MMP-2和PTEN蛋白在肝癌组织中的表达及临床意义

目的 探讨肝癌组织中MMP-2和PTEN的表达及两者与肝癌侵袭性的关系.方法 用免疫组化SP法检测61例肝癌组织和17例正常肝组织中MMP-2和PTEN蛋白质的表达,并分析两者与肝癌临床病理特征,侵袭性的关系.结果 HCC中PTEN阳性表达率明显弱于正常肝组织,而MMP-2阳性表达率明显高于正常肝组织,且两者呈负相关;PTEN蛋白阳性表达率与HCC组织分化程度、侵袭性有关(P＜0.05),而与性别、术前AFP水平无关(P＞0.05);MMP-2阳性表达率与HCC组织侵袭性有关(P＞0.05),而与HCC组织分化程度、性别、术前AFP水平无关(P＜0.05).结论 PTEN、MMP-2的表达一定程度上反映HCC侵袭性强弱;PTEN缺失可引起MMP-2表达增加,在HCC侵袭、转移中发挥作用.     

肝癌发生过程中的PTEN基因表达

目的 探讨肝癌PTEN基因的表达及与肝癌发生的关系.方法 应用RT-PCR法检测37例肝癌癌灶及非癌组织中PTEN基因mRNA表达,并通过凝胶图像系统分析其相对表达量.同时采用免疫组化方法分析肝癌组织中PTEN蛋白的定位.结果 癌灶PTEN mRNA表达率为48.6%(18/37),非癌组织为100.0%(37/37),癌灶组织中TEN基因表达显著低于非癌(P<0.01).免疫组化检测肝癌癌灶组织中PTEN蛋白表达率为37.8%(14/37),显著低于非癌组织中的94.6%(35/37)(P<0.01).结论 PTEN基因下调或缺失可能与肝癌的发生发展有密切相关.     

P21-activated kinase 5 plays essential roles in the proliferation and tumorigenicity of human hepatocellular carcinoma

Aim： To investigate the roles of P21-activated kinase 5 （PAK5） in proliferation and tumorigenicity of human hepatocellular carcinoma （HCC）.
Methods： HCC and matched paraneoplastictis tissue samples were obtained from 30 patients. Human HCC cell lines SMMC7721, HepG2, Hep3B, SK-HEP-1, Huh-7, and liver cell line HL-7702 were examined. The expression of PAK5 gene was studied using real-time qPCR and Western blotting. Cell proliferation was quantified with the MTT assay. Cell cycle was analyzed with flow cytometry. The
tumorigenicity of Lv-shRNA-transfected HepG2 cells was evaluated in BALB/cA nude mice.

Results： The mRNA level of PAK5 was significantly higher in 25 out of 30 HCC samples compared to the matched paraneoplastic
tissues. The HCC cell lines showed varying expression of PAK5 protein, and the highest level was found in the HepG2 cells. PAK5 gene silencing in HepG2 cells markedly reduced the cell proliferation and colony formation, and induced cell cycle arrest in the G1 phase. Furthermore, PAK5 gene silencing suppressed the tumor formation in nude mice, and significantly decreased the expression of HCC-related genes Cyclin D1 and beta-catenin.

Conclusion： PAK5 may play essential roles in the initiation and progression of human HCC. Thus, it may be an effective therapeutic target or perhaps serve as a clinical diagnostic or prognostic marker in human HCC.     

Galactose-modified selenium nanoparticles for targeted delivery of doxorubicin to hepatocellular carcinoma

Galactose-modified selenium nanoparticles (GA-SeNPs) loading with doxorubicin (DOX) for hepatocellular carcinoma (HCC) therapy was investigated in this paper. Selenium nanoparticles (SeNPs) were modified with galactose as tumor targeting moiety to fabricate tumor-targeted delivery carrier GA-SeNPs, then doxorubicin was loaded onto the surface of GA-SeNPs for improving antitumor efficacy of DOX in HCC therapy. Chemical structure characterization of GA-Se@DOX showed that DOX was successfully loaded to the surface of GA-SeNPs to prepare functionalized antitumor drug delivery system GA-Se@DOX. GA-Se@DOX exhibited effective cellular uptake in HepG2 cells and entered HepG2 cells mainly by clathrin-mediated endocytosis pathway. GA-Se@DOX showed significant activity to induce the apoptosis of HepG2 cells in vitro. The western blotting result indicated that GA-Se@DOX induced HepG2 cells apoptosis via activating caspase signaling and Bcl-2 family proteins. Moreover, active targeting delivery system GA-Se@DOX exhibited excellent antitumor efficacy in vivo in comparison with passive targeting delivery system Se@DOX. Histology analysis showed that GA-Se@DOX exhibited no obvious damage to major organs including heart, liver, spleen, lung, and kidney under the experimental condition. Taken together, GA-Se@DOX may be one novel promising nanoscale drug candidate for HCC therapy.     

