[1]赵亮,高蕊,刘菁,等.异位表达FOXO4对胃癌细胞凋亡的影响[J].第三军医大学学报,2015,37(18):1830-1836.
 Zhao Liang,Gao Rui,Liu Jing,et al.Effects of ectopic expression of Forkhead box O4 on cell apoptosis in human gastric cancer cells[J].J Third Mil Med Univ,2015,37(18):1830-1836.
点击复制

异位表达FOXO4对胃癌细胞凋亡的影响(/HTML )
分享到:

《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
37卷
期数:
2015年第18期
页码:
1830-1836
栏目:
论著
出版日期:
2015-09-30

文章信息/Info

Title:
Effects of ectopic expression of Forkhead box O4 on cell apoptosis in human gastric cancer cells
作者:
赵亮高蕊刘菁黄慧哲
重庆医科大学基础医学院:组织胚胎学教研室,发育生物学研究室,生物化学与分子生物学教研室
Author(s):
Zhao Liang Gao Rui Liu Jing Huang Huizhe

Department of Histology and Embryology, Department of Developmental Biology, Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Chongqing Medical University, Chongqing, 400016, China

关键词:
FOXO4基因过表达RNA干扰细胞凋亡胃癌细胞
Keywords:
Forkhead box O4 gene over-expression RNA interfering cell apoptosis gastric cancer cells
分类号:
R394-33;R730.23;R735.2
文献标志码:
A
摘要:

目的      应用pCMV5-Flag和pLL3.7真核表达载体构建Forkhead box O4 (FOXO4)基因的过表达质粒和干扰RNA表达质粒,研究其对胃癌细胞凋亡的影响。      方法      将FOXO4基因的开放阅读框克隆入pCMV5-Flag载体,将靶向FOXO4基因的siRNA克隆入pLL3.7载体,获得FOXO4的表达质粒Flag-FOXO4和干扰质粒FOXO4-shRNA。将获得的质粒及对照质粒通过脂质体转染MKN45和SGC7901胃癌细胞,Western blot检测其对细胞中FOXO4蛋白的表达和沉默效率。FCM检测其对细胞凋亡的影响,Western blot检测Cleaved Caspase-3的表达以及应用Real-time PCR检测其对促凋亡转录因子BCL-6的表达的变化。      结果      成功构建FOXO4的表达质粒Flag-FOXO4和干扰质粒FOXO4-shRNA。Western blot结果表明,Flag-FOXO4可以明显增加细胞FOXO4的表达,FOXO4-shRNA明显抑制细胞FOXO4的表达。FCM结果显示,Flag-FOXO4组与Control组相比较,MKN45细胞和SGC7901细胞凋亡显著增加了16.54%(P<0.001)和26.23%(P<0.001)。Western blot结果显示Cleaved Caspase-3表达与FCM结果一致。Real-time PCR结果表明Flag-FOXO4组细胞FOXO4下游促凋亡基因BCL-6的表达显著增加(P<0.01),FOXO4-shRNA明显抑制了细胞BCL-6的表达(P<0.01)。      结论      FOXO4的过表达可能通过增加FOXO4下游促凋亡基因BCL-6的表达促进了胃癌细胞的凋亡。FOXO4的沉默降低了BCL-6的表达。

Abstract:

Objective      To construct the expression vector and the small interfering RNA containing human Forkhead box O4 gene (FOXO4) by using the pCMV5-Flag and pLL3.7 eukaryon expression vector, and determine the effects on the apoptosis of gastric cancer cells after transfected with the vectors.        Methods      The open reading frame sequence of FOXO4 gene was cloned into the plasmid pCMV5-Flag, and the siRNA sequence targeting FOXO4 gene was cloned into the plasmid pLL3.7 in order to generate the expression plasmid Flag-FOXO4 and interference plasmid FOXO4-shRNA. The plasmids and control of the plasmids were transfected into the gastric cancer MKN45 and SGC7901 cells respectively by lipofectamine-mediated transfection. The expression and silencing efficiency were determined by Western blotting. Cell apoptosis were detected by flow cytometry, and the protein levels of cleaved Caspase-3 was detected by Western blotting. Real-time PCR was employed to measure the mRNA expression of apoptosis promoting transcription factor BCL-6 (a downstream gene of FOXO4 gene) in the gastric cancer cells after transfection with the vectors.       Results      The expression plasmid (Flag-FOXO4) and interference plasmid (FOXO4-shRNA) were successfully constructed respectively. The protein level of FOXO4 was significantly increased in the gastric cancer MKN45 and SGC7901 cells after plasmid Flag-FOXO4 transfection, and was obviously decreased in the cells transfected with FOXO4-shRNA vector. Flow cytometry showed that the Flag-FOXO4 transfection enhanced the apoptotic rates of the MKN45 cells and SGC7901 cells by 16.54% (P<0.01) and 26.23% (P<0.01) respectively when compared with the cells without transfection. Western blot result showed the expression level of cleaved Caspase-3 was also increased. Real-time PCR showed that ,the mRNA expression level of BCL-6 was increased in the gastric cancer cells with Flag-FOXO4 transfection than the cells without (P<0.01), and it was decreased in the cells with FOXO4-shRNA transfection (P<0.01).       Conclusion      Over-expression of FOXO4 enhances the apoptosis in gastric cancer cells through up-regulating the expression of its downstream pro-apopotic gene BCL-6, while, silencing of FOXO4 decreases the expression of BCL-6.

