TFPI or uPA-PAI-1 complex affect cell function through expression variation of type II very low density lipoprotein receptor

Very low density lipoprotein receptors (VLDLR) including type I and type II are known to affect cell functions by binding to its extracellular ligands. However, the effect of these ligands on VLDLR expression remains elusive. Tissue factor pathway inhibitor (TFPI) and urokinase plasminogen activator and plasminogen activator inhibitor 1 (uPA-PAI-1) complex, two ligands of VLDLR, were used to examine their effects on VLDLR expression. TFPI treatment decreased type II VLDLR expression, inhibited cell proliferation and migration, and degradated β-catenin in SGC7901 cells. However, uPA-PAI-1 complex, increased type II VLDLR expression with promoted cell proliferation and migration and stabilization of β-catenin. These results indicated that extracellular ligands can change the expression of type II VLDLR to affect cell proliferation and migration.     

MiR-935通过其靶基因Notch1调控胃癌SGC7901细胞的发生发展

目的:探究Mi R-935调控胃癌SGC7901细胞的增殖和浸润与Notch1基因表达的关系。方法:分别检测40例正常人胃粘膜组织与40例胃印戒细胞癌的Notch1表达情况,并分析胃印戒细胞癌组织中Notch1表达与患者年龄、性别、组织进展程度、TNM分期、有无淋巴结转移的关系;采用Mi R-935转染体外培养的SGC7901细胞系,检测Notch1的表达情况,其后采用Mi R-935抑制剂处理,通过Transwell实验检测胃癌细胞的侵袭能力,细胞划痕实验检测胃癌细胞迁移能力。结果:正常人胃粘膜组织中Notch1表达呈阴性,而胃印戒细胞癌组织中Notch1表达呈阳性;Notch1的表达与胃印戒细胞癌的TNM分期、有无淋巴结转移存在着显著的相关性;转染Mi R-935的SGC7901细胞Notch1表达明显上调,采用Mi R-935抑制剂处理后,Notch1的表达显著下降。结论:Mi R-935可能通过调控Notch1的表达调控胃癌的扩增和浸润。     

Silencing of lncRNA ZFAS1 inhibits malignancies by blocking Wnt/β-catenin signaling in gastric cancer cells

Gastric cancer is a common malignancy with high mortality. Long noncoding RNA (lncRNA) zinc finger antisense (ZFAS)1 is upregulated in gastric cancer specimens compared with the para-carcinoma tissues. The silencing of ZFAS1 inhibited the growth, proliferation, cell cycle progress, migration, invasion and epithelial-mesenchymal transition (EMT), and enhanced the sensitivity to cis-platinum or paclitaxel in SGC7901 cells, as evidenced by the expression changes of proliferating cell nuclear antigen, Cyclin D1, Cyclin E, Cyclin B1, E-cadherin, N-cadherin, vimentin, matrix metalloproteinase (MMP)-2 and MMP-14. The ZFAS1 also activated the Wnt/β-catenin signaling. Subsequently, the ZFAS1 knockdown-induced the inhibition of migration, invasion, EMT and resistance to chemotherapeutic reagens was reversed by the overexpression of β-catenin. In summary, the silencing of ZFAS1 inhibited the growth, proliferation, cell cycle progress, migration, invasion, EMT and chemotherapeutic tolerance by blocking the Wnt/β-catenin signaling in gastric cancer cells.     

