SIRT2沉默对胃癌SGC-7901细胞增殖、迁移和侵袭的影响
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摘要
目的探究沉默信息调节蛋白2(SIRT2)对人胃癌SGC-7901细胞增殖、迁移和侵袭的影响及机制。方法采用实时荧光定量PCR(q RT-PCR)检测SIRT2在胃癌SGC-7901细胞和正常胃上皮GES-1细胞中的表达。采用RNA干扰技术处理SGC-7901细胞,SIRT2沉默组转染靶向SIRT2的干扰小RNA(SIRT2-siRNA1组、SIRT2-siRNA2组),阴性对照组转染SIRT2-si RNA阴性对照序列,空白对照组不转染。CKK-8法和平板细胞克隆形成实验检测SGC-7901细胞增殖, Transwell法检测SGC-7901细胞侵袭迁移能力,蛋白印迹检测SGC-7901细胞中SIRT2、丝氨酸/苏氨酸蛋白激酶(Akt)和磷脂酰肌醇-3激酶(PI3K)蛋白表达。结果人胃癌细胞SNU-1、KATO III、SGC-7901中SIRT2m RNA和蛋白水平均高于正常胃上皮GES-1细胞(P<0.05)。转染SIRT2-si RNA的SGC-7901细胞中SIRT2m RNA和蛋白水平均低于阴性及空白对照组(P<0.05);转染SIRT2-si RNA后,SGC-7901细胞增殖能力、克隆形成能力、迁移和侵袭能力均降低(P<0.05);转染SIRT2-siRNA后SGC-7901细胞中Akt、PI3K蛋白磷酸化程度显著下降(P <0.05)。结论 SIRT2在胃癌细胞中表达上调,沉默SIRT2可能通过调节PI3K/Akt通路抑制胃癌细胞增殖、迁移和侵袭,推测SIRT2可能作为潜在靶标应用于胃癌的基因治疗。
Objective To investigate the influences and mechanisms of silencing sirtuin-2(SIRT2) on proliferation, migration and invasion of human gastric cancer SGC-7901 cells. Methods The expressions of SIRT2 in gastric cancer SGC-7901 cells and normal gastric epithelial GES-1 cells was detected by real-time fluorescence quantitative PCR(qRT-PCR). The SGC-7901 cells were treated with RNA interference technique, the SIRT2 silence group was transfected with interfering small RNAs targeting SIRT2(group SIRT2-siRNA1, group SIRT2-siRNA2), the negative control group was transfected with SIRT2-siRNA negative control sequence, blank control group was not transfected. CKK-8 assays and panel cell colony formation experiments were used to detect the proliferation of SGC-7901 cells. Transwell assay was applied to detect the invasion and migration abilities of SGC-7901 cells. The expressions of SIRT2, serine/threonine rotein kinase(Akt) and phosphatidylinositol-3 kinase(PI3K) in SGC-7901 cells were detected by Western blot. Results SIRT2 mRNA and protein levels in human gastric cancer cells SNU-1, KATO III and SGC-7901 were higher than those in normal gastric epithelial GES-1 cells(P < 0.05). The levels of SIRT2 m RNA and protein in SGC-7901 cells transfected with SIRT2-siRNA were lower than those in negative and blank control groups(P < 0.05); after SIRT2-siRNA transfection, the proliferation, clonality, migration and invasion of SGC-7901 cells were decreased(P < 0.05); after transfection of SIRT2-siRNA, the phosphorylation of Akt and PI3K proteins in SGC-7901 cells were decreased significantly(P < 0.05). Conclusion SIRT2 is up-regulated in gastric cancer cells and SIRT2 silence may inhibit proliferation, migration and invasion of gastric cancer cells by regulating PI3K/Akt pathway, on which it is speculated that SIRT2 may be used as a potential target for gene therapy of gastric cancer.
