8-氯腺苷通过ADAR1调控miR-335-5p抑制乳腺癌细胞的增殖、迁移和侵袭
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摘要
该文探讨了8-氯腺苷(8-chloro-adenosine, 8-Cl-Ado)调控RNA编辑酶1(adenosine deaminases acting on RNA1, ADAR1)对乳腺癌细胞增殖、迁移和侵袭的影响,确定了ADAR1与miR-335-5p表达的相关性。10μmol/L的8-Cl-Ado作用于乳腺癌细胞后(不同时间点),采用CCK-8检测细胞的增殖情况; Transwell检测细胞的迁移和侵袭情况; Western blot检测ADAR1蛋白的表达水平;CCK-8检测ADAR1过表达对8-Cl-Ado抑制乳腺癌细胞增殖的影响; Transwell检测ADAR1过表达对8-Cl-Ado抑制乳腺癌细胞迁移和侵袭的影响; miRNA芯片筛选8-Cl-Ado处理后乳腺癌细胞中上调的miRNA, qRT-PCR验证其与ADAR1的相关性。结果显示, 8-Cl-Ado能抑制乳腺癌细胞的增殖、迁移和侵袭; ADAR1蛋白的表达量随8-Cl-Ado处理时间的增加而逐渐降低; ADAR1的过表达能减弱8-Cl-Ado对乳腺癌细胞增殖、迁移和侵袭的抑制作用; miR-335-5p经8-Cl-Ado处理后表达水平上调,与ADAR1呈负向调控关系。以上结果表明, 8-Cl-Ado下调ADAR1抑制乳腺癌细胞增殖、迁移和侵袭,其作用机制可能与ADAR1下调mi R-335-5p有关。
The aim of this study was to investigate the effects of 8-chloro-adenosine(8-Cl-Ado) on proliferation, migration and invasion of breast cancer cells by regulating adenosine deaminases acting on RNA1(ADAR1),and the correlation of expression between ADAR1 and miR-335-5 p. After the treatment of breast cancer cells with10 μmol/L of 8-Cl-Ado at different time points or overexpression of ADAR1 in the cells for 48 h, proliferation was examined by CCK-8 while migration and invasion were determined by Transwell, and the expression levels of ADAR1 were measured by Western blot. Up-regulated miRNAs were screened by miRNA microarray and confirmed by qRT-PCR. The results showed that 8-Cl-Ado inhibited the proliferation, migration and invasion of breast cancer cells. The protein expression of ADAR1 gradually decreased in a time dependent manner. ADAR1 overexpression reduced the inhibition of proliferation, migration and invasion caused by 8-Cl-Ado. MiR-335-5 p was highly expressed after 8-Cl-Ado treatment, and it was negatively regulated by ADAR1. In summary, 8-Cl-Ado inhibits proliferation, migration and invasion of breast cancer cells through down-regulating ADAR1, and its mechanism may relate to inhibition of miR-335-5 p by ADAR1.
The aim of this study was to investigate the effects of 8-chloro-adenosine(8-Cl-Ado) on proliferation, migration and invasion of breast cancer cells by regulating adenosine deaminases acting on RNA1(ADAR1),and the correlation of expression between ADAR1 and miR-335-5 p. After the treatment of breast cancer cells with10 μmol/L of 8-Cl-Ado at different time points or overexpression of ADAR1 in the cells for 48 h, proliferation was examined by CCK-8 while migration and invasion were determined by Transwell, and the expression levels of ADAR1 were measured by Western blot. Up-regulated miRNAs were screened by miRNA microarray and confirmed by qRT-PCR. The results showed that 8-Cl-Ado inhibited the proliferation, migration and invasion of breast cancer cells. The protein expression of ADAR1 gradually decreased in a time dependent manner. ADAR1 overexpression reduced the inhibition of proliferation, migration and invasion caused by 8-Cl-Ado. MiR-335-5 p was highly expressed after 8-Cl-Ado treatment, and it was negatively regulated by ADAR1. In summary, 8-Cl-Ado inhibits proliferation, migration and invasion of breast cancer cells through down-regulating ADAR1, and its mechanism may relate to inhibition of miR-335-5 p by ADAR1.