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。     

人多肽N-乙酰氨基半乳糖转移酶2基因反义RNA表达质粒的构建及其功能的初步研究

反义封闭人多肽N-乙酰氨基半乳糖转移酶2 (pp-GalNAc-T2)的基因表达, 对胃癌细胞SGC7901中转化生长因子-β1(TGF-β1)与基质金属蛋白酶2 (MMP2)基因表达及细胞增殖有影响.在对几株肿瘤细胞的pp-GalNAc-T2基因表达水平进行分析后, 以高表达pp-GalNAc-T2的人胃癌细胞株SGC7901的总RNA为模板, 利用RT-PCR方法扩增两段不同长度pp-GalNAc-T2基因片段, 构建反义表达载体转染胃癌细胞SGC7901, 通过Ｇ418筛选, 建立一系列旨在封闭胃癌细胞SGC7901 ppGalNAc-T2基因表达的亚细胞克隆.通过流式细胞术、荧光显微镜、RT-PCR及Western印迹检测反义封闭pp-GalNAc-T2基因RNA表达后胃癌细胞SGC7901增殖以及TGF-β1、MMP2表达水平的变化. 反义封闭pp-GalNAc-T2基因表达后, 胃癌细胞SGC7901 pp-GalNAc-T2的表达水平明显降低, 细胞分裂增殖减慢, 表明反义封闭pp-GalNAc-T2基因表达对胃癌细胞SGC7901的生长增殖有影响.结果还显示, 反义封闭pp-GalNAc-T2基因表达可使TGF-β1、MMP2基因在mRNA与蛋白质表达水平均增加, 提示pp-GalNAc-T2基因表达可能对胃癌细胞SGC7901浸润转移产生影响.以上结果表明, pp-GalNAc-T2基因在肿瘤细胞中广泛表达, 并可能与肿瘤的增殖及浸润转移相关.     

Nampt及高糖高胰岛素微环境对人顺铂耐药肺癌细胞株增殖和转移的影响

摘要 目的：探究烟酰胺磷酸核糖转移酶（Nampt）及高糖高胰岛素（HG+HI）微环境对人顺铂耐药肺癌细胞增殖和转移的影响。方法：将人顺铂耐药细胞株A549/DDP分为6组（n=6）：对照组（control）、高糖高胰岛素干预组（HH，使用添加30 mmol/L的葡萄糖和500 mU/L的胰岛素的培养基培养72 h）、分别转染sh-NC和sh-Nampt组（sh-NC和sh-Nampt，使用Lipofectamine 2000将sh-NC和sh-Nampt分别转染到细胞中，转染时间为48 h）、HH干预sh-NC和sh-Nampt组（HH+sh-NC和HH+sh-Nampt）。每组6个重复样本。qRT-PCR检测转染效率，MTT法检测细胞增殖，流式细胞仪检测细胞凋亡，Transwell检测细胞迁移和侵袭，qRT-PCR检测Nampt mRNA，Western blot检测Nampt、Bcl-2、Bax、MMP-2、MMP-9、p-PI3K、PI3K、p-AKT和AKT蛋白表达。结果：与对照组和sh-NC组比较，sh-Nampt组的Nampt mRNA和蛋白表达水平、相对细胞活力、迁移和侵袭细胞数量降低，而细胞凋亡率升高，Bcl-2、MMP-2、MMP-9、Nampt、p-PI3K和p-AKT蛋白表达水平降低，Bax蛋白表达水平升高（P<0.005）。与对照组和sh-NC组比较，HH组和HH+sh-NC组的Nampt mRNA和蛋白表达水平、相对细胞活力、迁移和侵袭细胞数量升高，Bcl-2、MMP-2、MMP-9、Nampt、p-PI3K和p-AKT蛋白表达水平升高，Bax蛋白表达水平降低（P<0.005）。与HH组和HH+sh-NC组比较，HH+sh-Nampt组的Nampt mRNA和蛋白表达水平、相对细胞活力、迁移和侵袭细胞数量降低，细胞凋亡率升高，Bcl-2、MMP-2、MMP-9、Nampt、p-PI3K和p-AKT蛋白表达水平降低，Bax蛋白表达水平升高（P<0.005）。结论：高糖高胰岛素微环境可能通过上调Nampt/PI3K/AKT信号通路诱导人顺铂耐药肺癌细胞的增殖和转移。     

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.     

