人胃癌肿瘤相关成纤维细胞的原代培养和鉴定

目的 探讨人胃癌肿瘤相关成纤维细胞(carcinoma-associated fibroblasts,CAFs)的培养和鉴定方法,并分析其与胃正常成纤维细胞(normal fibroblasts,NFs)在生物学特性和蛋白表达方面的差异,为其功能研究奠定基础.方法 采用组织块法和酶消化法进行人胃癌CAFs和NFs的原代培养,通过酶消化法进行人工纯化,通过形态学观察和多种蛋白的免疫细胞化学染色对其进行鉴定.结果 获得纯化的人胃癌CAFs和NFs.胃癌CAFs呈长梭形,大小不等,生长密集,排列紊乱.除表达vimentin这一成纤维细胞的标记物外,还表达FAP、SMA,但不表达CK、desmin.结论 只要给细胞创造良好的生长条件并规范操作,组织块法和消化法均可成功的培养出人胃癌CAFs和人胃NFs.胃癌CAFs与NFs在形态结构、生长方式和蛋白表达等方面均存在明显差异.     

肿瘤相关成纤维细胞激活Thy-1调控胃癌细胞发生间质转化及迁移能力的研究

目的：探讨肿瘤相关成纤维细胞（CAFs）在调控胃癌细胞发生上皮间质转化（EMT）及迁移能力中的作用。方法：培养胃癌细胞系（SNU-1和MKN45）和原代成纤维细胞，分离和鉴定原代成纤维细胞；Western blot和实时定量PCR技术检测细胞内胸腺细胞分化抗原（Thy-1）的表达情况；并且建立胃癌细胞和CAFs共培养体系，将细胞分为胃癌细胞组、共培养体系组（空白对照组）、共培养体系+同型IgG抗体组（阴性对照组）、共培养体系+抗Thy-1抗体组。Transwell实验检测Thy-1对共培养体系中胃癌细胞的迁移能力的影响；Western blot技术检测Thy-1对共培养体系胃癌细胞株的上皮（E-cad）及间质(N-cad和ZEB2)表型标记蛋白表达的影响。结果：成功分离CAFs，与正常成纤维细胞的形态并无明显差异，均出现纺锤形态，发现CAFs中α-平滑肌肌动蛋白（α-SMA）的表达比配对NFs细胞的表达量显著增加，提示分离出的CAFs可以作为后续相关实验研究；Western blot和实时定量PCR技术检测提示，CAFs中Thy-1的mRNA和蛋白质高表达，这与配对的NFs相比具有明显的统计学意义，且Thy-1在胃癌细胞系中低表达；CAFs与胃癌细胞共培养后，与胃癌细胞组相比，胃癌细胞系的迁移能力明显增强，且E-cad的表达显著降低，N-cad和ZEB2的表达明显升高，但共培养体系中CAFs预先孵育Thy-1抗体后，发现与阴性对照组相比，胃癌细胞系的迁移能力和EMT效应明显减弱。结论：CAFs细胞内过表达的Thy-1增强体外共培养体系中胃癌细胞的迁移能力和EMT效应。     

胃癌相关纤维母细胞中miRNAs的表达及对癌细胞迁移和侵袭的影响

目的观察胃癌相关纤维母细胞(cancer associated fibroblasts,CAFs)中多种微小RNAs(microRNAs,miRNAs)的表达水平,并探讨CAFs中miRNA-214的表达对胃癌细胞迁移和侵袭能力的影响。方法原代培养人胃黏膜正常纤维母细胞(normal fibroblasts,NFs)和胃CAFs,采用RT-PCR检测NFs和CAFs中多种miRNAs表达水平;利用Transwell小室建立CAFs与胃癌细胞MGC-803相互作用的体外模型,分析CAFs中miRNA-214对MGC-803迁移和侵袭能力的影响。结果与NFs相比,18种miRNAs在CAFs中的表达均有差异。其中miRNA-214在NFs和CAFs中的表达差异最显著,且在CAFs中低表达。Transwell迁移和侵袭实验结果显示,上调CAFs中miRNA-214的表达水平明显抑制胃癌细胞MGC-803迁移和侵袭能力(P0.01)。结论人胃癌微环境中CAFs和NFs的miRNAs表达差异有显著性,其中miRNA-214在CAFs中的表达明显降低;过表达CAFs中的miRNA-214可以显著降低胃癌细胞的迁移和侵袭能力,在胃癌进展中发挥抑制作用。     