引文
1 Dennison JB,Ayres ML,Kaluarachchi K,Plunkett W,Gandhi V.Intracellular succinylation of 8-chloroadenosine and its effect on fumarate levels.J Biol Chem 2010;285(11):8022-30.
2 Stellrecht CM,Chen LS,Ayres ML,Dennison JB,Shentu S,Chen Y,et al.Chlorinated adenosine analogue induces AMPKand autophagy in chronic lymphocytic leukaemia cells during therapy.Br J Haematol 2017;179(2):266-71.
3 Stellrecht CM,Vangapandu HV,Le XF,Mao W,Shentu S.ATPdirected agent,8-chloro-adenosine,induces AMP activated protein kinase activity,leading to autophagic cell death in breast cancer cells.J Hematol Oncol 2014;7(1):23.
4 Han YY,Zhou Z,Cao JX,,Jin YQ,Li SY,Ni JH,et al.E2F1-mediated DNA damage is implicated in 8-Cl-adenosine-induced chromosome missegregation and apoptosis in human lung cancer H1299 cells.Mol Cell Biochem 2013;384(1/2):187-96.
5 Jiao YY,Wang XQ,Lu WL,Yang ZJ,Zhang Q.A novel approach to improve the pharmacokinetic properties of 8-chloro-adenosine by the dual combination of lipophilic derivatisation and liposome formulation.Eur J Pharm Sci 2013;48(1/2):249-58.
6 Duan HY,Cao JX,Qi JJ,Wu GS,Li SY,An GS,et al.E2F1 enhances 8-chloro-adenosine-induced G2/M arrest and apoptosis in A549 and H1299 lung cancer cells.Biochemistry(Mosc)2012;77(3):261-9.
7谢凤,莫小梅,杨生永,李梨.8-氯腺苷调节RNA编辑酶ADAR1对乳腺癌SK-BR-3细胞增殖和迁移的影响.重庆医科大学学报(Xie Feng,Mo Xiaomei,Yang Shengyong,Li Li.Effects of 8-chloroadenosine regulating RNA editing deaminase ADAR1 on proliferation and migration of SK-BR-3 breast cancer cells.Journal of Chongqing Medical University)2017;42(11):1411-6.
8 Dong XQ,Chen G,Cai ZX,Li ZL,Qiu LM,Xu HP,et al.CDK13RNA over-editing mediated by ADAR1 associates with poor prognosis of hepatocellular carcinoma patients.Cell Physiol Biochem 2018;47(6):2602-12.
9 Avesson L,Barry G.The emerging role of RNA and DNA editing in cancer.Biochim Biophys Acta 2014;1845(2):308-16.
10 Qi LH,Chan THM,Tenen DG,Chen LL.RNA editome imbalance in hepatocellular carcinoma.Cancer Res 2014;74(5):1301-6.
11 Steinman RA,Yang Q,Gasparetto M,Robinson LJ,Liu XP,Lenzner DE,et al.Deletion of the RNA-editing enzyme ADAR1causes regression of established chronic myelogenous leukemia in mice.Int J Cancer 2013;132(8):1741-50.
12 Nishikura K.A-to-I editing of coding and non-coding RNAs by ADARs.Nat Rev Mol Cell Biol 2016;17(2):83-96.
13 Tsai HP,Huang SF,Li CF,Chien HT,Chen SC.Differential microRNA expression in breast cancer with different onset age.PLoS One 2018;13(1):e0191195.
14 Yang SY,Deng P,Zhu ZW,Zhu JZ,Wang GL,Zhang LY,et al.Adenosine deaminase acting on RNA 1 limits RIG-I RNA detection and suppresses IFN production responding to viral and endogenous RNAs.J Immunol 2014;193(7):3436-45.
15 Stellrecht CM,Ayres M,Arya R,Gandhi V.A unique RNA-directed nucleoside analog is cytotoxic to breast cancer cells and depletes cyclin E levels.Breast Cancer Res Treat 2010;121(2):355-64.
16 Gandhi V,Chen W,Ayres M,Rhie JK,Madden TL,Newman RA.Plasma and cellular pharmacology of 8-chloro-adenosine in mice and rats.Cancer Chemother Pharmacol 2002;50(2):85-94.
