siRNA沉默Bmi-1基因对人肝癌细胞株MHCC97-H侵袭迁移能力的影响

目的:探讨RNA干扰技术沉默Bmi-1基因表达后,对人肝癌细胞株MHCC97-H侵袭迁移能力的影响.方法:设计并合成针对Bmi-1基因序列特异性的双链小干扰RNA(Bmi-1-siRNA),转染高转移性人肝癌细胞株MHCC97-H,用流式细胞仪观察转染效率,荧光实时定量PCR和Westernblot检测Bmi-1基因的mRNA和蛋白表达水平;通过体外Transwell小室基质侵袭和迁移实验,观察Bmi-1表达沉默后对人肝癌细胞株MHCC97-H侵袭和迁移能力的影响.结果:将针对Bmi-1基因序列特异性的小干扰RNA(Bmi-1-siRNA)转染高转移性人肝癌细胞株MHCC97-H后,流式细胞仪显示,转染效率可达到91%.与空白组、对照siRNA组相比,实验组Bmi-1-siRNA能有效抑制MHCC97-H细胞中Bmi-1基因的mRNA(F=56.199,P<0.05)和蛋白表达水平.通过Transwell小室基质侵袭和迁移实验,我们分析了不同组细胞的侵袭迁移能力.结果发现,与空白组、对照siRNA组相比,Bmi-1-siRNA转染的MHCC97-H细胞穿透能力明显降低(F=186.66,12.746,P<0....     

三氧化二砷对人肝癌SMMC-7721细胞增殖、迁移及侵袭能力的影响

目的 探讨三氧化二砷(As2O3)对人肝癌SMMC-7721细胞增殖、迁移和侵袭能力的影响及其分子机制.方法 利用MTS/PMS法检测As2O3对SMMC-7721细胞增殖的影响;划痕愈合和侵袭实验检测As2O3对SMMC-7721细胞迁移与侵袭能力的影响;实时定量PCR、Western印迹检测As2O3作用SMMC-7721细胞后MMP-9的表达.结果 2.0 μmol/L的As2O3对肿瘤细胞增殖有抑制作用;划痕实验结果显示,2.0μmol/LAs2O3处理SMMC-7721细胞12和24 h后,较未处理组划痕距离明显增加(P＜0.05),表明As2O3可抑制SMMC-7721细胞迁移.Transwell实验结果显示,2.0μmol/LAs2O3处理SMMC-7721细胞12h和24 h后,穿膜细胞数较未处理组明显减少(P＜0.05),表明As2O3可抑制SMMC-7721细胞侵袭能力.实时定量PCR和Western印迹结果显示,以2.0μmol/L As2O3处理SMMC-7721细胞后,MMP-9 mRNA和蛋白表达均明显下调(P＜0.05).结论 As2O3可通过抑制肿瘤细胞增殖、迁移及侵袭,抑制肿瘤的转移.     

circZFR promotes cell proliferation and migration by regulating miR-511/AKT1 axis in hepatocellular carcinoma

BackgroundEmerging data suggest the crucial regulatory roles of circular RNAs (circRNAs) in hepatocellular carcinoma (HCC). However, the pathophysiology role of circZFR in HCC remains largely unknown.AimsThis study aims to disclose the functions of circZFR in HCC progression and its potential molecular mechanism.MethodscircZFR and miR-511 were identified by qRT-PCR. Colony formation assay, wound-healing assay, transwell assay, and flow cytometry assay were performed to determine the cell proliferation, migration, invasion and apoptosis. Western blotting and immunohistochemistry (IHC) were utilized to evaluate the expression level of AKT1, GSK3β, β-catenin and cascades of proliferation-related proteins both in vitro and in vivo. Dual luciferase reporter assay was conducted to evaluate the interactions among circZFR, miR-511 and AKT1.ResultsThe expression of circZFR was enhanced and the expression of miR-511 was down-regulated in HCC tissues and cells. Functionally, circZFR silencing or miR-511 overexpression suppressed cell proliferation, migration and invasion, and induced apoptosis of HCC cells. Mechanistically, circZFR acted as a miR-511 sponge to up-regulate its target gene AKT1, which activated cascades of proliferation-related proteins (c-Myc, cyclin D1, Survivin and Bcl-2). Furthermore, depletion of circZFR inhibited tumorigenesis and decreased the expression level of AKT1 in xenograft models.ConclusioncircZFR promotes HCC progression by directly down-regulating miR-511 to activate AKT1 signaling, suggesting that circZFR is a potential target in HCC treatment. Targeting circZFR may provide therapeutic benefits for HCC.     