参考文献/References:

[1]Leja M, You W, Camargo M C, et al. Implementation of gastric cancer screening - the global experience[J]. Best Pract Res Clin Gastroenterol, 2014, 28(6): 1093-1106.
[2]Herrero R, Park J Y, Forman D. The fight against gastric cancer - the IARC Working Group report[J]. Best Pract Res Clin Gastroenterol, 2014, 28(6): 1107-1114.
[3]Zhang Y, Gan B, Liu D, et al. FoxO family members in cancer[J]. Cancer Biol Ther, 2011, 12(4): 253-259.
[4]张超, 王德荣, 曲飞, 等. 转录因子FOXO4与胃癌及Hp感染关系的临床研究[J]. 中国现代普通外科进展, 2013, 16(3): 210-213.
[5]Su L, Liu X, Chai N, et al. The transcription factor FOXO4 is down-regulated and inhibits tumor proliferation and metastasis in gastric cancer[J]. BMC Cancer, 2014, 14: 378.
[6]Srisuttee R, Koh S S, Park E H, et al. Up-regulation of Foxo4 mediated by hepatitis B virus X protein confers resistance to oxidative stress-induced cell death[J]. Int J Mol Med, 2011, 28(2): 255-260.
[7]Maiese K, Chong Z Z, Shang Y C, et al. A "FOXO" in sight: targeting Foxo proteins from conception to cancer[J]. Med Res Rev, 2009, 29(3): 395-418.
[8]Brunet A, Bonni A, Zigmond M J, et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor[J]. Cell, 1999, 96(6): 857-868.
[9]Glauser D A, Schlegel W. The emerging role of FOXO transcription factors in pancreatic beta cells[J]. J Endocrinol, 2007, 193(2): 195-207.
[10]甘立霞. FoxO转录因子在代谢调节及肿瘤抑制中的作用[J]. 第三军医大学学报, 2006, 28(12): 1347-1350.
[11]Kim S Y, Yoon J, Ko Y S, et al. Constitutive phosphorylation of the FOXO1 transcription factor in gastric cancer cells correlates with microvessel area and the expressions of angiogenesis-related molecules[J]. BMC Cancer, 2011, 11: 264.
[12]Yamamura Y, Lee W L, Inoue K, et al. RUNX3 cooperates with FoxO3a to induce apoptosis in gastric cancer cells[J]. J Biol Chem, 2006, 281(8): 5267-5276.
[13]Chen L, Tang Y, Wang J, et al. miR-421 induces cell proliferation and apoptosis resistance in human nasopharyngeal carcinoma via downregulation of FOXO4[J]. Biochem Biophys Res Commun, 2013, 435(4): 745-750.
[14]Chuang P Y, Dai Y, Liu R, et al. Alteration of forkhead box O (foxo4) acetylation mediates apoptosis of podocytes in diabetes mellitus[J]. PLoS One, 2011, 6(8): e23566.
[15]Nakayoshi T, Sasaki K, Kajimoto H, et al. FOXO4-knockdown suppresses oxidative stress-induced apoptosis of early pro-angiogenic cells and augments their neovascularization capacities in ischemic limbs[J]. PLoS One, 2014, 9(3): e92626.
[16]Tang T T, Dowbenko D, Jackson A, et al. The forkhead transcription factor AFX activates apoptosis by induction of the BCL-6 transcriptional repressor[J]. J Biol Chem, 2002, 277(16): 14255-14265.
[17]Ruggieri S, Tamma R, Marzullo A, et al. Translocation of the proto-oncogene Bcl-6 in human glioblastoma multiforme[J]. Cancer Lett, 2014, 353(1): 41-51.
[18]Zhang X, Tang N, Hadden T J, et al. Akt, FoxO and regulation of apoptosis[J]. Biochim Biophys Acta, 2011, 1813(11): 1978-1986.
[19]Sun Y, Tian H, Wang L, et al. The effects of silencing of PI3K p85alpha on 5-FU-induced colorectal cancer cells apoptosis[J]. Med Oncol, 2013, 30(4): 704.
[20]Tzivion G, Dobson M, Ramakrishnan G. FoxO transcription factors; Regulation by AKT and 14-3-3 proteins[J]. Biochim Biophys Acta, 2011, 1813(11): 1938-1945.
[21]Chiang C, Ihrie R A. Controlling COR competence: BCL-6 regulates neurogenesis and tumor suppression[J]. Cancer Cell, 2014, 26(6): 773-774.
[22]Lee E J, Kim J M, Lee M K, et al. Splice variants of the forkhead box protein AFX exhibit dominant negative activity and inhibit AFXalpha-mediated tumor cell apoptosis[J]. PLoS One, 2008, 3(7): e2743.