Objective To investigate the influences and mechanisms of silencing sirtuin-2(SIRT2) on proliferation, migration and invasion of human gastric cancer SGC-7901 cells. Methods The expressions of SIRT2 in gastric cancer SGC-7901 cells and normal gastric epithelial GES-1 cells was detected by real-time fluorescence quantitative PCR(qRT-PCR). The SGC-7901 cells were treated with RNA interference technique, the SIRT2 silence group was transfected with interfering small RNAs targeting SIRT2(group SIRT2-siRNA1, group SIRT2-siRNA2), the negative control group was transfected with SIRT2-siRNA negative control sequence, blank control group was not transfected. CKK-8 assays and panel cell colony formation experiments were used to detect the proliferation of SGC-7901 cells. Transwell assay was applied to detect the invasion and migration abilities of SGC-7901 cells. The expressions of SIRT2, serine/threonine rotein kinase(Akt) and phosphatidylinositol-3 kinase(PI3K) in SGC-7901 cells were detected by Western blot. Results SIRT2 mRNA and protein levels in human gastric cancer cells SNU-1, KATO III and SGC-7901 were higher than those in normal gastric epithelial GES-1 cells(P < 0.05). The levels of SIRT2 m RNA and protein in SGC-7901 cells transfected with SIRT2-siRNA were lower than those in negative and blank control groups(P < 0.05); after SIRT2-siRNA transfection, the proliferation, clonality, migration and invasion of SGC-7901 cells were decreased(P < 0.05); after transfection of SIRT2-siRNA, the phosphorylation of Akt and PI3K proteins in SGC-7901 cells were decreased significantly(P < 0.05). Conclusion SIRT2 is up-regulated in gastric cancer cells and SIRT2 silence may inhibit proliferation, migration and invasion of gastric cancer cells by regulating PI3K/Akt pathway, on which it is speculated that SIRT2 may be used as a potential target for gene therapy of gastric cancer.
引文
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[13]Li Y,Zhang M,Dorfman RG,et al.SIRT2 promotes the migration and invasion of gastric cancer through RAS/ERK/JNK/MMP-9 pathway by increasing PEPCK1-related metabolism.Neoplasia,2018,20(7):745-756.
[14]Pavlova NN,Thompson CB.The emerging hallmarks of cancer metabolism.Cell Metab,2016,23(1):27-47.
[15]Ran LK,Song CL,Li WW,et al.Effect of SIRT2 silencing on proliferation of hepatocellular carcinoma cells.J Third Milit Med Univ,2014,36(20):2098-2102.(in Chinese)冉龙宽,宋春丽,李宛蔚,等.SIRT2基因沉默后对肝癌细胞增殖的影响.第三军医大学学报,2014,36(20):2098-2102.
[16]Martini M,De Santis MC,Braccini L,et al.PI3K/AKT signaling pathway and cancer:an updated review.Ann Med,2014,46(6):372-383.
[17]Polivka JJ,Janku F.Molecular targets for cancer therapy in the PI3K/AKT/mTOR pathway.Pharmacol Ther,2014,142(2):164-175.
[18]Yang N,Hui L,Wang Y,et al.SOX2 promotes the migration and invasion of laryngeal cancer cells by induction of MMP-2 via the PI3K/Akt/mTOR pathway.Oncol Rep,2014,31(6):2651-2659.
[19]Yan LX,Liu YH,Xiang JW,et al.PIK3R1 targeting by miR-21suppresses tumor cell migration and invasion by reducing PI3K/AKTsignaling and reversing EMT,and predicts clinical outcome of breast cancer.Int J Oncol,2016,48(2):471-484.
[20]Ramakrishnan G,Davaakhuu G,Kaplun L,et al.Sirt2 deacetylase is a novel AKT binding partner critical for AKT activation by insulin.J Biol Chem,2014,289(9):6054-6066.
[2]Yoon H,Kim N.Diagnosis and management of high risk group for gastric cancer.Gut Liver,2015,9(1):5-17.
[3]Zhu T,Wang Q,Wu XR,et al.Long-term efficacy and prognostic factors of surgical treatment for gastric carcinoma with liver metastases:a Meta analysis.Chin J Dig Surg,2016,15(3):257-265.(in Chinese)朱婷,王琦,武希润,等.胃癌肝转移手术治疗远期疗效及预后因素的Meta分析.中华消化外科杂志,2016,15(3):257-265.