lncRNA-SNHG15 accelerates the development of hepatocellular carcinoma by targeting mi R-490-3p/histone deacetylase 2 axis

BACKGROUND Hepatocellular carcinoma(HCC) has become a great threat for people's health.Many long noncoding RNAs are involved in the pathogenesis of HCC.SNHG15,as a tissue specific long noncoding RNAs,has been studied in many human cancers,except HCC.AIM To explore the regulatory mechanism of SNHG15 in HCC METHODS In the present research,101 HCC patient samples,two HCC cell lines and one normal liver cell line were used.RT-qPCR and Western blot analysis were applied to detect SNHG15,miR-490-3 p and histone deacetylase 2(HD AC2)expression.The regulatory mechanism of SNHG15 was investigated using CCK8,Transwell and luciferase reporter assays.RESULTS Our research showed that up-regulation of SNHG15 was found in HCC and was related to aggressive behaviors in HCC patients.Moreover,knockdown of SNHG15 restrained HCC cell proliferation,migration and invasion.In addition,SNHG15 served as a molecular sponge for miR-490-3 p.Further,miR-490-3 p directly targets HDAC2.HD AC2 was involved in HCC progression by interacting with the SNHG15/miR-490-3 p axis.CONCLUSION In conclusion,long noncoding RNA SNHG15 promotes HCC progression by mediating the miR-490-3 p/HDAC2 axis in HCC.     

EZH2在肝癌发生发展中功能的研究进展

多种因素引起的原癌基因激活和抑癌基因失活是导致肝癌发生发展的主要原因.EZH2是新近发现的具有组蛋白甲基转移酶活性的癌相关基因,主要通过催化H3K27me3修饰介导基因沉默,参与X染色体失活、细胞分化和胚胎发育调节.近来发现,EZH2在多种机制调节下在肝癌组织中过度表达,通过多种机制调控其下游基因异常表达,从而参与肝癌...     

Connective tissue growth factor is overexpressed in human hepatocellular carcinoma and promotes cell invasion and growth

AIM: To determine the expression characteristics of connective tissue growth factor (CTGF/CCN2) in human hepatocellular carcinoma (HCC) in histology and to elucidate the roles of CCN2 on hepatoma cell cycle progression and metastasis in vitro.METHODS: Liver samples from 36 patients (who underwent hepatic resection for the first HCC between 2006 and 2011) and 6 normal individuals were examined for transforming growth factor β1 (TGF-β1) or CCN2 mRNA by in situ hybridization. Computer image analysis was performed to measure integrated optimal density of CCN2 mRNA-positive cells in carcinoma foci and the surrounding stroma. Fibroblast-specific protein-1 (FSP-1) and E-cadherin were examined to evaluate the process of epithelial to mesenchymal transition, α-smooth muscle actin and FSP-1 were detected to identify hepatic stellate cells, and CD34 was measured to evaluate the extent of vascularization in liver tissues by immunohistochemical staining. CCN2 was assessed for its stimulation of HepG2 cell migration and invasion using commercial kits while flow cytometry was used to determine CCN2 effects on HepG2 cell-cycle.RESULTS: In situ hybridization analysis showed that TGF-β1 mRNA was mainly detected in connective tissues and vasculature around carcinoma foci. In comparison to normal controls, CCN2 mRNA was enhanced 1.9-fold in carcinoma foci (12.36 ± 6.08 vs 6.42 ± 2.35) or 9.4-fold in the surrounding stroma (60.27 ± 28.71 vs 6.42 ± 2.35), with concomitant expression of CCN2 and TGF-β1 mRNA in those areas. Epithelial-mesenchymal transition phenotype related with CCN2 was detected in 12/36 (33.3%) of HCC liver samples at the edges between carcinoma foci and vasculature. Incubation of HepG2 cells with CCN2 (100 ng/mL) resulted in more of the cells transitioning into S phase (23.85 ± 2.35 vs 10.94 ± 0.23), and induced a significant migratory (4.0-fold) and invasive (5.7-fold) effect. TGF-β1-induced cell invasion was abrogated by a neutralizing CCN2 antibody showing that CCN2 is a downstream mediator of TGF-β1-induced hepatoma cell invasion.CONCLUSION: These data support a role for CCN2 in the growth and metastasis of HCC and highlight CCN2 as a potential novel therapeutic target.     

