Knockdown of BDNF suppressed invasion of HepG2 and HCCLM3 cells, a mechanism associated with inactivation of RhoA or Rac1 and actin skeleton disorganization

Brain-derived neurotrophic factor (BDNF) and its primary receptor tropomysin-related kinase B (TrkB) mediate critical signalings for supporting survival and growth of neurons. Even though we have previously confirmed that more expressions of BDNF and TrkB were closely correlated with multiple and advanced hepatocellular carcinoma (HCC), the exact mechanisms underlying have not been investigated. The expressions of BDNF and TrkB were examined by western blot and BDNF secretion was evaluated by ELISA in human HCC cell lines of HepG2 and HCCLM3 with high metastatic potential. BDNF knockdown was performed by specific BDNF-siRNA transfection in HCC cells, actin cytoskeleton was shown by FITC-phalloidin staining and the activations of RhoA, Rac1 or Cdc42 were determined using western blot. Cell apoptosis and invasion were examined by flow cytometry and transwell assay, respectively. More expressions of BDNF and TrkB were found in HCCLM3 than in HepG2 cells. Inhibited expression of BDNF by specific siRNA showed impaired actin polymerization and decreased activations of RhoA or Rac1 in both HepG2 and HCCLM3 cells. BDNF knockdown also induced apoptosis and suppressed invasion of both HepG2 and HCCLM3 cells. Our results suggested a role of BDNF/TrkB in confering HCCLM3 cells advantage of metastasis, and BDNF knockdown inhibited cell invasion probably through the blocked actin polymerization and the correlated inactivation of RhoA or Rac1. Aiming at BDNF/TrkB signaling interruption may be an effective strategy to prevent HCC progression.     

Annonaceous acetogenins reverses drug resistance of human hepatocellular carcinoma BEL-7402/5-FU and HepG2/ADM cell lines

Hepatocellular carcinoma (HCC) is the most common tumor in worldwide and chemotherapy resistant is a severe obstacle in HCC treatment. Annonaceous acetogenins was a nature compound from Uvaria accuminata and it has show the anti-tumor proliferation activity in many types cancer. In this study, we showed that annonaceous acetogenins is correlated with the drug resistance reversal in human hepatocellular carcinoma BEL-7402/5-FU and HepG2/ADM cell lines. We found that cell apoptosis was improved and cell cycle was arrested, further, multidrug-resistance proteins such as MDR1, MRP1, Topo-IIα, GST-π, cyclin D1, Survivin and bcl-2 are down-regulated, however, intracellular Rh-123 and caspase-3/8 was up-regulated by Annonaceous acetogenins treatment. We also found that there was a decreased activity of NF-κB and Akt in Annonaceous acetogenins treatment groups. Therefore, we demonstrate that Akt/NF-κB pathway was involved in Annonaceous acetogenins reverses drug resistance of human hepatocellular carcinoma cells.     

RPMI-1640和DMEM培养基中肝癌细胞系BEL-7402与HepG-2的生长状态比较

背景：DMEM和RPMI-1640是两种最常用的商品化培养基,二者对肝癌细胞生长的影响尚未见直接的对比研究。 目的：比较两种常用培养基对人肝癌BEL-7402与HepG-2细胞系体外生长和增殖效果的影响,从中选择更适合的培养基。 方法：分别应用高糖DMEM与RPMI-1640完全培养液培养BEL-7402和HepG-2细胞,于培养的0,24,48,72,96,  120 h用酸性磷酸酶检测法测定细胞生长和增殖速率,并于倒置显微镜下观察细胞的形态。 结果与结论：结果发现BEL-7402和HepG-2细胞在RPMI-1640培养基中的生长增殖速率均明显高于DMEM培养基 (P < 0.01)。镜下观察证实细胞在RPMI-1640培养基中的黏附和伸展状态更好。因此,建议首选RPMI-1640培养基进行肿瘤细胞体外培养。     

