收藏本站
收藏 | 手机打开
二维码
手机客户端打开本文

The Study on the Effects of FAK Expression to the Behaviors of Human Gastric Carcinoma Cells

杨艳丽  
【摘要】:Gastric cancer is the second most common cause of cancer-related death in the world. It is often asymptomatic or causes only nonspecific symptoms in its early stages. By the time symptoms occur, the cancer has often reached an advanced stage, one of the main reasons for its poor prognosis. Even patients who present in the most favorable condition and undergo curative surgical resection often die of recurrent disease. Currently, surgery is still the main theatment, in which the application of chemotherapy and radiotherapy is also combined. However, these theatments result in serious poisonous side effects and high metastasis and recurrence. With the development of cellular and molecular biology, as a new therapy method, gene therapy has become prominent, and it has achieved considerable progresses on cancer diagnosis, therapy and prognosis. RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing technique, which has a great significance in gene function research, and has received much consideration. Two types of small RNA molecules:microRNA (miRNA) and small interfering RNA (siRNA) are central to RNA interference. Most notably, siRNA is involved in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene. Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase involved in cancer cell survival, proliferation, apoptosis, and metastasis. To discover what roles FAK plays to regulate the malignant behaviors of gastric cancer cells, we studied its effects on gastric cancer proliferation, apoptosis and motility after FAK expression was inhibited in SGC-7901 cells (human gastric carcinoma cells) using RNAi. In addition, we detected Annexin A2 mRNA and protein levels after down-regulation of FAK, and to find if there is a relationship between them. It is concluded that this research can be used as the references for gastric cancer gene therapy research projects. Methods:1. Based on FAK variants mRNA sequences supplied by Genbank database, four siRNAs targeting to FAK variants were synthesized and constructed to recombinant plasmids pU6H1-GFP. Scramble control sequence (SCR) of human genome was also cloned to plasmid pU6H1-GFP.2. Recombinant plasmids pU6Hl-GFP-siFAK and pU6H1-GFP-siRNASCR were introduced into the human gastric cell, SGC-7901, using the optimized method. And six groups (NC, siRNASCR, siFAK-1, siFAK-2, siFAK-3 and siFAK-4) were included.3. FAK mRNA and protein levels were evaluated by semi-quantitative RT-PCR and western blotting at 24 h,48 h,72 h and 96 h post thansfection.4. The cell proliferation and motility were determined with MTT and wound healing assay after FAK down-regulation. Moreover, cell apoptosis at 96 h post transfection was detected by Hoechst 33258 staining and MitoViewTM 633 mitochondrial dye.5. Cancer related gene Annexin A2 mRNA and protein levels were evaluated by semi-quantitative RT-PCR and western blotting at 72 h after down-regulation of FAK. Results:1. The exogenous siRNAs were inserted into plasmid pU6Hl-GFP and siRNAs recombinants were constructed successfully, which were verified through enzyme digestion and gene sequencing.2. Tranfection condition was optimized and high efficiency was obtained. Following parameters were indicated as the optimal transfection conditions:60%-70% cultured cell cover ratio before transfection,8μg plasmid per 30 mm dish,30 mins complex formation time,16-18 h incubation time. 3. FAK mRNA and protein level were detected by semi-quantitative RT-PCR and western blotting. And the results showed, compared to NC group, FAK mRNA and protein expression were significantly down-regulated for every siFAK group at each time point post transfection (p0.05). The mRNA level inhibition was achieved maximal at 72 h post transfection with time dependent. Moreover, siFAK-2 and siFAK-3 decreased more significantly. However, siRNASCR indicated no significance with NC at each time point post transfection (p0.05).4. In MTT, and wound healing assay, tranfected with siFAK groups showed suppressed proliferation and motility rate (p0.05), and the inhibition efficiency was positively correlated with FAK knockdown rate. Evaluated apoptosis ability was observed in FAK silencing group at 96 h post transfection. The proliferation, apoptosis and motility ability of siRNASCR group indicated no significance with NC group (p0.05).5. Annexin A2 mRNA and protein levels were increased after down-regulation of FAK. Conclusion:Using RNAi, knockdown of FAK level in cultured SGC-7901 can suppress its proliferation and motility rate, and increase its apoptosis ability in vitro. However, there was no significant influence was indicated on proliferation, apoptosis and motility for siRNASCR.It is concluded that the expression of FAK in SGC-7901 is closely associated with the cell malignant behaviors, and siRNA can inhibit the FAK expression efficiently in SGC-7901 cells. Therefore, FAK can be used as gene therapy target and plasmid pU6Hl-GFP as safe clinical vector for gastric cancer gene therapy. The results indicate that FAK may serve as a potential therapeutic target for gantric cancer therapy.


