IGHMBP2过表达促进食管鳞癌细胞的侵袭和迁移

IGHMBP2(Immunoglobulin mu binding protein 2)基因编码一种解旋酶,参与DNA的复制和修复,并且作为转录调节因子在基因转录中发挥重要作用。IGHMBP2基因定位于11q13.2,该染色体区段在食管鳞癌中扩增频率较高。为了探讨IGHMBP2基因在食管鳞癌中的扩增情况及其在食管鳞癌中的作用,文章对本实验室前期报道的59例食管鳞癌原发肿瘤array-CGH数据进行分析,结果显示IGHMBP2基因扩增频率为28.9%(17/59)。进一步利用荧光原位杂交(FISH)和Western blot技术,发现食管鳞癌细胞系KYSE30、KYSE180、KYSE510和KYSE150中存在IGHMBP2基因扩增/增益以及蛋白高表达。敲降IGHMBP2后,KYSE30和KYSE150细胞的侵袭迁移能力明显降低(P<0.001),侵袭迁移相关蛋白E-cadherin的表达水平升高;敲降后转染IGHMBP2质粒,回复其蛋白表达后,细胞的侵袭迁移能力又得以恢复(P<0.01)。上述结果表明,IGHMBP2过表达可能通过降低E-cadherin的表达从而增强食管鳞癌细胞的侵袭迁移能力。     

Flot-2高表达促进食管鳞状细胞癌的生长和侵袭

目的探究flotillin-2(Flot-2)在人食管鳞状细胞癌中的表达及基本功能。方法利用免疫组织化学技术检测76例人食管鳞状细胞癌组织及相应癌旁组织中Flot-2的免疫反应阳性水平,并分析其与食管鳞状细胞癌的相关性。以小分子si RNA降低人食管鳞状细胞癌细胞系KYSE150细胞中Flot-2表达后,利用CKK-8及Transwell小室实验,检测细胞生长和侵袭能力的变化。结果 Flot-2在人食管鳞状细胞癌组织中免疫反应性明显升高,其在转移组织中的免疫反应水平显著高于非转移组织。敲低Flot-2表达后,KYSE150细胞的生长和侵袭能力明显降低。结论 Flot-2能促进细胞的生长和侵袭,参与人食管鳞状细胞癌的发生与转移过程。     

血清应答因子在食管鳞癌细胞侵袭转移中的作用

目的:研究血清应答因子(serum response factor,SRF)在人食管鳞癌细胞体外侵袭转移中的意义。方法:选用EC9706-H、EC9706-L和EC109-H、EC109-L两对高低转移细胞系,采用细胞划痕实验验证食管鳞癌高低转移细胞系体外侵袭转移能力的差异;Western blot检测SRF在两对食管鳞癌高低转移细胞系中的差异表达;在EC9706-H、EC109-H细胞中加入CCG(SRF抑制剂)抑制SRF的表达后,检测其侵袭转移能力的变化。结果:细胞划痕实验验证了两对食管鳞癌高低转移细胞系侵袭转移能力的差异;Western blot结果提示SRF在EC9706-H、EC109-H细胞中的表达水平显著高于EC9706-L、EC109-L细胞;在EC9706-H、EC109-H细胞中加入CCG抑制SRF的表达后,其侵袭转移能力明显减退。结论:SRF在高转移性食管鳞癌细胞系中呈现高表达,在低转移性食管鳞癌细胞系中呈现低表达,抑制高转移性食管鳞癌细胞系中SRF的表达后,其侵袭转移能力下降,提示SRF和食管鳞癌的侵袭转移能力呈正相关。     

新城疫病毒(NDV)D90抑制口腔鳞癌细胞系迁移和侵袭的研究

目的:证明NDV D90是否具有抑制口腔鳞癌细胞系迁移和侵袭的能力。方法:免疫荧光染色法检测D90对细胞微管和微丝形态的改变;Transwell法检测D90对HN-6细胞迁移和侵袭率的抑制作用;蛋白印迹法检测D90对于SP1、RECK、MMP-2和MMP-9表达的影响;明胶酶谱法用于检测MMP-2和MMP-9活性的改变。结果:实验结果表明,D90通过改变细胞的微管和微丝形态来抑制细胞的能动性;D90具有抑制HN-6细胞的迁移和侵袭的功能。同时,D90通过下调SP1和上调RECK的表达来抑制MMP-2和MMP-9的表达和活性。结论:NDV D90能够有效地抑制口腔鳞癌细胞系HN-6的迁移和侵袭,为一种新的抗肿瘤制剂提供了临床试验基础。     

