胃癌成纤维细胞中HAS2对胃癌细胞转移的影响

目的探讨胃癌成纤维细胞中透明质酸合成酶(HAS2)对胃癌细胞转移的影响。方法从手术切除的胃癌组织及癌旁正常组织中分别分离及培养胃成纤维细胞(CAFs)及正常成纤维细胞(NFs)。蛋白质免疫印迹(Western blot)及免疫荧光染色检测CAFs与NFs中α-平滑肌肌动蛋白(α-SMA)、波形蛋白(vimentin)、E-钙黏蛋白(E-cadherin)及HAS2的表达。利用siRNA下调CAFs中HAS2的表达,Western blot及免疫荧光染色检测CAFs中HAS2的表达,细胞迁移(Transwell)及划痕闭合实验检测胃癌细胞SGC7901的侵袭及迁移能力,Western blot检测SGC7901细胞上皮-间质转化相关蛋白E-cadherin、vimentin及N-钙黏蛋白(N-cadherin)的表达。结果α-SMA与HAS2在CAFs中高表达,vimentin在CAFs与NFs中均表达,而E-cadherin在CAFs与NFs中不表达。与si-NC组相比,si-HAS2组CAFs中HAS2表达降低,SGC7901细胞的侵袭及迁移能力降低,SGC7901细胞中上皮-间质转化相关蛋白E-cadherin表达升高,而Vimentin及N-cadherin表达降低。结论下调胃癌CAFs中HAS2的表达,可抑制胃癌细胞的侵袭、迁移及上皮-间质转化。     

核转录因子CDX2抑制胃癌细胞迁移、侵袭与逆转上皮-间质转化有关

背景:尾型同源盒转录因子2(CDX2)是一种肠上皮细胞特异性核转录因子,正常胃黏膜组织不表达CDX2,而肠化生、异型增生和胃癌组织中存在CDX2异位表达。近年研究发现CDX2在胃癌中起抑癌基因作用。上皮-间质转化(EMT)与恶性肿瘤的侵袭、转移密切相关。目的:探讨CDX2对胃癌细胞迁移、侵袭能力的影响与EMT的关系。方法:分别将CDX2过表达质粒和干扰质粒转染入低表达和高表达CDX2的人胃癌细胞株MKN-45和AGS中,以划痕试验检测各组细胞迁移能力,Transwell小室细胞侵袭实验检测细胞侵袭能力,蛋白质印迹法检测EMT标记物表达。结果:过表达CDX2能显著抑制MKN-45细胞的迁移、侵袭能力,并上调上皮标记物E-cadherin表达,下调间质标记物vimentin表达(P0.05);而沉默CDX2表达能显著增强AGS细胞的迁移、侵袭能力,下调E-cadherin表达,上调vimentin表达(P0.05)。结论:CDX2表达水平改变可影响胃癌细胞的迁移、侵袭能力,并参与EMT的调控,其对胃癌细胞迁移、侵袭的抑制作用可能与逆转EMT有关,具体分子机制有待深入研究。     

胃泌素对胃癌细胞EMT和Wnt/β-catenin信号通路相关基因表达的影响

目的探讨高表达胃泌素(gastrin)对胃癌细胞上皮间质转化(EMT)的影响及可能机制。方法用gastrin过表达质粒pcDNA3-gastrin及空载体pcDNA3.1转染胃癌细胞SGC-7901、MKN45,48 h后收集细胞蛋白,Western印迹检测鉴定gastrin表达,检测EMT相关标志物E-钙黏蛋白(cadherin)、N-cadherin、Snail及wnt/β-连环蛋白(catenin)信号通路相关基因wnt3α、β-catenin、c-myc的蛋白表达变化。结果在SGC-7901、MKN45细胞中成功过表达gastrin。与pcDNA3.1空载体转染组相比,pcDNA3.1-gastrin转染SGC-7901、MKN45细胞中上皮标志蛋白E-cadherin表达下调,间质标志蛋白N-cadherin、Snail表达上调,差异有统计学意义(P<0.05);同时Wnt/β-catenin通路蛋白相关蛋白wnt3α、β-catenin及其下游基因c-myc的表达上调,差异有统计学意义(P<0.05)。结论过表达gastrin能够激活Wnt/β-catenin信号通路和促进胃癌细胞SGC-7901和MKN45发生EMT。     

