,EGFR siRNA-1,EGFR siRNA-2 and EGFR siRNA-3 groups. Real-time RT-PCR and Western blot were used to detect degradation of EGFR after transfection with EGFR siRNA. In addition,A549 cells were treated with different EGF concentrations:0,10,25 and 50 ng/mL. Protein expression of EGFR was detected using Western blot. Furthermore,A549 cells were divided into four groups:EGFR siRNA,negative control siRNA,transfection reagent (only add equivalent transfection reagent) and untreated groups. All four groups also received 50 ng/mL EGF. Western blot was used to detect expression of LC3Ⅱ/LC3Ⅰ which was observed as the effect on silencing EGFR expression on autophagy activity. A549 cells were divided into EGFR siRNA transfection and negative control siRNA groups,and autophagosomes were observed by transmission electron microscope. RESULTS:After treatment of A549 cells with the three different EGFR siRNAs,the mRNA and protein expression of EGFR were significant reduced. Western blot indicates that increased LC3-Ⅱ/LC3-Ⅰ ratios were induced by down-regulation of EGFR. Transmission electron microscopy shows that EGFR siRNA treatment,compared with control siRNA treatment,induced marked formation of autophagsomes. CONCLUSION:Reducing the expression of EGFR increased autophagy activities of lung adenocarcinoma A549 cells.

Key words: EGFR, lung adenocarcinoma, autophagy, siRNA, LC3-Ⅱ/LC3-Ⅰ

中图分类号:

R730.2

马阳, 吴娟, 纪晓坤, 王蕊, 杜芸, 王珩, 郭晓, 张艳. 沉默表皮生长因子受体的表达对肺腺癌A549细胞自噬的影响[J]. 癌变·畸变·突变, 2019, 31(5): 347-351,358.

MA Yang, WU Juan, JI Xiaokun, WANG Rui, DU Yun, WANG Heng, GUO Xiao, ZHANG Yan. Silencing EGFR expression influenced autophagy activity in A549 cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2019, 31(5): 347-351,358.

参考文献

[1] FERLAY J, SOERJOMATARAM I, DIKSHIT R, et al. Cancer incidence and mortality worldwide:Sources, methods and major patterns in GLOBOCAN 2012[J]. Int J Cancer, 2015, 136(5):E359-E386.
[2] ROSKOSKI R J. The ErbB/HER family of protein-tyrosine kinases and cancer[J]. Pharmacol Res, 2014, 79(1):34-74.
[3] LEMMON M A, SCHLESSINGER J. Cell signaling by receptor tyrosine kinases[J]. Cell, 2010, 141(7):1117-1134.
[4] KOROLCHUK V I, SAIKI S, LICHTENBERG M, et al. Lysosomal positioning coordinates cellular nutrient responses[J]. Nat Cell Biol, 2011, 13(4):453-460.
[5] MENDELSOHN J, BASELGA J. Epidermal growth factor receptor targeting in cancer[J]. Semin Oncol, 2006, 33(4):369-385.
[6] READE C A, GANTI A K. EGFR targeted therapy in non-small cell lung cancer:potential role of cetuximab[J]. Biologics, 2009, 3:215-224.
[7] CHENG L, ZHANG S B, ALEXANDER R, et al. The landscape of EGFR pathways and personalized management of non-small-cell lung cancer[J]. Future Oncol, 2011, 7(4):519-541.
[8] ADAMS O, DISLICH B, BEREZOWSKA S, et al. Prognostic relevance of autophagy markers LC3B and p62 in esophageal adenocarcinomas[J]. Oncotarget, 2016, 7(26):39241-39255.
[9] MIZUSHIMA N. Methods for monitoring autophagy[J]. Int J Biochem Cell Biol, 2004, 36(12):2491-2502.
[10] WEI Y J, ZOU Z J, BECKER N, et al. EGFR-mediated Beclin 1 phosphorylation in autophagy suppression, tumor progression, and tumor chemoresistance[J]. Cell, 2013, 154(6):1269-1284.
[11] REN K H,JIN J H,BIAN C J,et al. MR-1 modulates proliferation and migration of human hepatoma HepG2 cells through MLC2/FAK/Akt signaling pathway[J]. J Biol Chem, 2008.
[12] TóTH S, NAGY K, PáLFIA Z, et al. Cellular autophagic capacity changes during azaserine-induced tumour progression in the rat pancreas. Up-regulation in all premalignant stages and down-regulation with loss of cycloheximide sensitivity of segregation along with malignant transformation[J]. Cell Tissue Res, 2002, 309(3):409-416.
[13] STROHECKER A M, GUO J Y, KARSLI-UZUNBAS G, et al. Autophagy sustains mitochondrial glutamine metabolism and growth of BrafV600E-driven lung tumors[J]. Cancer Discov, 2013, 3(11):1272-1285.
[14] MCMANUS M T, SHARP P A. Gene silencing in mammals by small interfering RNAs[J]. Nat Rev Genet, 2002, 3(10):737-747.
[15] ELBASHIR S M, LENDECKEL W, TUSCHL T. RNA interference is mediated by 21-and 22-nucleotide RNAs[J]. Genes Dev, 2001, 15(2):188-200.
[16] LEMMON M A, SCHLESSINGER J. Cell signaling by receptor tyrosine kinases[J]. Cell, 2010, 141(7):1117-1134.
[17] KUMA A, HATANO M, MATSUI M, et al. The role of autophagy during the early neonatal starvation period[J]. Nature, 2004, 432(7020):1032-1036.
[18] KLIONSKY D J. Autophagy:from phenomenology to molecular understanding in less than a decade[J]. Nat Rev Mol Cell Biol, 2007, 8(11):931-937.
[19] He H, Dang Y, Dai F, et al. Post-translational modifications of three members of the human MAP1LC3 family and detection of a novel type of modification for MAP1LC3B[J]. J Biol Chem, 2003, 278(31):29278-29287.
[20] CHEN Y Q, AZAD M B, GIBSON S B. Methods for detecting autophagy and determining autophagy-induced cell death[J]. Can J Physiol Pharmacol, 2010, 88(3):285-295.
[21] AMARAVADI R, KIMMELMAN A C, WHITE E. Recent insights into the function of autophagy in cancer[J]. Genes Dev, 2016, 30(17):1913-1930.
[22] WHITE E. Deconvoluting the context-dependent role for autophagy in cancer[J]. Nat Rev Cancer, 2012, 12(6):401-410.
[23] TOWERS C G, THORBURN A. Therapeutic targeting of autophagy[J]. EBio Medicine, 2016, 14:15-23.
[24] TóTH S, NAGY K, PáLFIA Z, et al. Cellular autophagic capacity changes during azaserine-induced tumour progression in the rat pancreas. Up-regulation in all premalignant stages and down-regulation with loss of cycloheximide sensitivity of segregation along with malignant transformation[J]. Cell Tissue Res, 2002, 309(3):409-416.
[25] STROHECKER A M, GUO J Y, KARSLI-UZUNBAS G, et al. Autophagy sustains mitochondrial glutamine metabolism and growth of BrafV600E-driven lung tumors[J]. Cancer Discov, 2013, 3(11):1272-1285.

沉默表皮生长因子受体的表达对肺腺癌A549细胞自噬的影响

Silencing EGFR expression influenced autophagy activity in A549 cells

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