[4]Xu Y,Li F,Lv L,et al.Oxidative stress activates SIRT2 to deacetylate and stimulate phosphoglycerate mutase.Cancer Res,2014,74(13):3630-3642.
[5]Narayan N,Lee IH,Borenstein R,et al.Retraction:the NAD-dependent deacetylase SIRT2 is required for programmed necrosis.Nature,2014,506(7489):516.
[6]Hoffmann G,Breitenbücher F,Schuler M,et al.A Novel Sirtuin 2(SIRT2)inhibitor with p53-dependent pro-apoptotic activity in non-small cell lung cancer.J Biol Chem,2014,289(8):5208-5216.
[7]Quan FT,Yang WZ.The value of lymph node metastasis in evaluating the prognosis of patients with gastric cancer.Chin J Clin Res,2017,30(6):804-806.(in Chinese)权峰涛,杨维桢.淋巴结转移率在评估胃癌患者预后中的价值.中国临床研究,2017,30(6):804-806.
[8]Chen L,Yan Y,Zhu L,et al.Systemic immune-inflammation index as a useful prognostic indicator predicts survival in patients with advanced gastric cancer treated with neoadjuvant chemotherapy.Cancer Manag Res,2017,9(1):849-867.
[9]Kozako T,Suzuki T,Yoshimitsu M,et al.Anticancer agents targeted to sirtuins.Molecules,2014,19(12):20295-20313.
[10]Poulose N,Raju R.Sirtuin regulation in aging and injury.Biochim Biophys Acta,2015,1852(11):2442-2455.
[11]Cheng F,Li S,Chao Y,et al.SIRT1 promotes epithelial-mesenchymal transition and metastasis in colorectal cancer by regulating Fra-1expression.Cancer Lett,2016,375(2):274-283.
[12]Grbesa I,Pajares MJ,Martínez-Terroba E,et al.Expression of sirtuin1 and 2 is associated with poor prognosis in non-small cell lung cancer patients.PLoS One,2015,10(4):e0124670.
[13]Li Y,Zhang M,Dorfman RG,et al.SIRT2 promotes the migration and invasion of gastric cancer through RAS/ERK/JNK/MMP-9 pathway by increasing PEPCK1-related metabolism.Neoplasia,2018,20(7):745-756.
[14]Pavlova NN,Thompson CB.The emerging hallmarks of cancer metabolism.Cell Metab,2016,23(1):27-47.
[15]Ran LK,Song CL,Li WW,et al.Effect of SIRT2 silencing on proliferation of hepatocellular carcinoma cells.J Third Milit Med Univ,2014,36(20):2098-2102.(in Chinese)冉龙宽,宋春丽,李宛蔚,等.SIRT2基因沉默后对肝癌细胞增殖的影响.第三军医大学学报,2014,36(20):2098-2102.
[16]Martini M,De Santis MC,Braccini L,et al.PI3K/AKT signaling pathway and cancer:an updated review.Ann Med,2014,46(6):372-383.
[17]Polivka JJ,Janku F.Molecular targets for cancer therapy in the PI3K/AKT/mTOR pathway.Pharmacol Ther,2014,142(2):164-175.
[18]Yang N,Hui L,Wang Y,et al.SOX2 promotes the migration and invasion of laryngeal cancer cells by induction of MMP-2 via the PI3K/Akt/mTOR pathway.Oncol Rep,2014,31(6):2651-2659.
[19]Yan LX,Liu YH,Xiang JW,et al.PIK3R1 targeting by miR-21suppresses tumor cell migration and invasion by reducing PI3K/AKTsignaling and reversing EMT,and predicts clinical outcome of breast cancer.Int J Oncol,2016,48(2):471-484.
[20]Ramakrishnan G,Davaakhuu G,Kaplun L,et al.Sirt2 deacetylase is a novel AKT binding partner critical for AKT activation by insulin.J Biol Chem,2014,289(9):6054-6066.