G蛋白偶联胆汁酸受体1促进胃癌细胞的增殖、迁移和侵袭的实验研究

目的:探讨G蛋白偶联胆汁酸受体1(G-protein coupled bile acid receptor 1,GPBAR1/TGR5)对胃癌细胞增殖、迁移和侵袭的影响。方法:免疫组织化学染色方法(Immunohistochemistry,IHC)检测胃癌及癌旁组织芯片中TGR5表达情况;qRT-PCR及Western blot检测胃癌细胞系中TGR5表达水平;小干扰RNA处理AGS、MKN-45胃癌细胞后构建TGR5敲减细胞系,慢病毒载体转染胃癌SGC-7901细胞构建TGR5过表达细胞系;CCK-8实验、平板克隆形成实验、裸鼠皮下移植瘤实验检测TGR5对细胞增殖的影响;流式细胞仪检测TGR5对细胞周期及凋亡的影响;Tanswell实验检测TGR5对胃癌细胞迁移及侵袭的影响;Western blot检测上皮间充质转化(Epithelial-mesenchymal transition,EMT)相关分子β-连环蛋白(β-catenin)、锌脂蛋白转录因子(Snail)、E盒结合锌指蛋白(Zinc finger E-box binding homeobox 1,ZEB)1在AGS、MKN-45及SGC-7901胃癌细胞中的表达。结果:TGR5在胃癌及癌旁组织中均有表达,胃癌组织TGR5高表达率(41.0%)显著高于癌旁组织(9.5%),伴肠化生癌旁组织TGR5高表达率(50%)显著高于不伴肠化生的癌旁组织(0%),胃癌组织TGR5表达与肿瘤大小相关。TGR5在正常人胃上皮永生化细胞株GES-1及各胃癌细胞系中均有表达。TGR5表达敲低的AGS和MKN-45细胞增殖能力减弱、凋亡率显著升高、侵袭和迁移能力显著降低。过表达TGR5的SGC-7901细胞增殖能力增强、克隆形成能力提高、凋亡率明显减低、侵袭和迁移能力显著升高。此外,TGR5过表达显著上调了间质细胞标志物β-catenin、Snail、ZEB1的表达水平。结论:TGR5能够增强胃癌细胞增殖及迁移能力,并抑制细胞凋亡。TGR5可能通过EMT途径介导胃癌细胞转移。     

钙周期素结合蛋白促进胃癌细胞迁移侵袭和MMP-2、MMP-9、p-ERK1/2、p-AKT水平上调

目的探讨钙周期素结合蛋白(calcyclin binding protein/Siah-1-interacting protein, CacyBP/SIP)对胃癌细胞侵袭迁移的影响和潜在机制。方法采用免疫组织化学和Western blot方法检测不同T分期胃癌组织中CacyBP/SIP水平;Western blot检测胃癌细胞中CacyBP/SIP水平;MKN-45细胞转染si-CacyBP/SIP与Ad-CacyBP/SIP后,细胞划痕实验检测细胞迁移情况,Transwell细胞侵袭实验检测细胞侵袭情况,Western blot检测MMP-2、MMP-9和p-ERK1/2、p-AKT水平。结果 CacyBP/SIP在胃癌组织和胃癌细胞中高表达;胃癌组织中CacyBP/SIP表达水平与T分期呈正相关;敲减CacyBP/SIP抑制MKN-45细胞的迁移侵袭能力和MMP-2、MMP-9、p-ERK1/2、p-AKT蛋白表达水平;过表达CacyBP/SIP促进MKN-45细胞迁移侵袭能力和MMP-2、MMP-9、p-ERK1/2、p-AKT蛋白表达水平。结论 CacyBP/SIP对胃癌转移侵袭能力的促进作用可能与其上调MMP-2、MMP-9、p-ERK1/2、p-AKT水平有关。     