氯化两面针碱对人胃癌细胞增殖、迁移的抑制作用

目的探讨氯化两面针碱(NC)对人胃癌SGC-7901细胞增殖、迁移的作用及其机制。方法 MTT实验检测NC对SGC-7901细胞的增殖作用;Transwell小室实验检测NC对SGC-7901细胞迁移和侵袭的影响;Western印迹检测NC对人胃癌SGC-7901细胞中Bax,Bcl-2,PCNA,MMP2,MMP1和GAPDH表达的影响。结果 MTT实验表明NC抑制人胃癌SGC-7901细胞增殖,并且呈时间、剂量依赖性(P<0.05);此外,NC显著抑制SGC-7901细胞中PCNA表达,呈剂量依赖性(P<0.05);Transwell小室实验表明NC显著抑制SGC7901细胞迁移和侵袭,呈剂量、时间依赖性(P<0.05);Western印迹表明NC上调凋亡蛋白Bax的表达,下调抗凋亡蛋白Bcl-2表达,抑制MMP1以及MMP2的表达(均P<0.05)。结论 NC通过诱导细胞凋亡抑制SGC-7901细胞增殖,通过降解MMP1和MMP2从而抑制SGC-7901细胞侵袭和迁移。     

miR-183调控Wnt/β-catenin信号通路影响胃癌SGC-7901细胞生物学特性的研究

背景 miR-183在胃癌、乳腺癌、膀胱癌等多种肿瘤组织中低表达,发挥抑癌基因的作用,但其在胃癌中作用机制目前尚不十分清楚.研究表明, miR-183可以通过调节Wnt/β-catenin信号通路抑制骨肉瘤细胞的生长、迁移和侵袭,而Wnt/β-catenin信号通路在胃癌中高度激活与胃癌的发生和转移密切相关.但miR-183是否调节Wnt/β-catenin信号通路影响胃癌细胞生物学特性尚不清楚.目的探讨miR-183调控Wnt/β-catenin信号通路对胃癌细胞生物学特性的影响.方法采用q RT-PCR检测miR-183在不同胃癌细胞株中的表达情况,在胃癌细胞SGC-7901中转染miR-183mimics或mimics对照,分别设为miR-183组和miR-NC组, qRT-PCR检测转染效率,噻唑蓝增殖实验检测SGC-7901细胞增殖变化,流式细胞仪检测SGC-7901细胞凋亡情况, Transwell实验检测SGC-7901细胞侵袭和迁移能力, Western blot法检测凋亡相关及Wnt/β-catenin信号通路相关蛋白表达水平.使用Wnt/β-catenin信号通路激动剂氯化锂处理过表达miR-183的SGC-7901细胞,观察SGC-7901细胞生物学特性的变化.结果与正常胃黏膜上皮GES-1细胞相比, 4株胃癌细胞中miR-183的表达水平明显降低(P 0.05).转染miR-183mimics后SGC-7901细胞中miR-183的表达水平显著升高(P0.05).过表达miR-183后SGC-7901细胞OD值降低(P0.05),凋亡率、Bax和Cleaved Caspase-3蛋白表达水平升高(P 0.05),侵袭和迁移细胞数减少(P0.05),β-catenin、p-GSK-3β和Cyclin D1蛋白表达水平下调(P0.05), GSK-3β蛋白表达水平上调(P 0.05).激活Wnt/β-catenin信号通路部分逆转了过表达miR-183对SGC-7901细胞增殖、侵袭和迁移的抑制作用及凋亡促进作用(P0.05).结论miR-183可能通过抑制Wnt/β-catenin信号通路阻碍人胃癌SGC-7901细胞增殖、侵袭和迁移能力,促进细胞凋亡.     