环吡酮胺对食管鳞癌细胞增殖和迁移的影响

环吡酮胺已被确认为潜在的抗肿瘤药物,但其对于食管鳞癌细胞的影响及其作用机制尚不完全明确。本研究应用MTT法确定了环吡酮胺对食管鳞癌细胞Eca109的IC_(50)和对细胞增殖能力的影响。通过Transwell迁移实验检测环吡酮胺对食管鳞癌细胞迁移能力的影响;Seahorse生物能量分析仪检测食管鳞癌细胞耗氧率;应用流式细胞术分析环吡酮胺对食管鳞癌细胞内活性氧、线粒体活性氧、线粒体膜电位、细胞周期以及细胞凋亡的影响;应用Western blot技术检测环吡酮胺对食管鳞癌细胞线粒体呼吸链酶复合物亚基、细胞周期以及细胞凋亡相关蛋白的影响。该研究显示,环吡酮胺能够破坏线粒体功能、促进食管鳞癌细胞内和线粒体活性氧的累积、诱导细胞凋亡、同时阻滞细胞周期在G1期,进而抑制食管鳞癌细胞的增殖和迁移。该研究结果提示,环吡酮胺具有一定的抑制食管鳞癌细胞增殖和迁移的能力,是一种潜在的治疗食管鳞癌的药物。     

CKS1 表达与食管癌细胞辐射敏感性的关系研究

目的:探讨CKS1表达对食管癌细胞辐射敏感性的影响,初步研究其分子机理.方法:用Western-blotting方法筛选CKS1低表达和高表达的食管癌细胞系;构建CKS1正义表达载体p-pcDNA 3.1/myc-His A-CKS1和RNA干扰载体CKS1 siRNA,分别转染CKS1低表达细胞和高表达细胞,用不同剂量γ-射线照射各组细胞,克隆形成实验检测细胞辐射敏感性的差异.结果:CKS1在四种食管癌细胞中的表达水平依次为EC9706＞ KYSE510＞KYSE450＞ KYSE150.用p-pcDNA 3.1/myc--His A-CKS1表达载体转染KYSE150细胞后CKS12表达升高,不同剂量γ-射线照射后细胞的克隆形成能力显著高于母系对照组(P＜0.01).RNA干扰载体转染KYSE510细胞后CKS1表达水平降低,不同剂量γ-射线照射后细胞的克隆形成能力显著低于母系对照组(P＜0.01).敲降CKS1表达后DNA损伤修复相关蛋白RAD51表达下降,KU70表达没有变化.CKS1过表达后RAD51表达升高,KU70表达没有变化.结论:CKS1表达与食管癌细胞的辐射敏感性密切相关,可能通过影响DNA损伤修复发挥作用.     

LncRNA MEG3抑制食管鳞癌组织及KYSE30细胞

本研究通过检测长链非编码RNA(lncRNA)MEG3在食管鳞癌组织及人KYSE30细胞中的表达情况,分析lncRNA MEG3在食管鳞癌发生发展中的作用。我们采集食管鳞癌手术肿瘤组织54例作为观察组,癌旁正常组织54例作为对照组,采用RT-PCR技术检测MEG3表达水平。进行MEG30过表达及抑制实验,检测KYSE30细胞增殖及侵袭活性的变化。研究结果显示观察组食管鳞癌组织中lncRNA MEG3表达低于对照组的正常组织,差异具有统计学意义(p0.05);lncRNA MEG3过表达后KYSE30细胞增殖及侵袭能力减弱,lncRNA MEG3表达抑制后,KYSE30细胞增殖和侵袭能力增强。结果表明,lncRNA MEG3对食管鳞癌的发生发展有抑制作用。     