二氢杨梅素对人胃癌MKN45细胞迁移和侵袭的影响及其机制*

目的:探讨二氢杨梅素(DHM )对人胃癌MKN45细胞迁移和侵袭的作用及其分子机制。方法:培养人低分化胃癌MKN45细胞,用不同浓度的DHM(0,10,20,30,40,50 μmol/L)分别处理细胞24及48 h,每组实验重复3次,采用CCK8实验检测癌细胞增殖活力;划痕实验检测细胞迁移能力;Transwell小室检测细胞侵袭能力;免疫印迹分析细胞迁移和侵袭相关蛋白表达情况。结果:不同浓度DHM干预可降低MKN45细胞活力。20,30及40 μmol/L的DHM处理48 h可明显抑制细胞的迁移能力(P＜0.01)和侵袭能力(P＜0.05及0.01)。20及30 μmol/L的DHM处理48 h可增加E-cadherin蛋白表达(P＜0.01)、降低Vimentin表达(P＜0.01),从而逆转EMT过程;10,20及30 μmol/L的DHM处理48 h可明显降低pJNK的活性表达水平(P＜0.05及0.01),及MMP-2蛋白表达(P＜ 0.01);JNK通路特异性抑制剂SP600125预处理可明显促进DHM对癌细胞侵袭能力的抑制作用(P＜0.01)及降低MMP-2表达(P＜0.01)。结论:DHM具有抑制人胃癌MKN45细胞的迁移及侵袭的作用,其机制可能与通过JNK通路下调MMP-2蛋白表达水平、逆转上皮间质转化有关。     

MMP2在胃癌侵袭和转移中的作用

目的 探索MMP2在胃癌侵袭和转移中的作用,以期为胃癌转移提供新的预测指标及治疗靶点。方法 选择胃癌细胞系MKN-1转染慢病毒,使MMP2低表达,获得细胞系sh-MMP2,并通过RT-PCR及Western Blot检测转染后MKN-1细胞中MMP2表达情况。进一步通过细胞划痕实验、Transwell、CCK8实验分别检测MMP2低表达后细胞迁移能力、侵袭能力、增殖能力变化。利用裸鼠建立sh-MMP2细胞系异种移植模型及转移模型,观察MMP2低表达后体内肿瘤生长变化及体内转移能力变化。通过RT-PCR及Western Blot检测MMP2对EMT及细胞凋亡的影响。结果 MMP2低表达后胃癌细胞迁移能力(P<0.05)、侵袭能力(P<0.01)、增殖能力(P<0.05)均下降。体内实验结果显示MMP2低表达可显著减缓肿瘤生长及体内转移(P<0.01)。进一步实验表明降低MMP2的表达,可抑制细胞EMT过程并增加细胞凋亡。结论 MMP2低表达可通过抑制EMT过程及增加细胞凋亡,减弱胃肿瘤细胞迁移能力、侵袭能力和增殖能力,抑制胃癌的发展。     

沉默胃泌素基因抑制胃癌细胞的增殖、迁移和侵袭

胃癌细胞分泌的胃泌素与胃癌的发生、发展密切相关.为了探讨胃泌素对胃癌细胞增殖、迁移和侵袭的影响,本文构建靶向胃泌素基因的siRNA表达载体, 转染胃癌细胞AGS, 成功获得沉默胃泌素基因的稳转胃癌细胞株AGS/Gas-siRNA. 用MTT实验、软琼脂集落形成实验、细胞伤愈实验、Transwell实验及ELISA检测沉默胃泌素基因后细胞的增殖、迁移、侵袭及转移相关蛋白基质金属蛋白酶-2（MMP-2）和血管内皮生长因子（VEGF）的含量. 结果显示: 与空载体转染的对照细胞比较, 沉默胃泌素基因的细胞, 其增殖率和克隆形成率显著降低,迁移和侵袭到Transwell下室的细胞数分别降低了31.6 %和34 %. 培养上清液中MMP-2和VEGF含量也低于对照细胞. 结果提示,沉默胃泌素基因的胃癌细胞,通过降低MMP 2和VEGF分泌,抑制了细胞的增殖、迁移和侵袭, 这可能是胃泌素促进胃癌侵袭转移的机制之一.     