比较NRP-1反义寡核苷酸与VEGFR-2反义寡核苷酸对人胃癌SGC7901细胞增殖和凋亡的影响

目的:研究比较神经纤毛蛋白1(NRP-1)反义寡核苷酸(ASODN)与血管内皮生长因子受体2(VEGFR-2)反义寡核苷酸(ASODN)对人胃癌SGC7901细胞增殖活性及凋亡水平的影响。 方法:分别及同时将不同浓度经硫代磷酸化修饰的NRP-1 ASODN 和 VEGFR-2 ASODN 转染入人胃癌SGC7901细胞,逆转录-聚合酶链反应(RT-PCR)检测NRP-1基因和VEGFR-2 基因mRNA的转录水平;MTT比色法测量细胞的增殖活性;流式细胞仪测量细胞的凋亡水平。 结果:转染NRP-1 ASODN和VEGFR-2 ASODN后,人胃癌SGC7901细胞NRP-1基因和VEGFR-2 基因mRNA的转录水平均出现降低;NRP-1 ASODN和VEGFR-2 ASODN对SGC7901细胞有明显抑制增殖和促进凋亡的作用,且随着ASODN浓度升高而增强;分别转染时其作用无显著差别,联合转染时其作用明显增强。结论:NRP-1 ASODN和VEGFR-2 ASODN可抑制人胃癌SGC7901细胞 NRP-1基因和VEGFR-2 基因mRNA的转录水平及细胞增殖活性,促进细胞凋亡;与分别转染相比,两者联合转染作用明显增强。     

Melatonin inhibits cell growth and migration,but promotes apoptosis in gastric cancer cell line,SGC7901

Abstract

The pineal hormone, melatonin (MLT), has been shown to have therapeutic effects in patients with gastric cancer; however, the mechanisms for the anti-cancer effects are unknown. We investigated the effects of melatonin on cell proliferation, apoptosis, colony formation and cell migration in the gastric adenocarcinoma cell line, SGC7901, using MTT assay, Hoechst 33258 staining, flow cytometry, western blot, caspase-3 activity assay, soft agar colony formation assay, and scratch-wound assay. Our results showed that melatonin could inhibit cell proliferation, colony formation and migration efficiency, and it promoted apoptosis of SGC7901 cells. Our findings suggest that the anti-cancer effects of melatonin may be due to both inhibition of tumor cell proliferation and reduction of the metastatic potential of tumor cells.     

Long non-coding RNA Linc00320 inhibits glioma cell proliferation through restraining Wnt/β-catenin signaling

Recent efforts have revealed that numerous oncogenic lncRNAs have been found play pivotal role in Glioma progression while there is little know about anti-oncogenic lncRNAs in Glioma. In current study, we found a HMGB1 regulated lncRNA, Linc00320, is significantly decreased in Glioma malignant tissues and its low expression predicts poor prognosis. Moreover, we found that the nucleus localized Linc00320 inhibits Glioma cell proliferation both in vitro and in vivo. In addition, we found that Linc00320 binds to β-catenin and inhibits the activity of Wnt/β-catenin signaling by disrupting β-catenin binds to TCF4 in Glioma cells. Taken together, we firstly demonstrated the tumor suppressive lncRNA, Linc00320, is down-regulated in Glioma tissues and inhibits Glioma cell proliferation by restraining Wnt/β-catenin signaling through segregating β-catenin and TCF4 and revealed the novel HMGB1/Linc00320/β-catenin axis in Glioma progression.     

Methylation of DACT2 Promotes Papillary Thyroid Cancer Metastasis by Activating Wnt Signaling

Thyroid cancer is the most common endocrine malignant disease and the incidence is increasing. DACT2 was found frequently methylated in human lung cancer and hepatocellular carcinoma. To explore the epigenetic change and the role of DACT2 in thyroid cancer, 7 thyroid cancer cell lines, 10 cases of non-cancerous thyroid tissue samples and 99 cases of primary thyroid cancer samples were involved in this study. DACT2 was expressed and unmethylated in K1, SW579, FTC-133, TT, W3 and 8505C cell lines. Loss of expression and complete methylation was found in TPC-1 cells. Restoration of DACT2 expression was induced by 5-aza-2′deoxycytidine treatment. It demonstrates that the expression of DACT2 was regulated by promoter region methylation. In human primary papillary thyroid cancer, 64.6% (64/99) was methylated and methylation of DACT2 was related to lymph node metastasis (p<0.01). Re-expression of DACT2 suppresses cell proliferation, invasion and migration in TPC-1 cells. The activity of TCF/LEF was inhibited by DACT2 in wild-type or mutant β-catenin cells. The activity of TCF/LEF was increased by co-transfecting DACT2 and Dvl2 in wild-type or mutant β-catenin cells. Overexpression of wild-type β-catenin promotes cell migration and invasion in DACT2 stably expressed cells. The expression of β-catenin, c-myc, cyclinD1 and MMP-9 were decreased and the level of phosphorylated β-catenin (p-β-catenin) was increased after restoration of DACT2 expression in TPC-1 cells. The expression of β-catenin, c-myc, cyclinD1 and MMP-9 were increased and the level of p-β-catenin was reduced after knockdown of DACT2 in W3 and SW579 cells. These results suggest that DACT2 suppresses human papillary thyroid cancer growth and metastasis by inhibiting Wnt signaling. In conclusion, DACT2 is frequently methylated in papillary thyroid cancer. DACT2 expression was regulated by promoter region methylation. DACT2 suppresses papillary thyroid cancer proliferation and metastasis by inhibiting Wnt signaling.     