LncRNA MIR31HG通过诱导细胞周期阻滞抑制食管鳞癌细胞增殖活性

本研究探讨lnc RNA MIR31HG对食管鳞癌细胞增殖活性的影响.利用定量PCR检测MIR31HG在食管鳞癌标本及其癌旁组织、人食管上皮细胞系Het-1A和食管鳞癌细胞系Eca-109、EC-1、KYSE30中的表达;采用过表达质粒pc DNA3.1-MIR31HG在食管鳞癌细胞系中过表达MIR31HG;MTT法和SRB法检测细胞增殖率;细胞周期分析试剂盒检测细胞周期进程;Caspase3活性检测试剂盒分析Caspase3活性;PCR和Western blot法检测p53、Caspase3及Bcl-2的m RNA和蛋白质表达水平.结果显示,食管癌组织中MIR31HG表达水平显著低于癌旁组织(P0.05);与Het-1A细胞相比,Eca-109、EC-1、KYSE30细胞中MIR31HG的表达均显著下调(P0.05),提示MIR31HG可能介导食管癌的发生发展.转染pc DNA3.1-MIR31HG可显著上调食管癌细胞中MIR31HG的m RNA表达(P0.01),且MIR31HG过表达可显著抑制食管癌细胞增殖活性(P0.05),减少S期细胞数(P0.05),增加G1期细胞数(P0.05),提示MIR31HG可能通过阻碍细胞周期G1期~S期进程抑制食管癌细胞增殖活性.此外,MIR31HG过表达显著增加Caspase3活性,增加Caspase3和p53的m RNA和蛋白质表达水平,同时抑制Bcl-2 m RNA和蛋白质表达水平.这表明,MIR31HG可通过抑制食管癌细胞的增殖活性阻碍食管癌的发生发展,这可能为食管癌的诊断和治疗提供新策略.     

LATS1在食管鳞癌中的表达及启动子甲基化状态研究

在我们以前的研究中,在食管鳞癌细胞株中过表达大肿瘤抑制基因-1(large tumor suppressor kinase 1,LATS1)能够明显抑制细胞增殖、细胞迁移和侵袭,同时能够促进细胞凋亡。为了进一步研究LATS1基因在食管鳞状细胞癌发生发展中的作用以及可能的机制,我们在食管鳞癌组织与配对癌旁组织中,应用免疫组化检测LATS1基因蛋白质表达情况,应用荧光定量PCR(qRT-PCR)检测配对组织LATS1基因mRNA表达差异。甲基化特异性PCR(MSP)和亚硫酸氢盐测序法(BGS)检测食管鳞癌组织及细胞中LATS1基因的CpG岛甲基化状态。我们发现LATS1基因在癌旁组织中广泛表达,而癌组织中LATS1基因表达明显下调。结果表明LATS1在食管鳞癌中起到抑癌基因的作用,并且与肿瘤的临床分期相关,而其下调与CpG岛的高甲基化无关。在食管鳞癌细胞中过表达LATS1抑制细胞增殖、细胞迁移和侵袭,同时能够促进细胞凋亡。因此,LATS1基因值得进一步探索作为食管鳞癌生物标志物的可能用途和未来分子诊断和治疗的目标。     

紫铆因对食管鳞癌细胞增殖和存活影响的初步研究

研究紫铆因对人食管鳞癌细胞增殖和存活的影响。通过MTS和软琼脂集落实验检测紫铆因对食管鳞癌增殖的抑制,生化分析仪检测紫铆因对食管鳞癌糖酵解的影响,并利用免疫印迹检测紫铆因对食管鳞癌细胞增殖和凋亡激活相关蛋白分子的表达。结果发现紫铆因剂量依赖性抑制KYSE150和Eca109细胞增殖,下调EGFR信号通路活化,抑制HK2的表达及糖酵解。一定浓度的紫铆因能诱导食管鳞癌细胞发生凋亡,caspase3和PARP被剪切,Bcl-2和Mcl-1表达下调,但Bcl-XL未见明显改变。结果证明紫铆因抑制食管鳞癌的增殖,可能与EGFR信号通路和糖酵解被抑制,及促存活蛋白Bcl-2和Mcl-1的表达下调有关。     