DZNep通过上调miR-200c表达延缓MGC-803胃癌细胞侵袭和迁移过程

本文旨在研究组蛋白甲基化修饰调控对胃癌细胞mi R-200c的表达调节以及对癌细胞的侵袭和迁移的作用。组蛋白甲基转移酶抑制剂DZNep(2.5μmol/L)处理MGC-803胃癌细胞系,用实时定量PCR(q RT-PCR)检测细胞mi R-200c的表达变化,用Western blot检测上皮间质转化相关蛋白、EZH2、EED、SUZ12、H3K27me3及MMP9的蛋白表达变化,用细胞划痕实验和Transwell法检测细胞迁移和侵袭。结果显示,与对照(DMSO处理)组相比,DZNep(2.5μmol/L)处理的MGC-803肿瘤细胞mi R-200c基因的表达显著提高,ZEB1、ZEB2、N-cadherin的表达显著下调,E-cadherin的表达上调,EZH2、EED、SUZ12、H3K27me3及MMP9的表达均显著降低,细胞迁移、侵袭能力均减弱。以上结果提示,DZNep通过上调mi R-200c的表达延缓胃癌细胞侵袭、迁移过程,其机制涉及对上皮间质转化相关蛋白和PRC2(polycomb repressive complex 2)的表达调节。     

RARβ在胃癌细胞生长调节中的作用

为探讨 RARβ受体介导全反式视黄酸 ( ATRA)抑制胃癌细胞生长的作用机理 ,用 Northern印迹测定 RARβ m RNA表达水平 ,脂质体介导的转染方法将含有 RARβ基因的表达载体转染MKN- 45细胞并稳定表达 ,MTT和软琼脂集落形成等实验测定细胞生长速率和生长状态 ,氯霉素乙酰转移酶活性 ( CAT)测定视黄酸应答元件βRARE的转录活性以及 AP- 1 ( activator protein- 1 )活性 .RARβ在 ATRA敏感细胞株 MGC80 - 3、BGC- 82 3和 SGC- 790 1中表达 ,而在 ATRA抗性细胞株 MKN- 45中不表达 .当 RARβ基因转染 MKN- 45细胞时 ,细胞变为 ATRA敏感 ,由此导致ATRA抑制 MKN- 45细胞生长和软琼脂集落形成 .ATRA可以加强诱导 MGC80 - 3、BGC- 82 3和SGC- 790 1细胞βRARE的转录活性 ,但对 MKN- 45细胞影响不大 ,不能抑制细胞 AP- 1活性 .当RARβ基因转染 MKN- 45细胞后 ,ATRA则能够诱导细胞 βRARE的转录活性 ,并抑制细胞的 AP-1活性 .RARβ表达与 ATRA抑制胃癌细胞生长密切相关 .ATRA诱导 βRARE转录活性和抑制AP- 1活性可能是其调控胃癌细胞生长的机制之一 .     

LncRNA MIR4435-2HG调控miR-138-5p/HMGA1轴对胃癌细胞的增殖、侵袭和迁移的影响

目的 探讨LncRNA MIR4435-2HG对胃癌细胞增殖、侵袭和迁移的影响及作用机制.方法 培养正常人胃黏膜上皮细胞GES-1和胃癌细胞系AGS、SGC7901、BGC823和BGC803,AGS细胞分为对照组(正常培养细胞)、si-con组(转染乱序无意义阴性序列)、si-MIR4435-2HG组(转染MIR44...     