γ-terpineol inhibits cell growth and induces apoptosis in human liver cancer BEL-7402 cells in vitro

To investigate the effect of γ-terpineol on cell proliferation and apoptosis of human hepatoma BEL-7402 cells to elucidate its molecular mechanism. Here, BEL-7402 cells were treated with various concentrations (40, 80, 160, 320 and 640 μg/ml) of γ-terpineol for 48 h, cell proliferation was determined by 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromides (MTT) assay. Cell colony inhibition was determined by soft agar assay. Apoptosis and possible molecular mechanisms were evaluated by morphological observation, flow cytometry analysis, and DNA fragmentation assay. The γ-terpineol significantly suppressed BEL-7402 cell proliferation in a dose-dependent manner. Characteristic morphological and biochemical changes associated with apoptosis such as cells shrinkage, deformation and vacuolization of mitochondria, nuclear chromatin condensation and fragmentation, formation of apoptotic bodies were observed after BEL-7402 cells treated with γ-terpineol for 24 h and 48 h. Cell cycle were displayed by flow cytometry analysis, the γ-terpineol treatment resulted in accumulation of cells at G₁ or S phase and a blockade of cell proliferation compared to control group. Treating BEL-7402 cells with 320 μg/ml of γ-terpineol for 36 h and 48 h, a typical apoptotic “DNA ladder” was observed using DNA fragmentation assay. The present study demonstrated that possible anti-cancer mechanism of γ-terpineol on human hematomas cells is through inducing cell apoptosis to suppress tumor cell growth.     

Long non-coding RNA CARLo-5 expression is associated with disease progression and predicts outcome in hepatocellular carcinoma patients

Recently, many studies show that long non-coding RNAs (lncRNAs) play important roles in cancer biology. Although its expression was reported dysregulated during tumorigenesis, the contributions of lncRNAs to hepatocellular carcinoma (HCC) are still largely unknown. In particular, the lncRNA CARLo-5 has a functional role in cell-cycle regulation in colon cancer, while the clinical significance and biological function of CARLo-5 in HCC remain unelucidated. In order to fill those study blanks, the expression level of CARLo-5 in human HCC specimens was tested, and its correlation with clinicopathologic features as well as the prognosis for patients with HCC was analyzed. Additionally, MTT, wound healing and transwell assays were employed to investigate the biological function of CARLo-5. The results showed that CARLo-5 levels were significantly overexpressed in HCC tissues compared to ANLT. Besides, high expression of CARLo-5 was associated with liver cirrhosis (P = 0.001), tumor number (P < 0.001), vascular invasion (P = 0.001), capsular formation (P = 0.014) and Edmondson–Steiner grade (P < 0.001), which proved that CARLo-5 was an independent risk factor for overall survival and disease-free survival. In addition, in highly metastatic HCC cell lines (HCCLM3 and MHCC97-L), CARLo-5 was up-regulated, but in lowly metastatic HCC cell lines (HepG2, SNU387), it showed down-regulated. Besides, by using gain and loss of function experiments in HCC cell lines (HCCLM3 and HepG2), the results showed that CARLo-5 overexpression significantly enhanced cell proliferation, migration and invasion in vitro. Our study also revealed that CARLo-5 was prominently up-regulated in HCC specimens and its high expression was associated with poor prognosis of HCC patients. Totally, those findings together indicate that CARLo-5 promotes proliferation and metastasis of HCC and potentially emerged as a novel therapeutic target.     