Differential LEF1 and TCF4 expression is involved in melanoma cell phenotype switching

Recent observations suggest that melanoma cells drive disease progression by switching back and forth between phenotypic states of proliferation and invasion. Phenotype switching has been linked to changes in Wnt signalling, and we therefore looked for cell phenotype-specific differences in the levels and activity of β-catenin and its LEF/TCF co-factors. We found that while cytosolic β-catenin distribution is phenotype-specific (membrane-associated in proliferative cells and cytosolic in invasive cells), its nuclear distribution and activity is not. Instead, the expression patterns of two β-catenin co-factors, LEF1 and TCF4, are both phenotype-specific and inversely correlated. LEF1 is preferentially expressed by differentiated/proliferative phenotype cells and TCF4 by dedifferentiated/invasive phenotype cells. Knock-down experiments confirmed that these co-factors are important for the phenotype-specific expression of M-MITF, WNT5A and other genes and that LEF1 suppresses TCF4 expression independently of β-catenin. Our data show that melanoma cell phenotype switching behaviour is regulated by differential LEF1/TCF4 activity.     

IFN-γ Induces Gastric Cancer Cell Proliferation and Metastasis Through Upregulation of Integrin β3-Mediated NF-κB Signaling

Interferon γ (IFN-γ), a multifunctional cytokine, was upregulated in the resected gastric cancer tissue. However, whether IFN-γ is involved in the regulation of gastric cancer has not been well elucidated. Herein, we aimed to investigate the effects and mechanism of IFN-γ on gastric cancer. In this study, we found a vital role of IFN-γ in enhancing proliferation, inhibiting apoptosis, and promoting cell migration and invasion in gastric cancer cells SGC-7901 and MGC-803. Additionally, IFN-γ activated nuclear factor κB (NF-κB) signaling pathway by upregulating the phosphorylation expression of p65 and IκBα, and induced the expression of integrin β3 in vitro. Therefore, to further investigate the relationship between IFN-γ and integrin β3, SGC-7901 cells were transfected with integrin β3 siRNA. And then cells expressed lower cell viability, migration, and invasion rates, while cell apoptosis was significantly enhanced. Meanwhile, expression of integrin β3, MMP-2, MMP-9, and NF-κB, including p65 and IκBα, and the nuclear translocation of NF-κB/p65 were dramatically repressed, whereas IFN-γ significantly improved the effects. Moreover, in vivo, the experiment of xenograft model and pulmonary metastasis model also retarded in integrin β3 siRNA group. And the expression of integrin β3, MMP-2, MMP-9, and NF-κB was repressed. However, the treatment with IFN-γ improved tumor volume, lung/total weight, tumor nodules, and the protein expression described above compared with integrin β3 siRNA group. Overall, the results indicated that IFN-γ induces gastric cancer cell proliferation and metastasis partially through the upregulation of integrin β3-mediated NF-κB signaling. Hence, the inhibition of IFN-γ or integrin β3 may be the key for the treatment of gastric cancer.     