HIF-2α/Notch3通路介导CoCl_2诱导的乳腺癌MCF-7细胞迁移和侵袭

本研究旨在探究HIF-2α和Notch3在CoCl_2诱导的乳腺癌MCF-7细胞迁移和侵袭中的作用。使用CoCl_2于体外建立化学性低氧模型;采用sh RNA技术沉默MCF-7细胞中HIF-2α和Notch3基因;采用RT-PCR法检测MCF-7细胞中HIF-2α、Notch3和Hey1的m RNA水平;采用Western blot方法检测MCF-7细胞中HIF-2α、Notch3、Hey1、Snail和E-cadherin蛋白的表达水平;采用划痕实验和Transwell实验分别检测CoCl_2诱导的MCF-7细胞迁移和侵袭。结果显示,CoCl_2处理可使MCF细胞中HIF-2α、Notch3、Hey1和Snail蛋白表达水平升高(P0.05),E-cadherin蛋白表达水平下降(P0.05),促进MCF-7细胞迁移和侵袭(P0.05);沉默HIF-2α基因表达可抑制CoCl_2诱导的Notch3和Hey1的m RNA和蛋白表达(P0.05);抑制Notch3基因表达后,CoCl_2诱导的MCF-7细胞迁移和侵袭以及CoCl_2对Snail和E-cadherin蛋白表达的调节随之受到抑制(P0.05)。以上结果表明,在CoCl_2化学性低氧环境下,HIF-2α可通过强化Notch3信号通路,进而导致Snail蛋白水平升高和E-cadherin蛋白水平下降,最终提高乳腺癌MCF-7细胞的迁移和侵袭能力。     

人参皂苷IH901抗肝癌生长及其侵袭转移的作用机理

人参皂苷IH901是一种天然二醇组人参皂苷肠道细菌最终代谢产物.目前研究表明,IH901具有抗肝癌活性,然而其作用机理尚不清楚.采用人肝癌细胞株SMMC-7721,HepG2,MHCC97-H来建立体内外的抗肿瘤模型,以探讨人参皂苷IH901的抗肝癌活性及其作用机理.结果发现,人参皂苷IH901能够通过细胞周期中亚二倍体峰的增加、线粒体跨膜电位（ΔΨm）崩溃、DNA梯形条带的形成和提高Caspase-9/Caspase-3的表达量、促进凋亡来抑制肝癌细胞的生长.通过抑制细胞的迁移和降低VEGF/bFGF的表达来抑制肝癌细胞的侵袭转移.裸鼠体内实验均表明了人参皂苷在体内外的抗肝癌活性,并且在分子、细胞、动物水平初步揭示了IH901抗肝癌生长及其侵袭转移的作用机理,为其作为抗肝癌药物的开发提供理论支持.     

Angiogenic factor thymidine phosphorylase increases cancer cell invasion activity in patients with gastric adenocarcinoma

We investigated the biological role of thymidine phosphorylase (TP), an angiogenic factor, in gastric cancer cell migration and invasion and explored a therapeutic approach for high TP-expressing tumors using TP enzymatic inhibitor (TPI) and rapamycin. We established TP cDNA overexpressing gastric cancer cell lines (MKN-45/TP and YCC-3/TP) and did invasion and adhesion assays with Matrigel-coated transwell membranes. The related signal pathway using recombinant human TP (rhTP), deoxy-d-ribose (D-dRib), and signal pathway inhibitors (wortmannin, LY294002, and rapamycin) was investigated. First, AGS and MKN-1 gastric cancer cell lines showed dose-dependent up-regulation of invasiveness through Matrigel following treatment with rhTP or D-dRib. TP-overexpressing cancer cell lines displayed increased migration and invasion activity, which doubled with rhTP and D-dRib treatment. This activity depended on the enzymatic activity of TP, and TP stimulated the adhesion of cancer cells onto Matrigel and induced actin filament remodeling. Finally, we showed that this activity is related to increased phosphatidylinositol 3-kinase activity in TP-overexpressing cells and that combination treatment with rapamycin and TP enzymatic inhibitor produces an additive effect to abrogate TP-induced invasion. Taken together, TP increases the migration and invasion of gastric cancer cells, especially in TP-expressing cells. Therapies targeting TP might diminish the propensity for invasion and metastasis in gastric cancer.     