人核受体hLRH-1不同变异体在多种肿瘤组织和细胞系中的表达

目的:初步分析人核受体hLRH-1不同变异体在多种肿瘤组织和细胞系中的表达。方法:常规RT-PCR法分析hLRH-1v1和hLRH-1在肝癌等多种肿瘤组织和HepG2等肿瘤细胞系中的表达情况，实时定量PCR法分析hLRH-1v1，hLRH-1和hOct4在HepG2，SMMC-7721和BEL-7402三株肝癌细胞系的表达水平。结果:hLRH-1v1的表达见于所有检测的12种类型肿瘤和8种恶性肿瘤细胞系，hLRH-1的表达则仅在12种类型肿瘤中的6种肿瘤组织中可检测到，但8种恶性肿瘤细胞系均为hLRH-1阳性表达；在HepG2，SMMC-7721和BEL-7402三株肝癌细胞系中hLRH-1v1和hOct4的表达呈正相关。结论:hLRH-1v1在肿瘤中广泛表达，可能在维持肿瘤干细胞的自我更新中发挥重要作用。     

含蟾酥胶囊血清诱导人肝癌BEL-7402细胞凋亡并下调Bcl-2的表达

目的 探讨含蟾酥胶囊血清诱导人肝癌BEL-7402细胞凋亡的作用机制.方法 用中药血清药理学方法,不同浓度含药血清加入体外培养的人肝癌BEL-7402细胞,分别孵育24h、48h,显微镜下观察细胞形态学变化,MTT比色法检测细胞存活率,流式细胞仪检测细胞凋亡率,琼脂糖凝胶电泳测定DNA梯状条带,免疫细胞化学染色检测Bc...     

Erythropoietin and erythropoietin receptor in hepatocellular carcinoma: correlation with vasculogenic mimicry and poor prognosis

To evaluate erythropoietin (Epo) and erythropoietin receptor (EpoR) expression, its relationship with vasculogenic mimicry (VM) and its prognostic value in human hepatocellular carcinoma (HCC), we examined Epo/EpoR expression and VM formation using immunohistochemistry and CD31/PAS (periodic acid-Schiff) double staining on 92 HCC specimens. The correlation between Epo/EpoR expression and VM formation was analyzed using two-tailed Chi-square test and Spearman correlation analysis. Survival curves were generated using Kaplan-Meier method. Multivariate analysis was performed using Cox regression model to assess the prognostic values. Results showed positive correlation between Epo/EpoR expression and VM formation (P < 0.05). Patients with Epo or EpoR expression exhibited poorer overall survival (OS) than Epo-negative or EpoR-negative patients (P < 0.05). Epo-positive/VM-positive and EpoR-positive/VM-positive patients had the worst OS (P < 0.05). In multivariate survival analysis, age, Epo and EpoR were independent prognostic factors related to OS. These results will provide evidence for further research on HCC microcirculation patterns and also will provide new possible targets for HCC diagnosis and treatment.     

The Anti-Tumor Effect and Biological Activities of the Extract JMM6 from the Stem-Barks of the Chinese Juglans Mandshurica Maxim on Human Hepatoma Cell Line Bel-7402

Juglans mandshurica Maxim is a traditional herbal medicines in China, and its anti-tumor bioactivities are of research interest. Bioassay-guided fractionation method was employed to isolate anti-tumor compounds from the stem barks of the Juglans mandshurica Maxim. The anti-tumor effect and biological activities of the extracted compound JMM6 were studied in BEL-7402 cells by MTT, Cell cycle analysis, Hoechst 33342 staining, Annexin V-FITC/PI assay and Detection of mitochondrial membrane potential (ΔΨm). After treatment with the JMM6, the growth of BEL-7402 cells was inhibited and cells displayed typical morphological apoptotic characteristics. Further investigations revealed that treatment with JMM6 mainly caused G2/M cell cycle arrest and induced apoptosis in BEL-7402 cells. To evaluate the alteration of mitochondria in JMM6 induced apoptosis. The data showed that JMM6 decreased significantly the ΔΨm, causing the depolarization of the mitochondrial membrane. Our results show that the JMM6 will have a potential advantage of anti-tumor, less harmful to normal cells. This paper not only summarized the JMM6 pick-up technology from Juglans mandshurica Maxim and biological characteristic, but also may provide further evidence to exploit the potential medicine compounds from the stem-barks of the Chinese Juglans mandshurica Maxim.     