知网文化
【相似文献】
中国期刊全文数据库 前19条
1 Jia-Cheng Tang;Jing-Hua Liu;Xiao-Long Liu;Xiao Liang;Xiu-Jun Cai;;Effect of fibulin-5 on adhesion, migration and invasion of hepatocellular carcinoma cells via an integrin-dependent mechanism[J];World Journal of Gastroenterology;2015年39期
2 Xiu-Li Guo;Ya-Jie Wang;Pei-Lin Cui;Yan-Bin Wang;Pi-Xia Liang;Ya-Nan Zhang;You-Qing Xu;;Effect of BRM S1 expression on proliferation, migration and adhesion of mouse forestomach carcinoma[J];Asian Pacific Journal of Tropical Medicine;2015年09期
3 ;Cell surface activation of progelatinase A (proMMP-2) and cell migration[J];Cell Research;1998年03期
4 ;Construction of a eukaryotic expression vector pEGFP-C1-BMP-2 and its effect on cell migration[J];Journal of Zhejiang University-Science B(Biomedicine & Biotechnology);2012年05期
5 Zhao-Dong Du;Li-Ting Hu;Gui-Qiu Zhao;Qian Wang;Qiang Xu;Nan Jiang;Jing Lin;;Protein tyrosine phosphatase 1B regulates migration of ARPE-19 cells through EGFR/ERK signaling pathway[J];International Journal of Ophthalmology;2015年05期
6 Yu-Ping Zheng;Shao-Bo Zhang;Feng Wang;Hui Liu;Wen Zhang;Bin Song;Zi-Yao Liu;Lei Xiong;Ya-Zhi Fan;Ding-Ying Liao;;Effects of lentiviral RNA interference-mediated downregulation of integrin-linked kinase on biological behaviors of human lens epithelial cells[J];International Journal of Ophthalmology;2016年01期
7 Atsushi Shiozaki;Hiroki Shimizu;Daisuke Ichikawa;Hirotaka Konishi;Shuhei Komatsu;Takeshi Kubota;Hitoshi Fujiwara;Kazuma Okamoto;Daisuke Iitaka;Shingo Nakashima;Yoshito Nako;Mingyao Liu;Eigo Otsuji;;Claudin 1 mediates tumor necrosis factor alpha-induced cell migration in human gastric cancer cells[J];World Journal of Gastroenterology;2014年47期
8 Bao-Hua Ji;Bo Huo;;Probing the mechanosensitivity in cell adhesion and migration: Experiments and modeling[J];Acta Mechanica Sinica;2013年04期
9 Cheng Zhang;Ming-Hui Ma;Yu Liang;Kun-Zhe Wu;Dong-Qiu Dai;;Novel long non-coding RNA LINC02532 promotes gastric cancer cell proliferation, migration, and invasion in vitro[J];World Journal of Gastrointestinal Oncology;2019年02期
10 Gaigai Li;Yang Hu;Yanfang Chen;Zhouping Tang;;Strategies to improve the migration of mesenchymal stromal cells in cell therapy[J];Translational Neuroscience and Clinics;2017年03期
11 Ann De Becker;Ivan Van Riet;;Homing and migration of mesenchymal stromal cells: How to improve the efficacy of cell therapy?[J];World Journal of Stem Cells;2016年03期