Smoc2 potentiates proliferation of hepatocellular carcinoma cells via promotion of cell cycle progression

AIM To determine the influence of Smoc2 on hepatocellular carcinoma(HCC) cell proliferation and to find a possible new therapeutic target for preventing HCC progression.METHODS We detected expression of Smoc2 in HCC tissues and corresponding non-tumor liver(CNL) tissues using PCR, western blot, and immunohistochemistry methods. Subsequently, we down-regulated and upregulated Smoc2 expression using siR NA and lentivirus transfection assay, respectively. Then, we identified the effect of Smoc2 on cell proliferation and cell cycle using CCK-8 and flow cytometry, respectively. The common cell growth signaling influenced by Smoc2 was detected by western blot assay. RESULTS The expression of Smoc2 was significantly higher in HCC tissues compared with CNL tissues. Overexpression of Smoc2 promoted HCC cell proliferation and cell cycle progression. Down-regulation of Smoc2 led to inhibition of cell proliferation and cell cycle progression. Smoc2 had positive effect on ERK and AKT signaling.CONCLUSION Smoc2 promotes the proliferation of HCC cells through accelerating cell cycle progression and might act as an anti-cancer therapeutic target in the future.     

类泛素FAT10高表达肝癌细胞株Hep3B酵母双杂交cDNA文库的构建

目的:构建类泛素FAT10高表达肝癌细胞株Hep3B的酵母双杂交用cDNA文库.方法:从肝癌细胞Hep3B中提取总RNA,分离mRNA.利用反转录酶M-MLV与Oligo(dT)AnchorPrimer合成1stStrandcDNA,用E.coliDNAPolymerase与E.coliDNALigase将RNA链置换成DNA链,合成2ndStrandcDNA.将双链cDNA与EcoRⅠAdaptor连接,然后用EcoRⅠ/XhoⅠ进行酶切.使用SpinColumn除去短链cDNA与pGADT7载体连接,转化入E.coliDH10B,建成原始文库.然后对其进行扩增并随机挑取单菌落,酶切鉴定重组子插入片段大小.结果:提取的总RNA降解少且分子完整;RNA纯度高,相对分子质量为400-5000bp;成功合成双链cDNA,均符合建库要求;库容量达到1.03×106克隆,原始文库滴度为2.50×109cfu/L,扩增后的文库滴度为3.60×1012cfu/L.插入片段大小分布为0.5-3.5kb,平均长度约为2.0kb.结论:所构建文库的各项指标均达到要求,为筛选FAT10作用蛋白奠定了重要基础.     

CXCR4抑制剂AMD3465调节肺癌细胞株增殖、侵袭的实验研究

目的探讨CXCR4抑制剂AMD3465对肺癌细胞株增殖、侵袭的影响。方法培养肺癌细胞株A549并分为AMD3465组、对照组;分别用含有三种不同剂量(50、100、200 mg/L)AMD3465的DMEM处理以及用不含药物的DMEM处理。处理后12、24、48 h,检测细胞的增殖活力和侵袭数目;处理后48 h,检测细胞中增殖及侵袭基因的mRNA表达量。结果处理后12、24、48 h,三种不同剂量(50、100、200 mg/L)AMD3465组细胞的OD值均显著低于对照组、侵袭数目均显著少于对照组,AMD3456剂量较低时OD值、侵袭数目与对照组差距均大于AMD3456剂量较高时(P0.05);处理后48 h,50 mg/L AMD3465组、100 mg/L AMD3465组、200 mg/L AMD3465组细胞中Cyclin D1、PCNA、Ki-67、Cat B、MMP2、MMP9、MMP13的mRNA表达量均显著低于对照组;AMD3456剂量较低时细胞中Cyclin D1、PCNA、Ki-67、Cat B、MMP2、MMP9、MMP13的mRNA表达量与对照组差距均大于AMD3456剂量较高时(P0.05)。结论 CXCR4抑制剂AMD3465能够有效抑制肺癌细胞株的增殖、侵袭。     

MiR-451 inhibits proliferation of esophageal carcinoma cell line EC9706 by targeting CDKN2D and MAP3K1

AIM: To investigate the underlying molecular mechanisms of miR-451 to inhibit proliferation of esophageal carcinoma cell line EC9706.METHODS: Assays for cell growth, apoptosis and invasion were used to evaluate the effects of miR-451 expression on EC cells. Luciferase reporter and Western blot assays were used to test whether cyclin-dependent kinase inhibitor 2D(CDKN2D) and MAP3K1 act as major targets of miR-451.RESULTS: The results showed that CDKN2 D and MAP3K1 are direct targets of miR-451. CDKN2 D and MAP3K1 overexpression reversed the effect of miR-451.MiR-451 inhibited the proliferation of EC9706 by targeting CDKN2 D and MAP3K1.CONCLUSION: These findings suggest that miR-451 might be a novel prognostic biomarker and a potential target for the treatment of esophageal squamous cell carcinoma in the future.     