HOXB7对结直肠癌细胞侵袭、迁移和EMT作用及机制的研究

目的：研究过表达和敲减同源盒蛋白B7(homeobox protein B7, HOXB7)对结直肠癌细胞迁移、侵袭和上皮-间充质转化（epithelial-mesenchymal transition, EMT）的影响。方法：使用生存分析探究HOXB7与结直肠癌患者预后的关系,使用生物信息学分析HOXB7与Wnt/β-catenin通路之间的关系。构建HOXB7的过表达和敲减细胞模型,在多种功能实验中研究过表达或敲减HOXB7对结直肠癌细胞迁移和侵袭能力的影响。使用Western blot和免疫荧光染色检测结直肠癌细胞在不同HOXB7水平下EMT相关蛋白的表达量。在裸鼠体内实验验证了HOXB7表达水平对结直肠癌转移的影响。结果：生存分析表明,HOXB7高表达的结直肠癌患者预后不良（P<0.05）。生物信息学分析表明HOXB7可靶向激活Wnt/β-catenin通路（P<0.01）。Transwell实验和细胞划痕实验结果表明,过表达HOXB7可增强结直肠癌细胞的迁移和侵袭能力,敲减HOXB7则导致其迁移和侵袭能力下降（P<0.01）。Western blot和荧光染...     

过表达Oct4B1 诱导结直肠癌SW480 细胞发生上皮间质转化

目的:探讨Oct4B1 基因过表达是否诱导人结直肠癌SW480 细胞发生上皮间质转化(EMT)及其可能的机制。方法:用带G418 抗性的Oct4B1 基因过表达质粒及阴性对照质粒转染人结直肠癌SW480 细胞株,分别称为实验组(SW480-Oct4B1)及对照组(SW480-NC),转染成功后用G418 筛选建立稳定转染的细胞株,对两种稳定转染细胞进行如下检测: RT-qPCR 检测Oct4B1 的mRNA 水平;于划痕实验和Transwell 小室实验检测迁移和侵袭能力;盂Western blot 检测EMT 相关标记物E-cadherin、N-cadherin 及Vimentin 蛋白表达; RT-qPCR 和Western blot 检测EMT 转录因子Twist 的mRNA 及蛋白表达。结果:稳定转染细胞株建立后,实验组与对照组比较:Oct4B1 基因表达水平明显升高(P<0.01);细胞迁移明显增强(P<0.01);细胞侵袭能力明显提高(P<0.01);上皮标记物E-cadherin 蛋白表达量明显下降(P<0.01);而间质标记物N-cadherin 及Vimentin蛋白表达量明显上调(P<0.01);Twist 的mRNA 及蛋白表达量均明显升高(P<0.01)。结论:过表达Oct4B1 基因可诱导人结直肠癌SW480 细胞发生EMT,增强细胞迁移及侵袭能力,其分子机制可能与提高Twist 表达有关。     