miR-215对胃癌细胞侵袭转移能力的影响及机制

目的探讨miR-215对胃癌细胞侵袭转移能力的影响及作用机制。方法通过RT-PCR检测miR-215在高转移胃癌细胞株NCIN87,BGC-823,RF-48及低转移细胞株HGC-27及MKN-28中的表达;Transwell迁移及侵袭实验检测miR-215抑制剂对胃癌细胞迁移及侵袭能力的影响;Western印迹检测miR-215抑制剂对胃癌细胞活化白细胞黏附分子(ALCAM),基质金属蛋白酶(MMP)-2,MMP-9,上皮细胞-间充质转化(EMT)相关蛋白E-钙黏附素(E-cadherin),波形蛋白(Vimentin)表达量的影响。结果 RT-PCR结果证实miR-215在高转移胃癌细胞株中的表达高于低转移胃癌细胞中的表达,且miR-215在BGC-823细胞中的表达最高,因此选用BGC-823作为后续实验细胞株。miR-215抑制剂转染BGC-823细胞48 h后,发现下调miR-215表达能显著抑制BGC-823细胞迁移及侵袭能力,并能下调ALCAM,MMP-2,MMP-9及Vimentin表达,上调E-cadherin表达。结论miR-215低表达能显著抑制胃癌细胞BGC-823侵袭及转移能力,与下调MMPs及抑制EMT生成有关。     

塞来昔布干预对人胃癌细胞E-钙黏蛋白和基质金属蛋白酶-2表达的影响

目的探讨COX-2与E-cadherin、MMP-2之间的相互关系及其参与胃癌细胞侵袭迁移的可能机制。方法应用塞来昔布(celecoxib)对体外培养的人胃癌细胞SGC7901进行干预,采用实时荧光定量反转录PCR检测COX-2、E-cadherin、MMP-2 mRNA的表达;运用免疫荧光标记法结合激光共聚焦荧光显微镜分析E-cadherin的蛋白表达量;应用Transwell法检测细胞侵袭及迁移能力的变化。结果塞来昔布明显抑制体外培养的人胃癌细胞SGC-7901中COX-2 mRNA的表达,E-cadherin mRNA的表达随着COX-2的表达下降而呈浓度与时间依赖性升高,MMP-2 mRNA的表达随着COX-2的表达下降而呈浓度与时间依赖性降低。塞来昔布30μmol/L干预人胃癌细胞SGC7901 24、36、48 h后,激光共聚焦荧光显微镜检测E-cadherin的蛋白表达量明显升高。塞来昔布干预组细胞穿过Transwell小室的细胞数明显少于对照组。结论 COX-2特异性抑制剂塞来昔布通过抑制COX-2的表达,上调E-cadherin的表达,下调MMP-2的表达,抑制体外培养的胃癌细胞SGC7901的侵袭迁移能力。     

Kruppel样因子8通过转化生长因子-β1介导的上皮间质转化促进胃癌细胞的侵袭转移

目的探讨在人胃癌细胞中Kruppel样因子(KLF)8是否参与了转化生长因子(TGF)-β1介导的上皮间质转化(EMT)作用促进胃癌细胞侵袭转移。方法 TGF-β1处理胃癌细胞SGC7901和MKN45后,采用Western印迹和实时定量荧光PCR(qRT-PCR)检测细胞中KLF8、上皮标志蛋白——钙黏附蛋白E(E-cadherin)和间质标志蛋白——波形蛋白(Vimentin)表达水平;采用小干扰RNA(siRNA)技术降低胃癌细胞MKN45中KLF8的表达水平,细胞侵袭试验和划痕试验检测细胞的体外侵袭和迁移能力,并且采用高内涵细胞分析试验检测细胞的运动速度。结果在胃癌细胞株中,TGF-β1刺激后,能够下调E-cadherin和上调Vimentin的表达,进而促进细胞发生EMT;进一步研究发现,在胃癌细胞MKN45中,TGF-β1能够增加KLF8 mRNA和蛋白表达水平(P0.05);Western印迹和qRT-PCR实验发现,siRNA干扰KLF8表达后,能够阻断TGF-β1诱导的EMT,进而部分逆转了E-cadherin的降低和Vimentin的升高;同时,抑制KLF8后,能够降低TGF-β1促进的细胞侵袭、迁移和运动能力。结论在胃癌细胞中,KLF8参与了TGF-β1诱导EMT过程,并且有可能成为胃癌治疗中的新治疗靶标。     