17窦桂芳,楼雅卿,邹安庆.新抗肿瘤化合物8-氯腺苷代谢物在大鼠体内的药代动力学研究.中国新药杂志(Dou Guifang,Lou Yaqing,Zhou Anqing.Pharmacokinetics of 8-chloro-adenosine,a new anticancer compound in rats.Chinese Journal of New Drugs)1999;8(11):738-740.
18杨莉,齐宪荣,石靖,陈文倩,张强.8-氯腺苷长循环脂质体的制备及其在大鼠体内的药代动力学.药学学报(Yang Li,Qi Xianrong,Shi Jing,Chen Wenqian,Zhang Qiang.Preparation of8-chloro-adenosine long circulation liposomes and its pharmacokinetics in rats.Acta Pharmaceutica Sinica)2005;40(4):382-4.
19 Zipeto MA,Court AC,Sadarangani A,Delos Santos NP,Balaian L,Chun HJ,et al.ADAR1 activation drives leukemia stem cell self-renewal by impairing Let-7 biogenesis.Cell Stem Cell 2016;19(2):177-91.
20 Wang YM,Xu XY,Yu SX,Jeong KJ,Zhou ZC,Han L,et al.Systematic characterization of A-to-I RNA editing hotspots in microR-NAs across human cancers.Genome Res 2017;27(7):1112-25.
21 Wang Y,Yang T,Zhang Z,Lu M,Zhao W,Zeng XD,et al.Long non-coding RNA TUG1 promotes migration and invasion by acting as a ceRNA of miR-335-5p in osteosarcoma cells.Cancer Sci 2017;108(5):859-67.
22 Wang F,Li L,Piontek K,Sakaguchi M,Selaru FM.Exosome miR-335 as a novel therapeutic strategy in hepatocellular carcinoma.Hepatology 2018;67(3):940-54.
23 Luo L,Xia L,Zha BS,Zuo CL,Deng DT,Chen MW,et al.miR-335-5p targeting ICAM-1 inhibits invasion and metastasis of thyroid cancer cells.Biomed Pharmacother 2018;106:983-90.
24 Wang H,Zhang MX,Sun GP.Long non-coding RNA NEAT1regulates the proliferation,migration and invasion of gastric cancer cells via targeting miR-335-5p/ROCK1 axis.Pharmazie2018;73(3):150-5.
2 Stellrecht CM,Chen LS,Ayres ML,Dennison JB,Shentu S,Chen Y,et al.Chlorinated adenosine analogue induces AMPKand autophagy in chronic lymphocytic leukaemia cells during therapy.Br J Haematol 2017;179(2):266-71.
3 Stellrecht CM,Vangapandu HV,Le XF,Mao W,Shentu S.ATPdirected agent,8-chloro-adenosine,induces AMP activated protein kinase activity,leading to autophagic cell death in breast cancer cells.J Hematol Oncol 2014;7(1):23.
4 Han YY,Zhou Z,Cao JX,,Jin YQ,Li SY,Ni JH,et al.E2F1-mediated DNA damage is implicated in 8-Cl-adenosine-induced chromosome missegregation and apoptosis in human lung cancer H1299 cells.Mol Cell Biochem 2013;384(1/2):187-96.
5 Jiao YY,Wang XQ,Lu WL,Yang ZJ,Zhang Q.A novel approach to improve the pharmacokinetic properties of 8-chloro-adenosine by the dual combination of lipophilic derivatisation and liposome formulation.Eur J Pharm Sci 2013;48(1/2):249-58.
6 Duan HY,Cao JX,Qi JJ,Wu GS,Li SY,An GS,et al.E2F1 enhances 8-chloro-adenosine-induced G2/M arrest and apoptosis in A549 and H1299 lung cancer cells.Biochemistry(Mosc)2012;77(3):261-9.
7谢凤,莫小梅,杨生永,李梨.8-氯腺苷调节RNA编辑酶ADAR1对乳腺癌SK-BR-3细胞增殖和迁移的影响.重庆医科大学学报(Xie Feng,Mo Xiaomei,Yang Shengyong,Li Li.Effects of 8-chloroadenosine regulating RNA editing deaminase ADAR1 on proliferation and migration of SK-BR-3 breast cancer cells.Journal of Chongqing Medical University)2017;42(11):1411-6.