miR-210参与调控间充质干细胞向肿瘤相关成纤维细胞转化

目的探讨miR-210是否在乳腺癌细胞诱导人脂肪来源间充质干细胞(h AD-MSCs)向肿瘤相关成纤维细胞(CAFs)转化中发挥作用。方法 Transwell小室将乳腺癌细胞系MCF-7和MDA-MB-231分别与h AD-MSCs共培养,RT-q PCR检测不同时间点(0、3、6及9 d)收取乳腺癌细胞中miR-210的表达;RT-q PCR检测共培养后h AD-MSCs向CAFs转化及CAFs相关特征标志物平滑肌肌动蛋白-α(α-SMA)和腱生蛋白-c(tenascin-c)的基因表达,用Western blot检测α-SMA和成纤维细胞活化蛋白-α(FAPA)的蛋白表达;慢病毒分别向乳腺癌细胞传导miR-210抑制剂和miR-NC,然后与同比例的MSCs混合注射到裸鼠皮下,动态观察肿瘤的生长情况,4周后分离提取肿瘤组织中的CAFs,RT-q PCR和Western blot检测CAFs中α-SMA、FAPA和波形蛋白(vimentin)的表达。结果与h AD-MSCs共培养后,乳腺癌细胞MCF-7和MDA-MB-231细胞中miR-210表达都持续上调(P0.05)。共培养条件下抑制肿瘤细胞表达miR-210可以抑制MSCs向CAFs转化(P0.05)。抑制肿瘤细胞miR-210的表达可以降低肿瘤生长(P0.05)。结论 miR-210作为一种重要的信息分子介导了肿瘤微环境中MSCs向CAFs转化。     

抗DR5单抗CTB006联合5-FU对结直肠癌细胞系作用研究

目的探讨肿瘤坏死因子相关诱导配体受体（DR5）激动型单抗CTB006对人结直肠痛细胞系SW1116、SW480、SW620、Colo205的影响。方法采用ATPlite法检测CTB006组、5-FU组及两药物合用组作用后的细胞存活率,流式细胞仪技术检测细胞系表面DR5的表达水平,研究两者之间的关系。结果CTB006和5-FU对四种结直肠癌细胞增殖均表现出抑制作用。CTB006对早期细胞SW1116增殖抑制效果欠佳,对中晚期细胞SW480、SW620、Colo205有着良好的增殖抑制作用（P〈0．05）。当联合使用5-FU后,对后三者有明显的协同作用。流式细胞仪检测DR5表达水平,SW1116细胞表达（47．01±30．4）％,SW480细胞表达（76．11±15．1）％,SW620细胞表达（86．77±9.3）％,Col0205细胞表达（93．55±7．9）％。结论CTB006对中晚期结直肠癌杀伤作用较强,联用5-FU具有显著的协同作用。     

肿瘤微环境和口腔癌相关成纤维细胞

肿瘤微环境是一个有其自身特点的系统,它有别于正常细胞和其周围组织形成的环境。癌相关成纤维细胞(carcinoma-associated fibroblasts,CAFs)是肿瘤微环境中最主要的宿主细胞。口腔肿瘤微环境中的成纤维细胞有别于口腔正常成纤维细胞,在口腔肿瘤的发生、发展及治疗中起着重要作用。     

成纤维细胞与结肠癌细胞相互作用促进细胞外基质金属蛋白酶诱导因子的表达

目的研究成纤维细胞与结肠癌细胞相互作用对二者表达细胞外基质金属蛋白酶诱导因子（EMMPRIN）的影响,初步探讨肿瘤-基质相互作用在结肠癌侵袭转移中的作用。方法结肠癌SW480细胞与HELF成纤维细胞以RPMI1640培养液分别共培养0、12、24、48h,通过RT-PCR和免疫细胞化学方法检测SW480和HELF细胞中EMMPRIN的表达。结果共培养组SW480细胞EMMPRINmRNA和蛋白的表达均明显升高,HELF细胞本来不表达EMMPRIN,与SW480细胞共培养12、24、48h后检测到EMMPRIN表达,且随时间延长而表达增加。结论成纤维细胞与结肠癌细胞相互作用上调SW480细胞EMMPRIN的表达,并诱导HELF细胞表达EMMPRIN,有可能在结肠癌侵袭转移中发挥重要作用。     