阻断Sonic Hedgehog信号对不同人肝癌细胞生长的影响

 目的: 研究阻断Sonic Hedgehog (Shh)信号对不同人肝癌细胞生长的影响,探讨阻断Shh信号抑制肝癌细胞生长的机制。方法: RT-PCR法检测Shh信号分子在3株人肝癌细胞(BEL-7402、Huh7和HepG2)中的表达,并检测Shh阻断抗体作用后BEL-7402细胞Shh信号效应分子表达变化;MTT法检测人肝癌细胞增殖活性;流式细胞术检测人肝癌细胞凋亡;Western blot 检测凋亡相关蛋白表达。结果: Shh信号分子在3株人肝癌细胞中均有表达,Shh阻断抗体可以下调Shh信号效应分子patched (Ptch)、Gli1和Gli2的表达;Shh阻断抗体可以抑制3株肝癌细胞生长,增加G₀/G₁期细胞,并诱导细胞凋亡;Shh阻断抗体作用后,BEL-7402细胞pro-caspase-3、pro-caspase-8和pro-caspase-9蛋白表达水平下降,cleaved caspase-3、cleaved caspase-8和cleaved caspase-9蛋白表达水平升高。结论: 阻断Shh信号可抑制Shh高表达的人肝癌细胞生长,阻滞细胞周期于G₀/G₁期,并诱导肝癌细胞凋亡。     

三氧化二砷对肝癌细胞生长抑制作用差异性探讨

目的:观察三氧化二砷对肝癌SMMC-7721和BEL-7402细胞生长抑制作用差异性并且探讨其可能的作用机理。方法: 应用细胞培养和台盼蓝拒染法观察不同时间和不同浓度的三氧化二砷对肝癌细胞SMMC-7721和BEL-7402细胞生长的抑制作用,比较生长抑制率的差异并用谷胱甘肽检测试剂盒测定两种肝癌细胞内谷胱甘肽(GSH)的含量。结果: 三氧化二砷浓度为0.50 μmol/L、作用时间为24 h可显著抑制肝癌细胞系BEL-7402的生长,抑制作用呈时间、剂量依赖性;对SMMC-7721细胞,三氧化二砷浓度为2.00 μmol/L、作用时间为24 h才出现抑制细胞生长作用,二者的生长抑制率存在显著差异,0.25-2.00 μmol/L As₂O₃作用72 h后,BEL-7402细胞的生长抑制率均高于SMMC-7721细胞(P＜0.05)。检测SMMC-7721和BEL-7402细胞内GSH含量分别为(50.8±5.2)μmol/g protein和(18.7±1.4)μmol/g protein,存在明显差异。 结论:三氧化二砷抑制肝癌细胞SMMC-7721和BEL-7402的生长存在显著的差异;BEL-7402细胞对三氧化二砷非常敏感性,可能与其细胞内谷胱甘肽含量较低,细胞的氧化还原解毒系统不足有关。     

miR-195 is a key negative regulator of hepatocellular carcinoma metastasis by targeting FGF2 and VEGFA

Hepatocellular carcinoma (HCC) is the most common primary tumor of liver and the fifth most common cancer in the world. Lung is the most frequent site for extra hepatic metastasis from hepatocellular carcinoma, while the cause and mechanism of it is still poor understood. Here, we identify that the expression of miR-195 is markedly impaired in the lung metastasis cell lines of HCC. The result of Real-time PCR reveals the expression of miR-195 is significantly downregulated in 92 HCC tissues. Low expression of miR-195 is associated with tumor size, portal vein thrombosis, TNM stage and patients survival. Luciferase reporter and ELISA assay prove that hematogenous metastasis related genes including FGF2 and VEGFA are the target genes of miR-195. Overexpression of miR-195 in HCC cell line BEL-7402 markedly inhibits the capability of migration and invasion. Taken together, our results suggest that miR-195, a tumor suppressor miRNA, contributes to the lung metastasis of HCC by negatively regulating FGF2 and VEGFA, providing key implications of miR-195 for the therapeutic intervention of HCC.     