lncRNA CEBPA-AS1靶向miR-455-3p调控胃癌细胞生物学行为的分子机制研究

摘要 目的：探讨lncRNA CEBPA-AS1对胃癌细胞生物学行为的影响及其可能作用机制。方法：qRT-PCR法检测胃癌组织、癌旁组织与正常人胃上皮GES1细胞和人胃癌SNU-1、AGS、HS-746T细胞系中lncRNA CEBPA-AS1、miR-455-3p的表达量。si-NC、si-lncRNA CEBPA-AS1、miR-NC、miR-455-3p mimics、si-lncRNA CEBPA-AS1与anti-miR-NC、si-lncRNA CEBPA-AS1与anti-miR-455-3p分别转染至SNU-1细胞（分别命名为si-NC组、si-lncRNA CEBPA-AS1组、miR-NC组、miR-455-3p组、si-lncRNA CEBPA-AS1+anti-miR-NC组和si-lncRNA CEBPA-AS1+anti-miR-455-3p组）后,MTT实验与平板克隆形成实验分别检测细胞增殖及克隆形成能力,Transwell小室实验检测细胞迁移及侵袭能力,双荧光素酶报告基因实验与qRT-PCR实验验证lncRNA CEBPA-AS1与miR-455-3p的靶向调控关系,Western blot法检测MMP2、MMP9蛋白表达情况。结果：与癌旁组织比较,胃癌组织中lncRNA CEBPA-AS1的表达量显著升高,miR-455-3p的表达量显著降低,差异均有统计学意义（均P＜0.05）。与GES1细胞比较,SNU-1、AGS、HS-746T细胞中lncRNA CEBPA-AS1的表达量显著升高,miR-455-3p的表达量显著降低,其中SNU-1细胞的lncRNA CEBPA-AS1表达量最高,差异均有统计学意义（均P＜0.05）。与si-NC组比较,si-lncRNA CEBPA-AS1组细胞活力降低,细胞克隆形成数、迁移及侵袭细胞数减少,MMP2、MMP9蛋白表达水平降低,差异均有统计学意义（P＜0.05）。与miR-NC组比较,miR-455-3p组细胞活力降低,细胞克隆形成数、迁移及侵袭细胞数减少,MMP2、MMP9蛋白表达水平降低,差异均有统计学意义（P＜0.05）。lncRNA CEBPA-AS1可靶向结合miR-455-3p,并可负调控miR-455-3p的表达。与si-lncRNA CEBPA-AS1+anti-miR-NC组比较,si-lncRNA CEBPA-AS1+anti-miR-455-3p组细胞活力升高,细胞克隆形成数、迁移及侵袭细胞数增多,MMP2、MMP9蛋白表达水平升高,差异均有统计学意义（P＜0.05）。结论：干扰lncRNA CEBPA-AS1表达可通过靶向调控miR-455-3p而抑制胃癌细胞增殖、克隆形成、迁移及侵袭。     

MicroRNA-137 Contributes to Dampened Tumorigenesis in Human Gastric Cancer by Targeting AKT2

MiRNAs play important roles in tumorigenesis. This study focused on exploring the effects and regulation mechanism of miRNA-137 on the biological behaviors of gastric cancer. Total RNA was extracted from tissues of 100 patients with gastric cancer and from four gastric cancer cell lines. Expression of miR-137 was detected by real-time PCR from 100 patients. The effects of miR-137 overexpression on gastric cancer cells’ proliferation, apoptosis, migration and invasion ability were investigated in vitro and in vivo. The target gene of miR-137 was predicted by Targetscan on line software, screened by dual luciferase reporter gene assay and demonstrated by western blot. As a result, the expression of miR-137 was significant reduced in gastric cancer cell line HGC-27, HGC-803, SGC-7901 and MKN-45 as well as in gastric cancer tissues compared with GES-1 cell or matched adjacent non-neoplastic tissues (p<0.001). The re-introduction of miR-137 into gastric cancer cells was able to inhibit cell proliferation, migration and invasion. The in vivo experiments demonstrated that the miR-137 overexpression can reduce the gastric cancer cell proliferation and metastasis. Bioinformatic and western blot analysis indicated that the miR-137 acted as tumor suppressor roles on gastric cancer cells through targeting AKT2 and further affecting the Bad and GSK-3β. In conclusion, the miR-137 which is frequently down-regulated in gastric cancer is potentially involved in gastric cancer tumorigenesis and metastasis by regulating AKT2 related signal pathways.     