Targeted Knockdown of IQGAP1 Inhibits the Progression of Esophageal Squamous Cell Carcinoma In Vitro and In Vivo

IQGAP1 is a scaffolding protein that can regulate several distinct signaling pathways. The accumulating evidence has demonstrated that IQGAP1 plays an important role in tumorigenesis and tumor progression. However, the function of IQGAP1 in esophageal squamous cell carcinoma (ESCC) has not been thoroughly investigated. In the present study, we showed that IQGAP1 was overexpressed in ESCC tumor tissues, and its overexpression was correlated with the invasion depth of ESCC. Importantly, by using RNA interference (RNAi) technology we successfully silenced IQGAP1 gene in two ESCC cell lines, EC9706 and KYSE150, and for the first time found that suppressing IQGAP1 expression not only obviously reduced the tumor cell growth, migration and invasion in vitro but also markedly inhibited the tumor growth, invasion, lymph node and lung metastasis in xenograft mice. Furthermore, Knockdown of IQGAP1 expression in ESCC cell lines led to a reversion of epithelial to mesenchymal transition (EMT) progress. These results suggest that IQGAP1 plays crucial roles in regulating ESCC occurrence and progression. IQGAP1 silencing may therefore develop into a promising novel anticancer therapy.     

miR-32 promotes esophageal squamous cell carcinoma metastasis by targeting CXXC5

MicroRNA-32 (miR-32) functioned as a tumor oncogene in some cancer, which control genes involved in important biological and pathological functions and facilitate the tumor growth and metastasis. However, the role of miR-32 modulates esophageal squamous cell carcinoma (ESCC) malignant transformation has not been clarified. Here, we focused on the function and the underlying molecular mechanism of miR-32 in ESCC. Results discovered a significant increased expression of miR-32 in ESCC tissues and cells. Downregulation of miR-32 inhibited the migration, invasion, adhesion of ESCC cell lines (EC9706 and KYSE450), and the levels of EMT protein in vitro. In vivo, miR-32 inhibitors decrease tumor size, tumor weight, and the number of metastatic nodules. Hematoxylin and eosin (H&E) results revealed that inhibition of miR-32 attenuate lung metastasis. Immunohistochemistry and immunofluorescence assay showed increased level of E-cadherin and decreased level of N-cadherin and Vimentin with treatment of miR-32 inhibitors. Furthermore, miR-32 targeted the 3′-untranslated region (3′-UTR) of CXXC5, and inhibited the level of mRNA and protein of CXXC5. There is a negative correlation between the expressions of CXXC5 and miR-32. Then, after EC9706 and KYSE450 cells cotransfected with si-CXXC5 and miR-32 inhibitors, the ability of cell migration, invasion, and adhesion was significantly reduced. In addition, the protein expression of EMT and TGF-β signaling was also depressed. Collectively, these data supply an insight into the positive role of miR-32 in ESCC progression and metastasis, and its biological effects may attribute the inhibition of TGF-β signaling mediated by CXXC5.     

Chloride intracellular channel 1 promotes esophageal squamous cell carcinoma proliferation via mTOR signalling

ObjectivesTo investigate the clinical significance of Chloride Intracellular Channel 1 (CLIC1) expression in esophageal squamous cell carcinoma (ESCC) and its functional contribution and molecular mechanisms to the progression of ESCC.MethodsCLIC1 expression was analyzed by immunohistochemistry (IHC) in a cohort of 86 ESCC tissue specimens and paired normal adjacent esophageal tissues. Associations between clinicopathological features of ESCC and CLIC1 expression were determined. In vitro analyses examined CLIC1 expression in the ESCC cell lines KYSE150 and TE1 using RT-PCR and Western blotting. The downstream pathways of CLIC1 were detected by lentiviral shRNA knockdown and subsequent proteomic analyses. CLIC1 siRNA knockdown was performed in ESCC cell lines KYSE150 and TE1 and the functional effects of CLIC1 on the growth and proliferation of ESCC cells were evaluated combined with cell viability and colony formation assays; the mTOR signaling pathway-related proteins were detected by Western blotting based on the previous proteomic data.ResultsCLIC1 expression was significantly increased in ex vivo ESCC tissues compared with corresponding normal tissues, and the up-regulation was associated with clinical tumor node metastasis (TNM) classifications. Knockdown of CLIC1 inhibited in vitro cell proliferation of ESCC cell lines KYSE150 and TE1. CLIC1 knockdown down-regulated the protein expression of p-mTOR and the downstream targets Rictor and p-4EBP1 in both KYSE150 and TE1 cell lines. And the CLIC1 knockdown induced inhibition of cell proliferation on ESCC cells could be rescued by mTOR overexpression.ConclusionsCLIC1 expression increases during esophageal carcinogenesis and it may functionally contribute to the progression of ESCC through growth promotion effects by promoting the mTOR and downstream signaling pathway. CLIC1 therefore constitutes a candidate molecular biomarker of ESCC.     