12 ;CREG regulates vascular endothelial cell migration mediated by ILK-β-parvin signal pathway[J];岭南心血管病杂志;2011年S1期
13 ;Mechanics of mechanosensitivity of cell[J];医用生物力学;2010年S1期
14 Pei-Cai Fu;Rong-Hua Tang;Zhi-Yuan Yu;Min-Jie Xie;Wei Wang;Xiang Luo;;The Rho-associated kinase inhibitors Y27632 and fasudil promote microglial migration in the spinal cord via the ERK signaling pathway[J];Neural Regeneration Research;2018年04期
15 Zhi-ping Qi;Guo-xiang Wang;Peng Xia;Ting-ting Hou;Hong-li Zhou;Tie-jun Wang;Xiao-yu Yang;;Effects of microtubule-associated protein tau expression on neural stem cell migration after spinal cord injury[J];Neural Regeneration Research;2016年02期
16 ;CD151 gene delivery increases eNOS activity and induces ECV304 migration, proliferation and tube formation[J];Acta Pharmacologica Sinica;2007年01期
17 ;Size dependence of grain boundary migration in metals under mechanical loading[J];Science Foundation in China;2019年02期
18 Louis WC Chow;Ka-Shun Cheng;Kar-Lok Wong;Yuk-Man Leung;;Voltage-gated K~+ channels promote BT-474 breast cancer cell migration[J];Chinese Journal of Cancer Research;2018年06期
19 钟睿;侯氢;马超琼;付宝勤;汪俊;;Temperature dependence of migration features of self-interstitials in zirconium[J];Chinese Physics B;2017年12期
中国重要会议论文全文数据库 前10条
1 赵莹;魏旭格;李培;张晨旭;崔颖;秦元华;金敏丽;高颖;;Recombinant heat shock protein 60 stimulates the migration of vascular smooth muscle cells via the Toll-like receptor 4 and ERK MAPK activation[A];中国生物化学与分子生物学会第十一次会员代表大会暨2014年全国学术会议论文集——专题报告七[C];2014年
2 Li Cai-hong;Xu Peng;Cheng Dong-kai;Sun Xiao-ling;Yu Hong-jun;Liu Ji;Li Chun-yi;Li Bao-shan;;EGF activation of p21 activated kinase-1(Pak1) is critical for human amnion mesenchymal stem cell migration in vitro[A];中国生理学会生殖科学专业委员会—中国动物学会生殖生物学分会第二次联合学术年会暨“生殖科学专业委员会第二次学术交流会”和“生殖生物学分会第十六次学术年会”论文集[C];2017年
3 Yuliang Zhou;Shenghan Zhou;Silin Qian;Wenbing Chang;;The study on optimization model of emergency resource migration cost in Multi-cycle[A];第30届中国控制与决策会议论文集(2)[C];2018年
4 Feng Yang;Hong Liang;;In vivo epidermal migration requires focal adhesion targeting of ACF7[A];中国晶体学会第六届学术年会暨会员代表大会(大分子晶体学分会)论文摘要集[C];2016年
5 Chen Jiong;LI Guohong;Zhang Wei;Li Lingling;Huang Yingxue;Zhao Shanting;Yin Yupeng;Chen Shulin;;Macrophage migration inhibitory factor antagonist,ISO-1,lead to cell cycle arrest in G2/M and facilitate migration in CHO cells[A];中国畜牧兽医学会动物解剖及组织胚胎学分会第十九次学术研讨会论文集[C];2016年