IMP3 基因沉默介导Notch 信号通路对结直肠癌上皮间质转化及血管生成的影响

目的:探究胰岛素样生长因子ⅡmRNA结合蛋白3(IMP3)基因沉默介导Notch信号通路对结直肠癌上皮间质转化(EMT)及血管生成的调控机制。方法:收集我院73例结直肠癌患者肿瘤切除术后癌组织及癌旁组织。免疫组化检测结直肠癌组织及癌旁组织中IMP3蛋白表达。将人结肠癌细胞系SW620细胞进行IMP3沉默或Notch通路抑制处理,设置阴性对照。Western blot检测处理后的细胞中Notch1、Hes1、Vimentin、E-cadherin、N-cadherin、VEGF、TNF-α和TGF-β蛋白表达。MTT比色法检测各组细胞活力。Transwell实验测定各组细胞侵袭能力。结果:免疫组化实验发现IMP3在癌组织中高表达。细胞实验表明IMP3沉默可下调Notch通路相关蛋白Notch1及Hes1表达。与阴性对照组相比,沉默IMP3或抑制Notch通路后,SW620细胞活力、转移能力、EMT管生成明显受到抑制(P0.05)。细胞经沉默IMP3联合抑制Notch通路处理,对上述指标的抑制效果较单独处理(沉默IMP3或抑制Notch通路)更加显著(P0.05)。结论:IMP3基因沉默能够抑制Notch通路活化进而抑制结直肠癌EMT及血管生成。     

LncRNA OGFRP1 promotes angiogenesis and epithelial-mesenchymal transition in colorectal cancer cells through miR-423-5p/CTCF axis

ObjectiveTo investigate the mechanism of lncRNA OGFRP1 affecting angiogenesis and epithelial-mesenchymal transition (EMT) in colorectal cancer (CRC) and provide a new target for the treatment of CRC.MethodsThe expressions of OGFRP1, miR-423-5p, and CTCF were measured in CRC cell lines (HT29, LoVo, HCT116, SW620, and SW480) and normal colonic epithelial cells NCM460. Gain and loss of function experiments were performed on HCT116 and SW620 cells, after which the proliferation, apoptosis, EMT, invasion, and migration of the cells were measured using CCK-8 and colony formation assays, flow cytometry, Western blotting, Transwell, and scratch assay. The transfected cells were incubated with human umbilical vein endothelial cells (HUVECs) to assess angiogenesis using tube formation assay. ELISA was performed to detect VEGF in the conditioned medium of HCT116 and SW620 cells. The interactions among OGFRP1, CTCF and miR-423-5p were validated by dual-luciferase reporter assay.ResultsCRC cell lines had increased expression levels of OGFRP1 and CTCF and a suppressed expression level of miR-423-5p when compared with NCM460 cells. Suppression on OGFRP1 or CTCF and overexpression of miR-423-5p led to inhibited proliferation, EMT, invasion and migration and increased apoptosis of HCT116 and SW620 cells. HUVECs incubated with cells transfected with si-OGFRP1, si-CTCF or miR-423-5p mimic had suppressed angiogenesis ability. The effect of OGFRP1 suppression in CRC cells could be counteracted by miR-423-5p inhibition. Both CTCF and OGFRP1 could bind to miR-423-5p.ConclusionOGFRP1 promotes proliferation, EMT, and angiogenesis in CRC through miR-423-5p/CTCF axis.     

CD10 inhibits cell motility but expression is associated with advanced stage disease in colorectal cancer

Introduction

CD10 is a cell membrane-bound endopeptidase which is expressed in normal small bowel but not in normal colon. It is aberrantly expressed in a small proportion of colorectal cancers (CRC) and this has been associated with liver metastasis and poor prognosis. We sought to investigate the mechanism of CD10 activity and its association with clinicopathological features.

Effects of the fibroblast activation protein on the invasion and migration of gastric cancer

Objective

Cancer-associated fibroblasts (CAFs) are one of the most important components of tumor microenvironment. CAFs are believed to play an important role in tumor invasion and metastasis. Recently, fibroblast activation protein (FAP), a type II integral membrane glycoprotein belonging to the serine protease family, has emerged as a specific marker of CAFs. FAP was overexpressed in stromal fibroblasts of solid malignancies, however, the role of FAP on the process of invasion and metastasis of gastric carcinomas is still unknown.