COPB2表达对胃癌细胞增殖、迁移和侵袭的影响

背景外被体蛋白复合物β2亚基(coatomer protein complex subunitbeta2,COPB2)可参与调节多种肿瘤细胞的恶性生物学行为,而其在胃癌中表达和临床意义仍不完全明确.目的探究COPB2对胃癌细胞增殖、侵袭和迁移能力的影响及其机制.方法采用免疫组化法观察COPB2在胃癌组织和癌旁组织中的表达情况.采用Western blot检测胃癌组织和胃癌细胞系(SGC-7901、MKN45和AGS)中COPB2蛋白的表达情况.将COPB2-sh RNA及其相应的阴性对照(Con-sh RNA)、pc DNA-COPB2及其相应的阴性对照(pc DNA-Con)转染到SGC-7901细胞后,采用CCK-8法、细胞集落形成法和Transwell法分析敲低或过表达COPB2对胃癌细胞增殖、集落形成、迁移和侵袭能力的影响;采用Western blot检测敲低或过表达COPB2对胃癌细胞中Akt信号的影响.建立肿瘤异体移植模型,检测敲低COPB2对瘤体生长能力的影响.结果相对于癌旁组织和正常人胃上皮细胞GES-1,胃癌组织和胃癌细胞系(SGC-7901、MKN45和AGS)中COPB2蛋白表达均显著升高.敲低COPB2能抑制SGC-7901增殖、集落形成、迁移与侵袭的能力和p-Akt的蛋白表达,而过表达COPB2则呈现相反作用.另外,肿瘤异体移植模型实验证实敲低COPB2能抑制SGC-7901细胞在体内的生长.结论敲低COPB2表达可抑制胃癌细胞增殖、侵袭转移,且这一作用可能与其抑制Akt信号活性相关.     

葛花解酲方对肝癌细胞增殖、侵袭、迁移、凋亡及上皮间质转化的影响

目的 探讨葛花解酲方含药血清对肝癌细胞侵袭、迁移和上皮间质转化的影响及其可能的作用机制。方法 以稳定高表达、高侵袭的HepG2肝癌细胞为体外研究模型,用不同浓度的葛花解酲方含药血清进行干预。CCK8法检测含药血清对HepG2细胞增殖的抑制作用,Transwell法观察含药血清对HepG2细胞的迁移和侵袭能力的影响,Western印迹法检测HepG2细胞中上皮型钙黏蛋白(E-cadherin)和神经型钙黏蛋白(N-cadherin)表达水平。结果 CCK8法检测结果显示含药血清可显著抑制HepG2细胞的增殖,抑制作用呈时间和剂量依赖性(P<0.05)。不同浓度的含药血清可显著抑制HepG2细胞的迁移和侵袭,显著下调肝癌细胞中N-cadherin的表达,显著上调E-cadherin的表达(P<0.05)。结论 葛花解酲方含药血清能抑制肝癌细胞的侵袭、迁移,其作用机制可能与抑制上皮间质转化有关。     

Interaction between cyclooxygenase-2,Snail,and E-cadherin in gastric cancer cells