相似文献/References:

[1]周恒宇,邓华聪,郑宏庭,等.磷脂酶Cβ1过表达对葡萄糖刺激胰岛素分泌的影响[J].第三军医大学学报,2009,31(15):1471.
 ZHOU Heng-yu,DENG Hua-cong,ZHENG Hong-ting,et al.Overexpression of phospholipase cβ1 improves glucose-stimulated insulin secretion in INS cells[J].J Third Mil Med Univ,2009,31(18):1471.
[2]杨春秀,陈建斌,张树君,等.Notch1过表达对K562细胞增殖及其周期的影响[J].第三军医大学学报,2011,33(06):574.
 Yang Chunxiu,Chen Jianbin,Zhang Shujun,et al.Exogenous Notch1 overexpression inhibits proliferation of K562 cells by arresting at G1 phase[J].J Third Mil Med Univ,2011,33(18):574.
[3]李林峪,张园园,舒茂琴.Geminin基因过表达对VSMCs表型转化的影响[J].第三军医大学学报,2012,34(05):378.
 Li Linyu,Zhang Yuanyuan,Shu Maoqin.Geminin over-expression promotes differentiation in rat vascular smooth muscle cells from dedifferentiation[J].J Third Mil Med Univ,2012,34(18):378.
[4]尹晓玲,陈应果,张建红,等.人IL-12对结肠癌干细胞生物学特性的影响及其机制[J].第三军医大学学报,2013,35(12):1215.
 Yin Xiaoling,Chen Yingguo,Zhang Jianhong,et al.Effect and mechanism of human interleukin-12 on biological property of colon cancer stem cells[J].J Third Mil Med Univ,2013,35(18):1215.
[5]李海玉,宋方洲,卜友泉,等.过表达Bmi-1基因对乳腺癌细胞株BT474侵袭转移能力的影响[J].第三军医大学学报,2014,36(21):2187.
 Li Haiyu,Song Fangzhou,Bu Youquan,et al.Effects of over-expression of Bmi-1 on invasion and metastasis in human mammary carcinoma cell line BT474[J].J Third Mil Med Univ,2014,36(18):2187.
[6]杨佳佳,刘苹,徐倩,等.过表达KLF4对小鼠成骨细胞成骨分化的影响[J].第三军医大学学报,2017,39(07):628.
 Yang Jiajia,Liu Ping,Xu Qian,et al.Effects of adenovirusmediated KLF4 overexpression on osteogenic differentiation of osteoblasts in vitro[J].J Third Mil Med Univ,2017,39(18):628.
[7]先艺,李冲,黎刚.睾丸间质细胞Prl3c1过表达转基因小鼠建立与生物学功能初步研究[J].第三军医大学学报,2017,39(15):1556.
 XIAN Yi,LI Chong,LI Gang.Establishment and biological functions of a transgenic mouse with Prl3c1 over-expression in testicular Leydig cells[J].J Third Mil Med Univ,2017,39(18):1556.
[8]常海平,宋淑芳,郑健.过表达TPX2对人宫颈癌HeLa细胞侵袭和凋亡的影响[J].第三军医大学学报,2018,40(03):216.
 CHANG Haiping,SONG Shufang,ZHENG.Effect of overexpression of TPX2 gene on apoptosis and invasion in human cervical cancer HeLa cells[J].J Third Mil Med Univ,2018,40(18):216.
[9]伏旭,李培武,马莉,等.过表达Nrf2基因对急性胰腺炎大鼠的保护机制研究[J].第三军医大学学报,2019,41(16):1552.
 Protective effect of nuclear factor E-related factor overexpression against acute pancreatitis in rats.Protective effect of nuclear factor E2-related factor 2 overexpression against acute pancreatitis in rats [J].J Third Mil Med Univ,2019,41(18):1552.

更新日期/Last Update: 2015-09-07