LncRNA PVT1 epigenetically silences miR-195 and modulates EMT and chemoresistance in cervical cancer cells

The plasmacytoma variant translocation 1 gene (PVT1) is an oncogenic lncRNA with regulative effect on chemosensitivity in cervical cancer. However, the underlying mechanisms were not fully understood. In this study, HPV16 positive CaSki and SiHa cells were used as in vitro cell model. Knockdown of HPV16 E7 significantly inhibited PVT1 and restored miR-195 expression. PVT1 directly interacts with EZH2 and the complex anchors in the promoter region of miR-195. PVT1 overexpression resulted in increased H3K27me3 levels in the miR-195 promoter region, while PVT1 knockdown decreased H3K27me3 levels in the promoter region. In addition, PVT1 could competitively bind with miR-195. MiR-195 overexpression suppressed PVT1 expression in the cancer cells. Both PVT1 and miR-195 could inhibit paclitaxel (PTX) induced epithelial-to-mesenchymal transition (EMT) and also sensitize CaSki cells to PTX. Based on these findings, we infer that PVT1 could decrease miR-195 expression via enhancing histone H3K27me3 in the miR-195 promoter region and also via direct sponging of miR-195. In addition, the PVT1/miR-195 axis can modulate responses of the cancer cells to PTX via regulating EMT.     

PUMA基因在肝癌组织的表达及意义

目的研究p53正向细胞凋亡调控因子(PUMA)基因mRNA及其功能蛋白在肝癌组织中的表达及其临床意义。方法用半定量RT-PCR和Western blot方法检测42例肝癌患者的癌组织及其癌旁组织和42肝良性病变组织的PUMA mRNA及其编码蛋白表达。结果癌组织、癌旁组织和肝良性病变PUMA mRNA的表达量分别为0.81±0.07、0.97±0.04和1.76±0.06,PUMA蛋白的表达量分别为0.48±0.19、0.61±0.12和1.11±0.34;肝癌组织及癌旁组织的PUMAmRNA及其蛋白表达量明显低于肝良性病变组织高于(P<0.05)。结论 PUMA基因在肝癌组织中的表达下调,与癌的发生呈负相关。     

Evaluation of anti-invasion effect of cannabinoids on human hepatocarcinoma cells

Context: Cancer is a disease characterized by abnormal growth of cells. One of the most common types of liver cancers is called hepatocellular carcinoma (HCC) which is highly metastatic. As most of cannabinoids have shown anticancer effect against different cell lines in a number of reports, a biological investigation of two cannabinoids, CB65 (CB2 receptor agonist) and ACEA (CB1 receptor agonist) was carried out in this study.

Objective: In an attempt to find natural products as a new solution of cancer, this study was designed to investigate the potential antitumoral and anti-invasive activity of cannabinoids on HepG2 cells and the possible roles of matrix metalloproteinase-2 (MMP-2) and MMP-9 in its action.

Materials and methods: The researchers examined the effect of various concentrations of CB65 (CB2 receptor agonist) and ACEA (CB1 receptor agonist), on the cell proliferation, viability, and invasion as well as expression of MMP-2 and MMP-9 in HepG2 cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay, matrigel invasion assay, and western blotting method.

Results: The results revealed that both cannabinoids reduce cell viability, cell invasion as well as MMP-2 and MMP-9 expression in higher dose of 20?nM. Furthermore, higher concentrations of examined cannabinoids were more effective.

Discussion: These data suggest ACEA and CB65 as an option for novel treatment of hepatocellular cancer.

Conclusion: Our findings may contribute to design of new therapeutic strategies for the management of HCC.     

胃癌组织中PTEN和MMP-9的表达与腹腔游离癌细胞的相关性

目的 观察PTEN和基质金属蛋白酶9（MMP-9）在胃癌组织中的表达,探讨其在癌变机制中的作用。方法 采用SP法检测65例胃癌组织中PTEN、MMP-9的表达,同时应用实时荧光RT-PCR方法检测胃癌术中腹腔灌洗液中游离癌细胞。结果 （1）PTEN、MMP-9阳性表达率在有无腹腔游离癌细胞组之间的差异均有显著性（P〈0．05）;（2）PTEN与MMP-9的表达呈负相关。结论 PTEN、MMP-9可以反映腹腔游离癌细胞是否存在,为评估预后及指导化疗提供参考。     

Argonaute 2 promotes angiogenesis via the PTEN/VEGF signaling pathway in human hepatocellular carcinoma

Aim:

Argonaute2 (AGO2) protein is the active part of RNA-induced silencing complex, cleaving the target mRNA strand complementary to their bound siRNA. An increasing number of miRNAs has been identified as essential to angiogenesis of hepatocellular carcinoma (HCC). In this study we investigated how AGO2 affected HCC angiogenesis.