Methods

Expression of FAP level was detected by immunohistochemistry in 60 gastric cancer surgical specimens (28 with omentum metastasis and 32 without), 20 normal human gastric tissues and omentum of 10 non-neoplastic gastric diseases. Fibroblasts were isolated from patient's tissues in the distal normal zones and tumor zones respectively, which were correspondingly designated as normal zone fibroblasts (NFs) and cancer-associated fibroblasts (CAFs). To explore the effects of FAP on NFs or CAFs, fibroblasts were co-cultured with human gastric cancer cell line MGC-803 cells. The ability of invasion and migration of MGC-803 cells was evaluated after transfecting FAP siRNA into CAFs of gastric carcinomas.

Results

We investigated the level of expression of FAP in surgical specimens, and found overexpressed in CAFs and non-expressed in NFs. Expression of FAP level in CAFs is significantly associated with Lauren classification, the degree of differentiation, depth of tumor invasion and TNM stage, but it is not correlated to age and gender in gastric carcinoma patients. There was positive correlation between the FAP level with metastasis to the omentum (p < 0.05, R² = 0.2736, p < 0.05, R² = 0.1479). In addition, the invasion and migration abilities of MGC-803 cells were significantly increased when cells were co-cultured with CAFs. On the other hand, invasion and migration abilities were significantly decreased by 46.9 and 50.3%, respectively, after knocking down FAP in CAFs. Further, NFs did not have appreciable effect on the invasion and migration of MGC-803 cells.

Conclusions

Our findings showed that FAP was overexpressed in CAFs of gastric carcinomas, and siRNA-mediated knock down of FAP significantly suppressed invasion and migration of MGC-803 cells. FAP may be an important regulator in the invasion and migration of gastric cancer and may provide a novel therapeutic target in gastric carcinomas.     

miR-126 对结肠癌细胞生物学行为的影响及其在调控结肠癌EMT 转化中的作用

目的:观察miR-126 在不同转移潜能的人结肠癌细胞系中的表达情况及其对结肠癌细胞增殖、侵袭转移能力的影响并探讨可能的作用机制。方法:采用实时荧光定量PCR 检测结肠癌细胞系(SW480、SW620 及HCT116)中miR-126 的表达量。通过脂质体瞬时转染法将miR-126 过表达(miR-126 mimics),并设置阴性对照组,然后采用CCK8 法检测细胞的增殖能力,细胞划痕实验检测细胞的迁移能力,Transwell 侵袭小室实验检测细胞的侵袭能力,Western blot 实验检测E-cadherin 和Vimentin 蛋白表达量的变化。结果:相对于低转移潜能的结肠癌细胞株SW480,miR-126 在高转移潜能的SW620 和HCT116细胞中的表达降低。过表达miR-126 可使SW620 细胞增殖、迁移和侵袭能力降低,E-cadherin 蛋白表达增加,Vimentin 蛋白表达降低,差异具有统计学意义(P<0.05)。结论:低表达的miR-126 与结肠癌的转移密切相关,miR-126 影响结肠癌细胞生物学行为的作用可能是通过调控EMT 进程实现的。     

Cancer associated fibroblasts exploit reactive oxygen species through a proinflammatory signature leading to epithelial mesenchymal transition and stemness

Cancer-associated fibroblasts (CAFs) are key determinants in the malignant progression of cancer, supporting tumorigenesis and metastasis. CAFs also mediate epithelial mesenchymal transition (EMT) of tumor cells and their achievement of stem cell traits. We demonstrate that CAFs induce EMT and stemness through a proinflammatory signature, which exploits reactive oxygen species to drive a migratory and aggressive phenotype of prostate carcinoma cells. CAFs exert their propelling role for EMT in strict dependence on cycloxygenase-2 (COX-2), nuclear factor-κB, and hypoxia-inducible factor-1. CAF-secreted metalloproteases elicit in carcinoma cells a Rac1b/COX-2-mediated release of reactive oxygen species, which is mandatory for EMT, stemness, and dissemination of metastatic cells. Tumor growth is abolished, and metastasis formation is severely impaired by RNA interfering-mediated targeting of the proinflammatory signature, thereby supporting the therapeutic targeting of the circuitry COX-2/nuclear factor-κB /hypoxia-inducible factor-1 as a valuable antimetastatic tool affecting cancer cell malignancy.