茵陈蒿汤抑制JAK2/STAT3信号通路调控肝癌细胞增殖与凋亡

目的 探索茵陈蒿汤的抗肝癌作用及其机制.方法 体外培养HepG2肝癌细胞,分为对照组和茵陈蒿汤低、中、高浓度(12.5、25、50μg/mL)组.通过CCK-8检测细胞活力、EdU染色观察茵陈蒿汤对HepG2肝癌细胞增殖的影响,钙黄绿素/PI细胞染色观察茵陈蒿汤对肝癌细胞存活能力的影响.通过Western blot检测...     

抗氧化剂PDTC对肝癌细胞株Hep3B增殖的影响及机制

目的探讨抗氧化剂二硫代氨基甲酸毗咯烷（PDTC）在肝细胞性肝癌化学预防中的作用及机制。方法将不同浓度PDTC、阿霉素及PDTC联用阿霉素作用于Hep3B细胞。采用MTT法检测细胞存活率;流式细胞术（FCM）检测凋亡率;电泳迁移率变动分析（EMSA）检测NF—KB活性。结果PDTC作用后Hep3B存活率明显降低,呈浓度依赖性,P〈0．01。阿霉素联用PDTC的细胞生长抑制率明显高于单用阿霉素（P〈0．01）。PDTC为10μmol／L时的细胞凋亡率明显低于50μmol／L时（P〈0．01）。EMSA示PDTC作用后NF-κB表达降低,不同浓度PDTC作用两两相比P〈0．01。结论PDTC能抑制肝癌细胞株Hep3B的增殖、促进细胞凋亡、增强阿霉素的细胞毒作用;其作用机制主要是抑制NF-κB激活。PDTC可用于肝癌的化学预防,联用阿霉素可进一步提高疗效。     

Knockdown of lncRNAXLOC＿001659 inhibits proliferation and invasion of esophageal squamous cell carcinoma cells

BACKGROUND Studies have shown that long non-coding RNAs(lnc RNAs) play a key role in almost all key physiological and pathological processes, including different types of malignant tumors. Our previous lnc RNA microarray results have shown that lnc RNA XLOC_001659 is upregulated in esophageal cancer(EC) tissues, with a fold change of 20.9 relative to normal esophageal tissues. But its effect and the molecular biological mechanisms on proliferation and invasion of EC cells remain unclear.AIM To investigate the effect of lnc RNA XLOC_001659 on esophageal squamous cell carcinoma(ESCC) cells and explore the molecular biological mechanisms involved.METHODS RT-q PCR assay was used to quantify the expression levels of lnc RNAXLOC-001659 and mi R-490-5 p. The proliferative capacity of the cells was determined using CCK8 and colony formation assays, and the effect of lnc RNAXLOC-001659 on the invasion of ESCC cells was determined by Transwell assay. Dualluciferase reporter assay was used to detect the target genes of lnc RNAXLOC-001659 and mi R-490-5 p.RESULTS The results of RT-q PCR showed that the expression of lnc RNAXLOC_001659 was upregulated in ESCC cells. CCK-8 assay showed that knockdown of lnc RNAXLOC_001659 significantly inhibited ESCC cell proliferation. Colony formation and Transwell invasion assays showed that knockdown of lnc RNAXLOC_001659 or overexpression of mi R-490-5 p significantly inhibited ESCC cell growth and invasion. Furthermore, lnc RNAXLOC_001659 acts as an endogenous sponge by competitively binding to mi R-490-5 p to downregulate mi R-490-5 p. Further results confirmed that mi R-490-5 p targeted PIK3 CA, and therecovery of PIK3 CA rescued lnc RNAXLOC_001659 knockdown or mi R-490-5 p overexpression-mediated inhibition of cell proliferation and invasion, which suggested the presence of an lnc RNAXLOC_001659/mi R-490-5 p/PIK3 CA regulatory axis.CONCLUSION Knockdown of lnc RNA XLOC_001659 inhibits proliferation and invasion of ESCC cells via regulation of mi R-490-5 p/PIK3 CA, suggesting that it may play a role in ESCC tumorigenesis and progression.     