6 Xu Bo Duan;Lu Ye Qin;Kai Gao;Jiang Shan Zhan;Albert Cheung Hoi Yu;Lina Li;;Macropinocytosis speeds up cell migration[A];中国神经科学学会第十二届全国学术会议论文集[C];2017年
7 Qingru Yun;Yingchao Zhang;Shuang Wu;Alatan Gaole;;LPA promotes migration and invasion of human ovarian cancer cells through LPA receptors[A];第五届泛环渤海生物化学与分子生物学会学术交流会论文集[C];2015年
8 Mi Jun;Zou Yongxin;Lu Juanjuan;Lin Xiaohua;liu Xiaochen;Zhao Hui;Ye Xiang;Hu Huili;Jiang Baichun;Han Bo;Shao Changshun;Gong Yaoqin;;CUL4B promote proliferation and migration of non-small-ce ll lung cells through epigenetically repression of miR-194[A];中华医学会第十五次全国医学遗传学学术会议暨中国医师协会医学遗传医师分会第一届全国学术会议暨2016年浙江省医学遗传学年会论文汇编[C];2016年
9 Weiwei Liu;Fusheng Wan;;Overexpression of miRNA-206 activates apoptosis,induces cell cycle arrest and inhibits migration of human hepatocellular carcinoma HepG2 cells[A];第八届全国医学生物化学与分子生物学第五届全国临床应用生物化学与分子生物学2013华东六省一市生物化学与分子生物学联合学术研讨会论文汇编[C];2013年
10 Wu Shuang;Wang Yue-wu;Shadman Khan;Alatangaole;;Involvement of LPA receptors in LPA-induced gastric cancercell migration[A];2017第七届泛环渤海生物化学与分子生物学会学术交流会论文集[C];2017年
中国博士学位论文全文数据库 前10条
1 周陆扬;[D];中国海洋大学;2009年
2 高蕊(SIHAM GOURIDA);发展中国家的城乡劳动力流动—对中国的研究并与阿拉伯国家的比较[D];南昌大学;2015年
3 徐慧;人口流动与流出地的互动研究[D];华东师范大学;2012年
4 张恒;L1-siRNAs在猪早期胚胎中的功能研究及猪早期胚胎单细胞转录组分析[D];东北农业大学;2018年
5 唐勇;[D];广西医科大学;2010年
6 张力军;关于Cofilin的不对称分布和磷酸化调控协同作用促进细胞定向迁移的研究[D];南京大学;2011年
7 杰瑞(RAKOTONIRINA Jeremy Desiré);马达加斯加乡—城迁移的动因与策略研究[D];中国地质大学;2016年
8 刘冰冰;Integrin β1-Rac1对乳腺浸润性微乳头状癌细胞极性倒转的影响[D];天津医科大学;2017年
9 倪永梁;Plectin通过抑制integrin α6β4/FAK/p38MAPK通路减轻阿霉素诱导的足细胞凋亡及F-actin细胞骨架紊乱[D];山东大学;2018年
10 张中玉;β1整合素和CD146在半乳凝集素-3诱导血管内皮细胞迁移中的作用和机制[D];东北师范大学;2018年
中国硕士学位论文全文数据库 前10条
1 杨艳丽;[D];陕西师范大学;2011年
2 Virag Katies;浅析匈牙利难民问题及其对欧匈关系的影响[D];上海外国语大学;2018年
3 Mohammed Salam Mohammed Ali;[D];上海交通大学;2015年
4 Shamshina Olga(莉娅);[D];山东理工大学;2015年
5 安德烈;当代中国城乡劳动力流动的原因及其社会背景因素分析[D];浙江大学;2013年
6 INSENG Souliphone;[D];复旦大学;2010年
7 HONG LIMTONG;[D];南京农业大学;2016年
8 Ammar Mohammed Al_Moalmi;[D];湖南大学;2014年
9 耿丽娟;包容与排斥[D];上海外国语大学;2008年
10 赵伟;路就是全部[D];武汉大学;2005年
中国重要报纸全文数据库 前3条
1 宋燕编译自《WSR-88D多普勒雷达培训教程》,杨福全校对;鸟群?降雨?[N];中国气象报;2002年
2 记者 王俊鸣;多国科学家合作研制抗SARS新药获进展[N];科技日报;2005年
3 余志平;siRNA治疗研究异彩纷呈(上)[N];中国医药报;2007年
 快捷付款方式  订购知网充值卡  订购热线  帮助中心
  • 400-819-9993
  • 010-62982499
  • 010-62783978