Methods:

Human HCC cell lines HepG2, Hep3B, Huh7, SMMC-7721, Bel-7404, MHCC97-H and LM-3, and human umbilical vein endothelial cells (HUVEC) were tested. The expression of AGO2 in HCC cells was knocked down with siRNA and restored using recombinant adenovirus expressing Ago2. The levels of relevant mRNAs and proteins were examined using RT-PCR, Western blot and EILSA. Nude mice were implanted with Huh7 or SMMC-7721 cells, and tumor volumes were measured. After the mice were euthanized, the xenograft tumors were used for immunohistological analysis.

Results:

In 6 HCC cell lines, AGO2 protein expression was significantly correlated with VEGF expression (r=+0.79), and with VEGF secretion (r=+0.852). Knockdown of Ago2 in Huh7 cells and SMMC-7721 cells substantially decreased VEGF expression, whereas the restoration of AGO2 reversed both VEGF expression and secretion. Furthermore, knockdown of Ago2 significantly up-regulated the expression of PTEN (a tumor suppressor involved in the inhibition of HCC angiogenesis), and vice versa. Moreover, the specific PTEN inhibitor bisperoxovanadate (7, 14, 28 nmol/L) dose-dependently restored the expression of VEGF and the capacity of HCC cells to induce HUVECs to form capillary tubule structures. In the xenograft nude mice, knockdown of Ago2 markedly suppressed the tumor growth and decreased PTEN expression and CD31-positive microvascular in the xenograft tumors.

Conclusion:

A direct relationship exists between the miRNA processing machinery AGO2 and HCC angiogenesis that is mediated by the AGO2/PTEN/VEGF signaling pathway. The results suggest the high value of Ago2 knockdown in anti-angiogenesis therapy for HCC.     

Hepatitis B virus X protein promotes hepatoma cell proliferation via upregulation of MEKK2

Aim:

To investigate the mechanism underlying the increase of hepatoma cell proliferation by hepatitis B virus X protein (HBx).

Methods:

HepG2, H7402 and HepG2.2.15 cells, which constitutively replicated hepatitis B virus were used. The effects of HBx on hepatoma cell proliferation were examined using 5-ethynyl-2-deoxyuridine (EdU) incorporation assay and MTT assay. The expression level of MEKK2 was measured using RT-PCR, Western blot and luciferase reporter gene assay. The activity of activator protein 1 (AP-1) was detected using luciferase reporter gene assay. The phosphorylation levels of JNK and c-Jun were measured using Western blot. The expression levels of HBx and MEKK2 in 11 clinical hepatocellular carcinoma (HCC) tissues were measured using real time PCR and Western blot. In addition, the expression of MEKK2 in 95 clinical HCC tissues was examined using immunohistochemistry.

Results:

HBx significantly enhanced HepG2-X cell proliferation. In HepG2-X, H7402-X and HepG2.2.15 cells, the expression level of MEKK2 was remarkably increased. In HepG2.2.15 cells, HBx was found to activate JNK and AP-1, which were the downstream effectors of MEKK2 in HepG2-X and HepG2.2.15 cells. In 11 clinical HCC tissues, both HBx and MEKK2 expression levels were remarkably increased, as compared to those in the corresponding peritumor tissues. In 95 clinical HCC tissues, the rate of detection of MEKK2 was 85.3%.

Conclusion:

HBx promotes hepatoma cell proliferation via upregulating MEKK2, which may be involved in hepatocarcinogenesis.     

Do animal models hold value in Autism spectrum disorder (ASD) drug discovery?

ABSTRACT

Introduction: Autism spectrum disorder (ASD) defines impairments in a broad range of behaviors in two domains, social communication and repetitive behaviors and/or restricted interests. Drug discovery is ongoing for ASD, but no drugs have been approved for the core behaviors. Animal models are invaluable for drug discovery, but are limited by the face, construct, and predictive validity for ASD. The genetic construct validity of animal models has provided potential targets including biological events early in development which are indeed challenging to treat pharmacologically.

Areas covered: The focus of this review is on the current models for ASD being used to test potential therapeutics. Drugs reviewed include sulforaphane, propranolol, oxytocin, vasopressin antagonists, arbaclofen, and bumetanide, that have been evaluated on behaviors with face validity for both the core behaviors of ASD, social and repetitive behaviors, and the modifying behaviors including learning and memory.

Expert opinion: Animal models for the core symptoms of ASD have suffered from the same problems hampering research in humans, including lack of a biomarker, heterogeneity of symptom severity, and appropriate endpoints for evaluation. Despite this, the data from animal models has allowed several drugs to move on to clinical testing.     