AIM:To investigate the mechanisms of how cyclooxygenase-2(COX-2)regulates E-cadherin in gastric cancer cells.METHODS:COX-2 expression in human gastric cancer cell lines SGC-7901,BGC-823,MGC-803 and AGS were measured at the mRNA and protein level.COX-2 rich cell line SGC-7901 was chosen for subsequent experiments.siRNA mediated gene knockdown was used to investigate the impact of COX-2 on nuclear factor-κB (NF-κB),Snail,and E-cadherin in gastric cancer cells.Gene expression was determined by Western blot and real-time polymerase chain reaction.To analyze whether NF-κB inhibition could interrupt the modulatory effect of COX-2 or prostaglandin E2(PGE2)on E-cadherin,gastric cancer cells were treated with celecoxib or PGE2,in the presence of NF-κB specific siRNA.RESULTS:Highest expression level of COX-2 was found in SGC-7901 cells,both at mRNA and protein levels.siRNA mediated down-regulation of COX-2 led to a reduced expression of NF-κB and Snail,but an increased expression of E-cadherin in SGC-7901 cells.siRNA mediated down-regulation of NF-κB also led to a reduced expression of E-cadherin and Snail in SGC-7901 cells.However,COX-2 expression did not alter after cells were treated with NF-κB specific siRNA in SGC-7901 cells.Treatment of SGC-7901 cells with celecoxib led to a reduced expression of Snail but an increased expression of E-cadherin.In contrast,treatment of SGC-7901 cells with PGE2 led to an increased Snail and a decreased E-cadherin.However,siRNAmediated knockdown of NF-κB partially abolished the effect of celecoxib and PGE2 on the regulation of E-cadherin and Snail in SGC-7901 cells.CONCLUSION:COX-2 likely functions upstream of NF-κB and regulates the expression of E-cadherin via NF-κB/Snail signaling pathway in gastric cancer cells.     

Xiaotan Sanjie decoction inhibits interleukin-8-induced metastatic potency in gastric cancer

AIM:To investigate the interaction between Xiaotan Sanjie(XTSJ) decoction and interleukin-8(IL-8) and its effect on adhesion,migration and invasion of SGC-7901 gastric cancer cells.METHODS:SGC-7901 gastric cancer cells were exposed to serum containing XTSJ decoction and/orIL-8(1 ng/m L).SGC-7901 cell adhesion to fibronectin,an extracellular matrix component,was detected using the Cell Counting Kit-8.Migration and invasion abilities of SGC-7901 cells were detected by scratch wound and Transwell chamber assays.Then,protein(immunofluorescence and Western blot) and m RNA levels(quantitative polymerase chain reaction) of cluster of differentiation 44(CD44),a cell adhesion molecule,were measured in 72-h-cultured SGC-7901 cells.RESULTS:Cell adhesion was promoted by IL-8(P = 0.001),but was inhibited by XTSJ decoction(P = 0.0001).Similarly,IL-8 promoted SGC-7901 cell invasion(P = 0.003),and XTSJ decoction inhibited cell invasion(P = 0.001).IL-8 induced SGC-7901 cell migration,but this was inhibited by XTSJ decoction.IL-8 up-regulated CD44 protein(P = 0.028) and m RNA expression(P = 0.002),whereas XTSJ decoction inhibited CD44 protein expression(P = 0.0001),but not m RNA expression(P = 0.275).An interaction between XTSJ decoction and IL-8 was confirmed in the invasion(P = 0.001) and CD44 m RNA expression of SGC-7901 cells(P = 0.010),but not in cell adhesion(P = 0.051).CONCLUSION:XTSJ decoction may inhibit adhesion,migration and invasion of gastric cancer cells,which is partly associated with down-regulation of IL-8.     