LncRNA MYU对胶质瘤细胞周期分布、增殖、转移和凋亡以及PI3K/Akt信号通路的影响

摘要 目的：探讨长链非编码RNA（LncRNA）MYU对胶质瘤细胞周期分布、细胞增殖、迁移、侵袭和凋亡的影响,并初步探讨其作用机制。方法：实时荧光定量PCR（RT-qPCR）检测人脑正常胶质细胞HEB和胶质瘤细胞（U-251MG、A172、SHG139）中LncRNA MYU的表达情况。选取SHG139细胞,分为正常对照（NC）组、si-con组、si-LncRNA MYU组进行转染实验,行RT-qPCR检测转染效果。分别采用流式细胞术、细胞计数试剂盒（CCK-8）、Transwell实验检测沉默LncRNA MYU对SHG139细胞周期分布和凋亡、细胞增殖、细胞迁移和侵袭的影响。蛋白免疫印迹（Western blot）法检测基质金属蛋白酶2（MMP-2）、MMP-9、裂解的半胱氨酸天冬氨酸蛋白酶3（Cleaved caspase-3）、Cleaved caspase-9以及磷脂酰肌醇-3-羟激酶/蛋白激酶B（PI3K/Akt）信号通路相关蛋白表达情况。结果：LncRNA MYU在胶质瘤细胞株中比人脑正常胶质细胞中的表达水平显著升高（P＜0.05）,因此选择表达量最高的SHG139细胞进行转染实验。沉默LncRNA MYU能够显著诱导SHG139细胞G0-G1期阻滞、抑制细胞增殖、迁移和侵袭并诱导细胞凋亡（P＜0.05）。沉默LncRNA MYU可显著抑制MMP-2、MMP-9、p-PI3K和p-AKT表达并促进Cleaved caspase-3、Cleaved caspase-9表达（P＜0.05）。结论：沉默LncRNA MYU可诱导胶质瘤细胞G0-G1期阻滞,抑制细胞增殖、迁移和侵袭,促进细胞凋亡,其机制可能与抑制PI3K/AKT信号通路有关。     

MicroRNA let-7f inhibits tumor invasion and metastasis by targeting MYH9 in human gastric cancer

Background

MicroRNAs (miRNAs) are important regulators that play key roles in tumorigenesis and tumor progression. A previous report has shown that let-7 family members can act as tumor suppressors in many cancers. Through miRNA array, we found that let-7f was downregulated in the highly metastatic potential gastric cancer cell lines GC9811-P and SGC7901-M, when compared with their parental cell lines, GC9811 and SGC7901-NM; however, the mechanism was not clear. In this study, we investigate whether let-7f acts as a tumor suppressor to inhibit invasion and metastasis in gastric cancers.

Methodology/Principal

Real-time PCR showed decreased levels of let-7f expression in metastatic gastric cancer tissues and cell lines that are potentially highly metastatic. Cell invasion and migration were significantly impaired in GC9811-P and SGC7901-M cell lines after transfection with let-7f-mimics. Nude mice with xenograft models of gastric cancer confirmed that let-7f could inhibit gastric cancer metastasis in vivo after transfection by the lentivirus pGCsil-GFP- let-7f. Luciferase reporter assays demonstrated that let-7f directly binds to the 3′UTR of MYH9, which codes for myosin IIA, and real-time PCR and Western blotting further indicated that let-7f downregulated the expression of myosin IIA at the mRNA and protein levels.

Conclusions/Significance

Our study demonstrated that overexpression of let-7f in gastric cancer could inhibit invasion and migration of gastric cancer cells through directly targeting the tumor metastasis-associated gene MYH9. These data suggest that let-7f may be a novel therapeutic candidate for gastric cancer, given its ability to reduce cell invasion and metastasis.     

KHC-4 inhibits β-catenin expression in prostate cancer cells

ABSTRACT

KHC-4 is a 2-phenyl-4-quinolone analogue that exhibits anticancer activity. Aberrant activation of β-catenin signaling contributes to prostate cancer development and progression. Therefore, targeting β-catenin expression could be a useful approach to treating prostate cancer. We found that KHC-4 can inhibit β-catenin expression and its signaling pathway in DU145 prostate cancer cells. Treatment with KHC-4 decreased total β-catenin expression and concomitantly decreased β-catenin levels in both the cytoplasm and nucleus of cells. KHC-4 treatment also inhibited β-catenin expression and that of its target proteins, PI3K, AKT, GSK3β and TBX3. We monitored the stability of β-catenin with the proteasomal inhibitor, MG132, in DU145 cells and found that MG132 reversed KHC-4-induced proteasomal β-catenin degradation. We verified CDK1/β-catenin expression in KHC-4 treated DU145 cells. We found that roscovitine treatment reversed cell proliferation by arresting the cell cycle at the G2/M phase and β-catenin expression caused by KHC-4 treatment. We suggest that KHC-4 inhibits β-catenin signaling in DU145 prostate cancer cells.