8 Dong XQ,Chen G,Cai ZX,Li ZL,Qiu LM,Xu HP,et al.CDK13RNA over-editing mediated by ADAR1 associates with poor prognosis of hepatocellular carcinoma patients.Cell Physiol Biochem 2018;47(6):2602-12.
9 Avesson L,Barry G.The emerging role of RNA and DNA editing in cancer.Biochim Biophys Acta 2014;1845(2):308-16.
10 Qi LH,Chan THM,Tenen DG,Chen LL.RNA editome imbalance in hepatocellular carcinoma.Cancer Res 2014;74(5):1301-6.
11 Steinman RA,Yang Q,Gasparetto M,Robinson LJ,Liu XP,Lenzner DE,et al.Deletion of the RNA-editing enzyme ADAR1causes regression of established chronic myelogenous leukemia in mice.Int J Cancer 2013;132(8):1741-50.
12 Nishikura K.A-to-I editing of coding and non-coding RNAs by ADARs.Nat Rev Mol Cell Biol 2016;17(2):83-96.
13 Tsai HP,Huang SF,Li CF,Chien HT,Chen SC.Differential microRNA expression in breast cancer with different onset age.PLoS One 2018;13(1):e0191195.
14 Yang SY,Deng P,Zhu ZW,Zhu JZ,Wang GL,Zhang LY,et al.Adenosine deaminase acting on RNA 1 limits RIG-I RNA detection and suppresses IFN production responding to viral and endogenous RNAs.J Immunol 2014;193(7):3436-45.
15 Stellrecht CM,Ayres M,Arya R,Gandhi V.A unique RNA-directed nucleoside analog is cytotoxic to breast cancer cells and depletes cyclin E levels.Breast Cancer Res Treat 2010;121(2):355-64.
16 Gandhi V,Chen W,Ayres M,Rhie JK,Madden TL,Newman RA.Plasma and cellular pharmacology of 8-chloro-adenosine in mice and rats.Cancer Chemother Pharmacol 2002;50(2):85-94.
17窦桂芳,楼雅卿,邹安庆.新抗肿瘤化合物8-氯腺苷代谢物在大鼠体内的药代动力学研究.中国新药杂志(Dou Guifang,Lou Yaqing,Zhou Anqing.Pharmacokinetics of 8-chloro-adenosine,a new anticancer compound in rats.Chinese Journal of New Drugs)1999;8(11):738-740.
18杨莉,齐宪荣,石靖,陈文倩,张强.8-氯腺苷长循环脂质体的制备及其在大鼠体内的药代动力学.药学学报(Yang Li,Qi Xianrong,Shi Jing,Chen Wenqian,Zhang Qiang.Preparation of8-chloro-adenosine long circulation liposomes and its pharmacokinetics in rats.Acta Pharmaceutica Sinica)2005;40(4):382-4.
19 Zipeto MA,Court AC,Sadarangani A,Delos Santos NP,Balaian L,Chun HJ,et al.ADAR1 activation drives leukemia stem cell self-renewal by impairing Let-7 biogenesis.Cell Stem Cell 2016;19(2):177-91.
20 Wang YM,Xu XY,Yu SX,Jeong KJ,Zhou ZC,Han L,et al.Systematic characterization of A-to-I RNA editing hotspots in microR-NAs across human cancers.Genome Res 2017;27(7):1112-25.
21 Wang Y,Yang T,Zhang Z,Lu M,Zhao W,Zeng XD,et al.Long non-coding RNA TUG1 promotes migration and invasion by acting as a ceRNA of miR-335-5p in osteosarcoma cells.Cancer Sci 2017;108(5):859-67.
22 Wang F,Li L,Piontek K,Sakaguchi M,Selaru FM.Exosome miR-335 as a novel therapeutic strategy in hepatocellular carcinoma.Hepatology 2018;67(3):940-54.
23 Luo L,Xia L,Zha BS,Zuo CL,Deng DT,Chen MW,et al.miR-335-5p targeting ICAM-1 inhibits invasion and metastasis of thyroid cancer cells.Biomed Pharmacother 2018;106:983-90.
24 Wang H,Zhang MX,Sun GP.Long non-coding RNA NEAT1regulates the proliferation,migration and invasion of gastric cancer cells via targeting miR-335-5p/ROCK1 axis.Pharmazie2018;73(3):150-5.