环氧合酶2抑制剂对肝肿瘤细胞的血管内皮生长因子表达的影响

目的探讨肝肿瘤细胞环氧合酶2(COX-2)和血管内皮生长因子(VEGF)关系。方法实验分为对照组和实验组,培养肝癌细胞株(BEL-7402)作为对照组,培养肝癌细胞株用不同浓度赛来昔布(celeccoxib商品名:西乐葆)处理作为实验组;提取mRNA,用ELISA方法检测细胞培养基中VEGF水平,用RT-PCR分别检测COX-2和VEGF的mRNA。结果正常培养的肝癌细胞能同时检测到COX-2和VEGF的mRNA;COX-2抑制剂赛来昔布能抑制VEGF的表达;COX-2在mRNA水平调控VEGF的表达。结论COX-2在VEGF的分泌与合成中起重要作用,从而影响肿瘤新生血管生成及肿瘤生长。     

NK细胞对不同人肝癌细胞株的杀伤作用

目的: 观察自然杀伤(NK)细胞对不同肝癌细胞株的体内外抑瘤作用,并检测肝癌细胞MHC-I类链相关蛋白(MIC蛋白)的表达。方法: 抽取志愿者外周血50 mL,分离单个核细胞,置入NK细胞试剂盒行孵化及逐级扩增。计算NK细胞对人白血病细胞株K562及人肝癌细胞株BEL7402、HepG2、SMMC7721的杀伤率。接种建立人肝癌细胞株裸鼠移植瘤,进行NK细胞瘤内及瘤周注射;计算各组裸鼠移植瘤的体积,绘制各组肿瘤生长曲线;处死裸鼠,称瘤重,计算NK细胞对各组肿瘤抑制率。检测人肝癌细胞表面 MIC蛋白表达。结果: NK细胞对K562细胞杀伤最强,BEL-7402次之,SMMC-7721细胞株杀伤敏感性最低。裸鼠抑瘤实验结果显示NK细胞对于BEL-7402细胞株的抑瘤效果最好,而对于SMMC-7721细胞株的抑瘤效果较差。BEL-7402及HepG2细胞株表面表达MIC,而SMMC-7721则很少表达。结论: NK细胞对于不同人肝癌细胞株体内外抑瘤作用不同,其差异可能和不同肝癌细胞株MIC的表达有关。     

蛋白激酶CβⅡ诱导上皮-间质转化及血管新生促进肝癌发展

目的 探讨蛋白激酶CβⅡ（PKCβⅡ）在肝细胞癌(HCC)发展中的作用机制。方法 免疫印迹法观察PKCβⅡ在肝细胞系L02和肝癌细胞系SK-hep1、HepG2、BEL-7404、7721、Hep3B和huh7中的表达，构建稳定高表达PKCβⅡ的细胞系，倒置相差显微镜下观察细胞形态变化，免疫荧光观察E-钙黏蛋白（E-cadherin）、N-钙黏蛋白（N-cadherin）表达的变化；通过免疫印迹法、实时定量聚合酶链反应(Real-time PCR)和放线菌酮（CHX）追踪实验，观察PKCβⅡ调控E-cadherin、N-cadherin和Snail表达的分子机制；小室迁移和侵袭实验(transwell assay)以及裸鼠尾静脉注射观察PKCβⅡ对肝癌细胞转移的影响；成管实验观察PKCβⅡ对人脐静脉内皮细胞（HUVECs）血管形成能力的影响，酶联免疫吸附法（ELISA）观察PKCβⅡ对肝癌细胞上清中血管内皮生长因子A（VEGFA）含量的影响。结果 PKCβⅡ在肝癌细胞系中的表达高于肝细胞系L02，PKCβⅡ促进肝癌细胞形态从鹅卵石样上皮细胞向梭行间质样细胞的转变，通过mRNA水平下调E-cadherin（P<0.05）和上调N-cadherin（P<0.01）的蛋白表达，通过翻译水平上调Snail蛋白表达，PKCβⅡ还促进了肝癌细胞的迁移、侵袭（P<0.01）以及VEGFA的分泌（P<0.01）和血管新生（P<0.01）。结论 蛋白激酶CβⅡ诱导上皮-间质转化及血管新生在肝癌的发展中有重要作用。     