Downregulation of microRNA-92b-3p suppresses proliferation,migration, and invasion of gastric cancer SGC-7901 cells by targeting Homeobox D10

To investigate the effect and mechanism of microRNA-92b-3p (miR-92b-3p) targeting Homeobox D10 (HOXD10) on proliferation, migration, and invasion of gastric cancer, we detected t he expression of miR-92b-3p and HOXD10 in SGC-7901 cells. The effects of miR-92b-3p or HOXD10 on proliferation, migration, invasion, and matrix metalloproteinase (MMP)-2/9 expression in SGC-7901 cells were measured by the Cell Counting Kit-8 assay, Transwell assay, and Western blot, respectively. The results showed that miR-92b-3p expression was increased, and HOXD10 expression was decreased in SGC-7901 cells, compared with human normal gastric epithelial cells GES-1. Functional experiments demonstrated that cell proliferation, migration, invasion, and expression of MMP-2/9 in SGC-7901 cells were significantly inhibited by miR-92b-3p silencing and HOXD10 overexpression. Moreover, HOXD10 was a potential target gene of miR-92b-3p as evidenced by the TargetScan software and double luciferase reporter assay. In the rescue experiment, knockdown of HOXD10, accompanied by higher expression of MMP-2/9, could significantly eliminate the inhibitory effects of miR-92b-3p silencing on cell proliferation, migration, and invasion. In conclusion, miR-92b-3p is highly expressed in gastric cancer SGC-7901 cells, and interfering with its expression might inhibit SGC-7901 cell proliferation, migration, and invasion via downregulating MMP-2/9 expression and targeting HOXD10.     

RGD肽对肺癌A549 细胞增殖侵袭能力的影响及其机制研究

目的:研究RGD肽对肺癌A549细胞增殖凋亡及侵袭迁移的影响,并探讨其作用机制。方法:不同浓度RGD肽处理肺癌A549细胞后,MTT检测肺癌细胞的增殖能力,流式细胞仪检测肺癌细胞凋亡及周期分布,Transwell检测其迁移及侵袭能力的变化,Western blot检测RGD肽对肺癌A549细胞MMP2、MMP9的表达水平影响。结果:当RGD肽浓度增加至50 mg/L时,肺癌A549细胞增殖明显受到抑制,且这种抑制作用呈剂量依赖关系;RGD肽组A549细胞G0/G1期细胞比例增高,细胞凋亡率由(6.1±0.1)%增至(15.2±0.5)%;在迁移和侵袭试验中,RGD肽组A549细胞的穿膜细胞数分别由123±10和43±10降至45±5和18±5;RGD肽组A549细胞MMP2、MMP9表达水平显著降低。结论:RGD肽对肺癌A549细胞的增殖有明显抑制作用,并促进其凋亡,可能与RGD肽改变其周期分布有关,RGD肽可明显抑制A549细胞的迁移及侵袭,可能与其下调MMP2、MMP9的表达相关。     

下调lncRNA MCF2L-AS1靶向miR-33b-5p抑制胃癌细胞增殖、迁移、侵袭并促进凋亡

摘要 目的：探讨lncRNA MCF2L-AS1对胃癌细胞恶性生物学行为的影响及分子机制。方法：选取45例胃癌患者的癌组织及癌旁正常组织,或培养胃黏膜上皮细胞GES-1、胃癌细胞HGC-27,采用RT-qPCR检测MCF2L-AS1和miR-33b-5p的表达水平。采用双荧光素酶报告实验检测MCF2L-AS1和miR-33b-5p的靶向关系。将HGC-27细胞分为si-NC组、si-MCF2L-AS1组、mimic NC组、miR-33b-5p mimic组、si-MCF2L-AS1+inhibitor NC组、si-MCF2L-AS1+miR-33b-5p inhibitor组,分别转染si-NC、si-MCF2L-AS1、mimic NC、miR-33b-5p mimic或共转染si-MCF2L-AS1+inhibitor NC、si-MCF2L-AS1+miR-33b-5p inhibitor。采用MTT实验检测细胞增殖情况,流式细胞术检测细胞凋亡率,克隆形成实验检测细胞克隆形成数,Transwell实验检测迁移和侵袭细胞数。结果：与癌旁正常组织或GES-1细胞相比,胃癌组织或HGC-27细胞中MCF2L-AS1表达水平升高、miR-33b-5p表达水平降低,差异均有统计学意义（P<0.05）。MCF2L-AS1可靶向调控miR-33b-5p。下调MCF2L-AS1或过表达miR-33b-5p,miR-33b-5p表达水平升高,HGC-27细胞凋亡率升高,但细胞增殖、克隆形成数、迁移和侵袭数均减少,差异均有统计学意义（P<0.05）。抑制miR-33b-5p可减弱下调MCF2L-AS1对HGC-27细胞的生物学作用。结论：下调MCF2L-AS1通过上调miR-33b-5p抑制胃癌细胞增殖、迁移、侵袭并促进凋亡;MCF2L-AS1通过靶向调控miR-33b-5p表达进而参与胃癌细胞的恶性生物学行为。     