大黄酸通过miR-29c-3p调节FSCN1的表达影响胃癌细胞的生物学行为

目的探讨大黄酸(Rhein)对人胃癌细胞(SGC-7901)增殖、迁移、侵袭、凋亡的影响及其机制。方法通过qRT-PCR检测大黄酸处理后细胞中miR-29c-3p的表达以及miR-29c-3p的转染效率;miR-29c-3p mimics组、NC mimics组、Rhein+miR-29c-3p inhibitor组、Rhein+NC mimics组均使用脂质体转染试剂转染至SGC-7901细胞中,再使用50μM的大黄酸处理48h;CCK-8法检测细胞增殖;Annexin V-FITC/PI和流式细胞术联合检测细胞凋亡;Transwell实验检测细胞迁移和侵袭能力;双荧光素酶报告实验检测细胞荧光活性;Western blot检测FSCN1蛋白表达。结果与NC组相比较,大黄酸处理后可抑制SGC-7901细胞增殖、迁移、侵袭,促进细胞凋亡;过表达miR-29c-3p可抑制细胞增殖、迁移、侵袭,促进细胞凋亡;抑制miR-29c-3p表达可逆转大黄酸对SGC-7901细胞增殖、迁移、侵袭的抑制和细胞凋亡促进作用。miR-29c-3p可靶向作用于FSCN1。结论大黄酸可抑制人胃癌细胞(SGC-7901)增殖、迁移、侵袭,促进细胞凋亡,其机制可能与miR-29c-3p/FSCN1有关,为大黄酸用于治疗胃癌提供一定的科学依据。     

胃癌细胞多药耐药相关基因的表达与其侵袭转移能力的相关性研究

目的探讨胃癌细胞株BGC-823、SGC-7901的多药耐药相关基因的表达与其侵袭转移能力的关系。方法采用实时荧光定量PCR技术检测胃癌细胞株BGC-823、SGC-7901的多药耐药相关基因(包括ABCB1、MMP2、CDH1、CD44)的mRNA表达水平。采用细胞划痕实验、Transwell迁徙实验评价两株胃癌细胞的侵袭转移能力,进而探讨胃癌细胞多药耐药相关基因的表达与侵袭转移能力的关系。结果荧光定量PCR实验发现胃癌细胞株BGC-823的ABCB1、CDH1、CD44基因表达较SGC-7901高,而MMP2基因的表达在SGC-7901中较高。细胞划痕实验及Transwell迁徙实验显示胃癌细胞株BGC-823的迁徙能力比SGC-7901强。结论胃癌细胞的多药耐药与侵袭转移有一定的关系,CD44的高表达在胃癌细胞的侵袭转移中可能起主要作用。     

The Down-Regulation of Notch1 Inhibits the Invasion and Migration of Hepatocellular Carcinoma Cells by Inactivating the Cyclooxygenase-2/Snail/E-cadherin Pathway In Vitro

Background

The Notch signaling pathway plays an important role in cancer, but the mechanism by which Notch1 participates in invasion and migration of hepatocellular carcinoma (HCC) cells is unclear.

Aims

Our purpose is to confirm the anti-invasion and anti-migration effects of the down-regulation of Notch1 in HCC cells.

Methods

The invasion and migration capacities of HCC cells were detected with Transwell cell culture chambers. The expressions of Notch1, Notch1 intracellular domain (N1ICD), E-cadherin, Snail, and cyclooxygenase-2 (COX-2) were analyzed by RT-PCR and/or western blotting. Notch1 and Snail were down-regulated by RNA interference, and COX-2 was inhibited by NS-398. Cell apoptosis was analyzed by MTT and flow cytometry.

Results

In HCC cells, Snail, Notch1, and COX-2 were up-regulated, and E-cadherin was down-regulated in mRNA and/or protein levels. The down-regulation of Snail or Notch1 or the inhibition of COX-2, respectively, can increase the mRNA and protein expressions of E-cadherin and decrease the invasion and migration capabilities of HCC cell. Down-regulated Notch1 or inhibited COX-2 can reduce the mRNA and protein expressions of Snail. The down-regulation of Notch1 can also reduce the protein expression of COX-2. However, exogenous PGE2 can reverse the role of down-regulated Notch1. The results of MTT and flow cytometry showed that down-regulated Notch1 did not affect HCC cell viability.

Conclusions

Down-regulated Notch1 may be an effective approach to inactivating Snail/E-cadherin by regulating COX-2, which results in inhibiting the invasion and migration of HCC cells. The inhibitory effects of down-regulated Notch1 on cell invasion and migration were independent of apoptosis.