Potential role of P2X7R in esophageal squamous cell carcinoma proliferation

Esophageal cancer is an aggressive tumor and is the sixth leading cause of cancer death worldwide. ATP is well known to regulate cancer progression in a variety of models by different mechanisms, including P2X7R activation. This study aimed to evaluate the role of P2X7R in esophageal squamous cell carcinoma (ESCC) proliferation. Our results show that treatment with high ATP concentrations induced a decrease in cell number, cell viability, number of polyclonal colonies, and reduced migration of ESCC. The treatment with the selective P2X7R antagonist A740003 or siRNA for P2X7 reverted this effect in the KYSE450 cell line. In addition, results showed that P2X7R is highly expressed, at mRNA and protein levels, in KYSE450 lineage. Additionally, KYSE450, KYSE30, and OE21 cells express P2X3R, P2X4R, P2X5R, P2X6R, and P2X7R genes. P2X1R is expressed by KYSE30 and KYSE450, and only KYSE450 expresses the P2X2R gene. Furthermore, esophageal cancer cell line KYSE450 presented higher expression of E-NTPDases 1 and 2 and of Ecto-5′-NT/CD73 when compared to normal cells. This cell line also exhibits ATPase, ADPase, and AMPase activity, although in different levels, and the co-treatment of apyrase was able to revert the antiproliferative effects of ATP. Moreover, results showed high immunostaining for P2X7R in biopsies of patients with esophageal carcinoma, indicating the involvement of this receptor in the growth of this type of cancer. The results suggest that P2X7R may be a potential pharmacological target to treat ESCC and can lead us to further investigate the effect of this receptor in cancer cell progression.     

长链非编码RNA BANCR在食管鳞癌中的表达及对细胞增殖和侵袭能力的影响

目的:探索长链非编码RNA BANCR与食管鳞癌(esophageal squamous cell carcinoma ESCC)临床病理特征以及预后的关系,以及对于ESCC细胞增殖,迁移和侵袭能力的影响。方法:使用实时荧光定量PCR(q RT-PCR)技术检测ESCC组织及多个细胞系中BANCR的表达水平,分析其与临床病理特征及预后的关联,用小干扰RNA(si RNA)干扰BANCR后用CCK8法检测其对ESCC细胞生长的影响,使用transwell法检测对细胞侵袭和转移能力的影响。结果:相对于癌旁组织,有86%(123/142)的癌组织中BANCR表达量升高,BANCR在癌组织中的相对表达水平与肿瘤的组织学分级、TNM分期和淋巴结转移数量相关(P均0.05)。BANCR在本文涉及的八株ESCC细胞中的表达量均高于正常食管上皮细胞(Het1A)。在TE10和KYSE30细胞中敲降BANCR后可明显降低细胞生长速率,并抑制细胞的侵袭和迁移能力(P0.01)。结论:BANCR在ESCC组织和细胞中表达显著上调。并能增强ESCC细胞的增殖和侵袭能力,有希望成为一种新的辅助ESCC早期诊断和预后判断的肿瘤分子标志物。     

Direct binding of occupied urokinase receptor (uPAR) to LDL receptor-related protein is required for endocytosis of uPAR and regulation of cell surface urokinase activity