Antiproliferative mechanism of a cannabinoid agonist by cell cycle arrest in human gastric cancer cells

For gastric cancers, the antineoplastic activity of cannabinoids has been investigated in only a few reports and knowledge regarding the mechanisms involved is limited. We have reported previously that treatment of gastric cancer cells with a cannabinoid agonist significantly decreased cell proliferation and induced apoptosis. Here, we evaluated the effects of cannabinoids on various cellular mediators involved in cell cycle arrest in gastric cancer cells. AGS and MKN-1 cell lines were used as human gastric cancer cells and WIN 55,212-2 as a cannabinoid agonist. Cell cycles were analyzed by flow cytometry and western blotting. Treatment with WIN 55,212-2 arrested the cell cycle in the G0/G1 phase. WIN 55,212-2 also upregulated phospho-ERK1/2, induced Kip1/p27 and Cip1/WAF1/p21 expression, decreased cyclin D1 and cyclin E expression, decreased Cdk 2, Cdk 4, and Cdk 6 expression levels, and decreased phospho-Rb and E2F-1 expression. ERK inhibitor decreased the proportion of G0/G1 phase which was induced by WIN 55,212-2. Inhibition of pAKT led to cell cycle arrest in gastric cancer cells. Cell cycle arrest preceded apoptotic response. Thus, this cannabinoid agonist can reduce gastric cancer cell proliferation via G1 phase cell cycle arrest, which is mediated via activation of the MAPK pathway and inhibition of pAKT.     

RASAL1 influences the proliferation and invasion of gastric cancer cells by regulating the RAS/ERK signaling pathway

The aim of this study was to investigate the biological characteristics of the RASAL1 gene in a well-differentiated gastric cancer cell line MKN-28 and a poorly differentiated gastric cancer cell line BGC-823 cells, using RNA interference and gene transfection technology, respectively. MKN-28 cells were transfected with the shRNA of RASAL1 and BGC-823 cells were transfected with the pcDNA 3.1 plasmid vector containing RASAL1. RT-PCR and western blotting were then used to detect the expression of RASAL1 mRNA and protein. The activities of RAS and extracellular signal-regulated kinase 1/2 were analyzed by the pull-down method and western blotting. The proliferate capacity, apoptosis rate, invasive and migratory potentials of MKN-28 or BGC-823 cells were also measured by Cell Counting Kit-8 cell proliferation assay, propidium iodide/Annexin V staining coupled with flow cytometry, and transwell chamber assays, respectively. Measurement of RASAL1 mRNA and protein expression in two cells revealed successful transfection of the shRNA of RASAL1 and RASAL1-pcDNA3.1 plasmid into these two cells. Moreover, decreased expression of RASAL1 in MKN-28 cells resulted in increased expression of RAS-GTP and p-ERK1/2. Interestingly, decreased expression of RASAL1 inhibited apoptosis and facilitated cell proliferation, invasion and migration. The increased expression of RASAL1 in BGC-823 cells caused declined expression of RAS-GTP and p-ERK1/2, as well as promoted apoptosis and restrained cell proliferation, invasion and migration. The down-regulation of RASAL1 promoted the proliferation, invasion and migration of gastric cancer MKN-28 cells, and up-regulation of RASAL1 inhibited the proliferation, invasion and migration of BGC-823 gastric cancer cells by regulating the RAS/ERK signaling pathway. Thus, our results suggest that RASAL1 may play an important role as a tumor suppressor gene in gastric cancer.