Proliferation and migration mediated by Dkk-1/Wnt/beta-catenin cascade in a model of hepatocellular carcinoma cells

Beta-catenin is a multifunctional protein acting as a key factor in the cadherin-mediated cell-cell adhesion system and in the Wnt signaling pathway. To demonstrate the molecular mechanisms of metastasis of hepatocellular carcinoma (HCC) cells, we established a metastatic subclone of human HCC H7402 cells, termed M-H7402, by isolating from transplantation of H7402 cells into severe combined immunodeficient (SCID) mice. Based on the 2 parallel cell lines, we investigated the roles of dickkopf-1 (Dkk-1) and Wnt/beta-catenin pathway in proliferation and migration of HCC cells. cDNA microarray showed that 24 genes were related to tumor metastasis differentially expressed between H7402 and M-H7402 cells. Western blot analysis revealed that the expression levels of beta-catenin, c-Myc, and cyclin D1 were upregulated, but Dkk-1 and nm23 were dramatically downregulated in M-H7402 cells, which suggests that the 2 cell lines were remarkably different in molecular events associated with metastasis. Furthermore, we found that overexpression of Dkk-1 by transfection was able to downregulate the expression of c-Myc and cyclin D1, and it also inhibited the growth and migration in M-H7402 cells. Although reduction of Dkk-1 expression by RNA interference was able to upregulate the expression of beta-catenin, c-Myc, and cyclin D1 in H7402 cells, it also promoted beta-catenin translocation from cytoplasm into nuclei and increased the migration of the cells. Therefore, we conclude that Dkk-1/Wnt/beta-catenin cascade may mediate the proliferation and migration of HCC cells during the metastasis process.     

UBE2O promotes hepatocellular carcinoma cell proliferation and invasion by regulating the AMPKα2/mTOR pathway

The ubiquitin-conjugating enzyme (E2) is a critical component of the ubiquitin-proteasome system and regulates hepatocarcinogenesis by controlling protein degradation. Ubiquitin-conjugating enzyme E2 O (UBE2O), a member of the E2 family, functions as an oncogene in human cancers. Nevertheless, the role of UBE2O in hepatocellular carcinoma (HCC) remains unknown yet. Here, we demonstrated that the UBE2O level was markedly upregulated in HCC compared with adjacent noncancerous tissues. UBE2O overexpression was also confirmed in HCC cell lines. UBE2O overexpression was prominently associated with advanced tumor stage, high tumor grade, venous infiltration, and reduced HCC patients'' survivals. UBE2O knockdown inhibited the migration, invasion, and proliferation of HCCLM3 cells. UBE2O overexpression enhanced the proliferation and mobility of Huh7 cells. Mechanistically, UBE2O mediated the ubiquitination and degradation of AMP-activated protein kinase α2 (AMPKα2) in HCC cells. UBE2O silencing prominently increased AMPKα2 level and reduced phosphorylated mechanistic target of rapamycin kinase (p-mTOR), MYC, Cyclin D1, HIF1α, and SREBP1 levels in HCCLM3 cells. UBE2O depletion markedly activated the AMPKα2/mTOR pathway in Huh7 cells. Moreover, AMPKα2 silencing reversed UBE2O downregulation-induced mTOR pathway inactivation. Rapamycin, an inhibitor of mTOR, remarkably abolished UBE2O-induced mTOR phosphorylation and HCC cell proliferation and mobility. To conclude, UBE2O was highly expressed in HCC and its overexpression conferred to the poor clinical outcomes of patients. UBE2O contributed to the malignant behaviors of HCC cells, including cell proliferation, migration, and invasion, by reducing AMPKα2 stability and activating the mTOR pathway.     