Low-density lipoprotein receptor-related protein (LRP) mediates internalization of urokinase:plasminogen activator inhibitor complexes (uPA:PAI-1) and the urokinase receptor (uPAR). Here we investigated whether direct interaction between uPAR, a glycosyl-phosphatidylinositol-anchored protein, and LRP, a transmembrane receptor, is required for clearance of uPA:PAI-1, regeneration of unoccupied uPAR, activation of plasminogen, and the ability of HT1080 cells to invade extracellular matrix. We found that in the absence of uPA:PAI-1, uPAR is randomly distributed along the plasma membrane, whereas uPA:PAI-1 promotes formation of uPAR-LRP complexes and initiates redistribution of occupied uPAR to clathrin-coated pits. uPAR-LRP complexes are endocytosed via clathrin-coated vesicles and traffic together to early endosomes (EE) because they can be coimmunoprecipitated from immunoisolated EE, and internalization is blocked by depletion of intracellular K(+). Direct binding of domain 3 (D3) of uPAR to LRP is required for clearance of uPA-PAI-1-occupied uPAR because internalization is blocked by incubation with recombinant D3. Moreover, uPA-dependent plasmin generation and the ability of HT1080 cells to migrate through Matrigel-coated invasion chambers are also inhibited in the presence of D3. These results demonstrate that GPI-anchored uPAR is endocytosed by piggybacking on LRP and that direct binding of occupied uPAR to LRP is essential for internalization of occupied uPAR, regeneration of unoccupied uPAR, plasmin generation, and invasion and migration through extracellular matrix.     

Low density lipoprotein (LDL) receptor-related protein 1B impairs urokinase receptor regeneration on the cell surface and inhibits cell migration

The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a newly identified member of the LDL receptor family and is closely related to LRP. It was discovered as a putative tumor suppressor and is frequently inactivated in lung cancer cells. In the present study, we used an LRP1B minireceptor (mLRP1B4), which mimics the function and trafficking of LRP1B, to explore the roles of LRP1B on the plasminogen activation system. We found that mLRP1B4 and urokinase plasminogen activator receptor (uPAR) form immunoprecipitable complexes on the cell surface in the presence of complexes of uPA and its inhibitor, plasminogen activator inhibitor type-1 (PAI-1). However, compared with cells expressing the analogous LRP minireceptor (mLRP4), cells expressing mLRP1B4 display a substantially slower rate of uPA.PAI-1 complex internalization. Expression of mLRP1B4, or an mLRP4 mutant deficient in endocytosis, leads to an accumulation of uPAR at the cell surface and increased cell-associated uPA and PAI-1 when compared with cells expressing mLRP4. In addition, we found that expression of mLRP1B or the mLRP4 endocytosis mutant impairs the regeneration of unoccupied uPAR on the cell surface and that this correlates with a diminished rate of cell migration. Taken together, these results demonstrate that LRP1B can function as a negative regulator of uPAR regeneration and cell migration.     

The LRP1-independent mechanism of PAI-1-inudced migration in CpG-ODN activated macrophages

CpG-oligodeoxynucleotides (CpG-ODNs) induces plasminogen activator inhibitor type-1 (PAI-1) expression in macrophages, leading to enhanced migration through vitronectin. However, the precise role of low-density lipoprotein receptor-related protein 1 (LRP1) in PAI-1 induced migration of macrophages in the inflammatory environment is not known. In this study, we elucidated a novel mechanism describing the altered role of LRP1 in macrophage migration depending on the activation state of the cells. Experimental evidence clearly shows that the blocking of LRP1 function inhibited the PAI-induced migration of resting RAW 264.7 cells through vitronectin but exerted a pro-migratory effect on CpG-ODN-activated cells. We also demonstrate that CpG-ODN downregulates the protein and mRNA levels of LRP1 both in vivo and in vitro, a function that depends on the NF-κB signaling pathway, resulting in reduced internalization of PAI-1. This work illustrates the distinct mechanism that PAI-1-induced migration of CpG-ODN-activated cells through vitronectin depends on the interaction of PAI-1 with vitronectin but not LRP1 unlike in the resting cells, where the migration is LRP1 dependent and vitronectin independent. In conclusion, our experimental results demonstrate the altered function of LRP1 in the migration of resting and activated macrophages in the context of microenvironmental extracellular matrix components.