NES1基因在肝细胞肝癌中的表达及临床意义

目的探讨NES1基因在HCC发生发展中的作用及意义。方法用逆转录聚合酶链反应(RT-PCR)技术检测30例HCC患者癌组织、癌旁肝组织以及10例正常肝组织中NES1mRNA表达情况。结果NES1mRNA在HCC组织中的表达水平显著低于在癌旁肝组织和正常肝组织中的表达水平;NES1mRNA的表达水平与肿瘤大小、肿瘤数为单个或多个、脉管癌栓及临床分期均呈负相关性(P<0.05)。结论NES1mRNA的低表达或表达缺失可能与肝细胞肝癌的发生、发展密切相关;NES1在HCC的发生发展中具有重要地位。     

Comparison of MeHg-induced toxicogenomic responses across in vivo and in vitro models used in developmental toxicology

Toxicogenomic evaluations may improve toxicity prediction of in vitro-based developmental models, such as whole embryo culture (WEC) and embryonic stem cells (ESC), by providing a robust mechanistic marker which can be linked with responses associated with developmental toxicity in vivo. While promising in theory, toxicogenomic comparisons between in vivo and in vitro models are complex due to inherent differences in model characteristics and experimental design. Determining factors which influence these global comparisons are critical in the identification of reliable mechanistic-based markers of developmental toxicity. In this study, we compared available toxicogenomic data assessing the impact of the known teratogen, methylmercury (MeHg) across a diverse set of in vitro and in vivo models to investigate the impact of experimental variables (i.e. model, dose, time) on our comparative assessments. We evaluated common and unique aspects at both the functional (Gene Ontology) and gene level of MeHg-induced response. At the functional level, we observed stronger similarity in MeHg-response between mouse embryos exposed in utero (2 studies), ESC, and WEC as compared to liver, brain and mouse embryonic fibroblast MeHg studies. These findings were strongly correlated to the presence of a MeHg-induced developmentally related gene signature. In addition, we identified specific MeHg-induced gene expression alterations associated with developmental signaling and heart development across WEC, ESC and in vivo systems. However, the significance of overlap between studies was highly dependent on traditional experimental variables (i.e. dose, time). In summary, we identify promising examples of unique gene expression responses which show in vitro–in vivo similarities supporting the relevance of in vitro developmental models for predicting in vivo developmental toxicity.     

三子颗粒下调miR-205-5p抑制小鼠脾虚型肠道腺瘤生长研究

目的 探讨三子颗粒通过降低微小核糖核酸-205-5p（miR-205-5p）水平抑制小鼠脾虚型肠道腺瘤生长的作用。方法 取70只4周龄的雄性C57BL/6J小鼠,采用对氧化偶氮甲烷（AOM）/葡聚糖硫酸钠（DSS）诱导小鼠结直肠腺瘤模型,将建模小鼠随机分为模型组、阿司匹林（200 mg·kg^-1）组、miR inhibitor-NC （2 mg·kg^-1）组、miR-205-5p inhibitor （2 mg·kg^-1）组和三子颗粒低、中、高剂量（1.7、3.4、6.8 g·kg^-1）组,造模期间ig给药,每天1次。比较各组小鼠肠道腺瘤的数量并测量腺瘤体积;苏木素-伊红（HE）染色观察小鼠肠道腺瘤的病理情况;CCK-8法检测各组小鼠肠道腺瘤细胞增殖活力;原位末端标记（TUNEL）法检测小鼠肠道腺瘤细胞凋亡;实时荧光定量PCR（qRT-PCR）法检测肠道腺瘤miR-205-5p表达量及磷酸酯酶与张力蛋白同源物（PTEN）、B淋巴细胞瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、Ki67 mRNA表达量;Western blotting法检测PTEN、Bcl-2、Bax、Ki67蛋白表达水平;荧光素酶活性实验验证miR-205-5p和PTEN的靶向关系。结果 与模型组比较,阿司匹林组和三子颗粒低、中、高剂量组小鼠肠道腺瘤数量、体积及细胞增殖活性均显著降低,凋亡率显著升高（P＜0.05）,miR-205-5p表达量、Bcl-2、Ki67 mRNA及蛋白表达量显著降低,PTEN、Bax mRNA及蛋白表达量显著升高（P＜0.05）,其中三子颗粒作用呈剂量相关性;与miR inhibitor-NC组比较,miR-205-5p inhibitor组小鼠肠道腺瘤数量、体积及细胞增殖活性均显著降低,凋亡率显著升高（P＜0.05）,miR-205-5p表达量、Bcl-2、Ki67 mRNA及蛋白表达量显著降低,PTEN、Bax mRNA及蛋白表达量显著升高（P＜0.05）;荧光素酶活性实验证实miR-205-5p可靶向调控PTEN。结论 三子颗粒可抑制小鼠脾虚型肠道腺瘤生长,可能是通过下调miR-205-5p,上调PTEN、Bax表达,下调Bcl-2、Ki67表达发挥作用的。