Increased LEF1 Expression and Decreased Notch2 Expression Are Strong Predictors of Poor Outcomes in Colorectal Cancer Patients

Background/Objective. We aimed to examine the expression of lymphoid enhancer factor 1 (LEF1) and Notch2 in colorectal cancer (CRC) and their association with clinicopathologic variables and CRC patients'' prognosis. Methods. Immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot analysis were performed to assess the expression of LEF1 and Notch2 in 184 patients with CRC. Results. We observed a strong negative correlation between LEF1 expression and Notch2 expression (P < 0.001). Both LEF1 mRNA and protein expression increased while the Notch2 mRNA and protein expression decreased in tumor specimens compared with the matched paratumorous normal tissue (P < 0.001). An increase in LEF1 protein expression was significantly associated with lymph node metastases, distant metastasis, advanced TNM (tumor-node-metastasis) stage, and shorter overall survival. A decrease in Notch2 protein expression was associated with poorly differentiated tumors, lymph node metastases, distant metastasis, advanced TNM stage, and shorter overall survival of patients. In the multivariate Cox regression analysis, the LEF1 protein expression (P < 0.001), Notch2 protein expression (P < 0.001), TNM stage (P < 0.001), and the combination of increased LEF1 protein coexpression and decreased Notch2 protein coexpression (P < 0.001) were found to be independent prognostic indicators in CRC. Conclusion. Our results suggest that increased LEF1 coexpression and decreased Notch2 coexpression represent a risk factor for poor overall survival of CRC patients.     

鞘氨醇激酶通过调控血管发生促进肝癌转移

目的确定鞘氨醇激酶(SPK)在肝癌转移中的作用。方法用Ad-SPK1腺病毒感染肝癌细胞LM-3使其SPK过表达,Western blot检测SPK1表达及激活情况;Transwell、划痕实验检测肝癌细胞迁移情况。体外管状结构形成实验检测SPK对脐静脉内皮细胞(HUVEC)管状化形成的影响。建立SPK过表达裸鼠模型,体内检测肝癌迁移情况。结果在体外SPK对肝癌细胞的迁移无显著影响;Ad-SPK能够促进HUVEC管状结构形成;裸鼠皮下移植瘤结果显示Ad-SPK促进肿瘤生长和转移。结论鞘氨醇激酶通过调控血管发生促进肝癌转移。     

Dual VEGF/PDGF knockdown suppresses vasculogenic mimicry formation in choroidal melanoma cells via the Wnt5a/β-catenin/AKT signaling pathway

ObjectiveThis study aimed to explore the effects of knocking down both vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) on vasculogenic mimicry (VM) formation in choroidal melanoma (CM) cells.MethodsCell counting Kit (CCK)?8, monoclonal formation, wound healing, transwell and flow cytometry assays were used to observe the cell effects in CM cell line, ocular choroidal melanoma-1 cells (OCM-1) with respect to proliferation, migration, invasion and apoptosis. Three-dimensional (3D) cultures were also used to characterize VM tube structural effects in OCM-1 cells and western blotting was used to characterize protein expression changes in VM-related markers.ResultsDual VEGF/PDGF knockdown suppressed cell proliferation, migration and invasion, but promoted cell apoptosis. It also reduced VM tube structures in OCM-1 cells. VM associated markers including, VE-cadherin, EphA2 and MT1-MMP were also down-regulated in OCM-1 cells. Similarly, Wnt5a, β-catenin and phosphorylated-AKT levels were also down-regulated. Western blotting and 3D cultures further demonstrated that combined Wnt5a silencing with dual VEGF/PDGF knockdown significantly decreased VE-cadherin and EphA2 levels and reduced VM tube structures in OCM-1 cells.ConclusionsDual VEGF/PDGF knockdown suppressed cell growth and metastasis in OCM-1 cells, and blocked the Wnt5a/β-catenin/AKT signaling pathway thereby inhibiting VM formation.