多梳基因EZH2与BMI-1在动脉粥样硬化大鼠主动脉中的过表达和EZH2的磷酸化

目的 检测EZH2与BMI-1在动脉粥样硬化大鼠主动脉中的表达改变,以及磷酸化蛋白激酶B-磷酸化EZH2通路在动脉粥样硬化大鼠主动脉中的表达变化,以探讨动脉粥样硬化发病中DNA甲基化修饰异常的上游表观遗传机制.方法 建立大鼠动脉粥样硬化模型,通过血脂生物化学分析以及主动脉HE染色切片鉴定动脉粥样硬化模型.取胸腹主动脉内膜、中膜为标本,逆转录聚合酶链反应检测EZH2和BMI-1的mRNA表达,免疫组织化学法检测蛋白激酶B和EZH2蛋白磷酸化在动脉粥样硬化主动脉中的变化.结果 模型组EZH2和BMI-1的mRNA表达明显高于正常对照组(P＜0.05和P＜0.01).模型组中蛋白激酶B和EZH2蛋白的磷酸化程度均明显高于正常对照组(P＜0.01).结论 EZH2和BMI-1的mRNA在动脉粥样硬化病损动脉中显示共同高表达;且磷酸化EZH2蛋白和磷酸化蛋白激酶B蛋白表达在动脉粥样硬化病损动脉中平行上调,提示EZH2和BMI-1的共同高表达和蛋白激酶B介导的EZH2磷酸化可能是动脉粥样硬化发病过程中的早期表观遗传机制紊乱.     

Human hepatitis B virus and hepatocellular carcinoma II. Experimental induction of hepatocellular carcinoma in tree shrews exposed to hepatitis B virus and aflatoxin B1

On the basis of the successful establishment of an animal model in tree shrews experimentally infected with human hepatitis B virus (HBV), a study on the hepatocarcinogenic effects of HBV and/or aflatoxin B1 (AFB1) was conducted. The results showed that the incidence of hepatocellular carcinoma (HCC) was significantly higher in the animals both infected with HBV and exposed to AFB1 (52.94%) than in those solely infected with HBV (11.11%) or exposed to AFB1 (12.50%). No HCC of precancerous lesions were found in the controls that were neither HBV-infected nor AFB1-exposed. Precancerous lesions, including liver cell dysplasia and enzyme-altered hyperplastic hepatocyte foci, were observed before the occurrence of HCC, and the frequency of their appearance correlated well with the incidence of HCC. HBV DNA and the protein it encodes were detected in the cancer cells and/or the surrounding hepatocytes. Integration of HBV DNA inot the host liver genome was found during hepatocarcinogenesis among the animals infected by HBV. These results suggest that exposure to HBV and AFB1 may play a synergistic role in the development of HCC, and support the viewpoint of an aetiological relationship between HBV and HCC.Abbreviations HCC hepatocellular carcinoma - HBV human hepatitis B virus - AFB1 aflatoxin B1 - GGT foci hyperplastic hepatocyte foci positive for -glutamyltranspeptidase - LCD liver cell dysplasia     

Axl glycosylation mediates tumor cell proliferation, invasion and lymphatic metastasis in murine hepatocellular carcinoma

AIM: To investigate the effects of Axl deglycosylation on tumor lymphatic metastases in mouse hepatocellular carcinoma cell lines. METHODS: Western blotting was used to analyze the expression profile of Axl glycoprotein in mouse hepa-tocellular carcinoma cell line Hca-F treated with tunicamycin and PNGase F 3-(4,5)-dimethylthiazol(-zyl)-3,5- diphenyltetrazolium bromide (MTT) assay, extracellular matrix (ECM) invasion assay (in vitro ) and tumor metastasis assay (in vivo ) were utilized to evaluate the effect of Axl deglycosylation on the Hca-F cell proliferation, invasion and lymphatic metastasis. RESULTS: Tunicamycin and PNGase F treatment markedly inhibited Axl glycoprotein synthesis and expression, proliferation, invasion, and lymphatic metastasis both in vitro and in vivo . In the MTT assay, proliferation was apparent in untreated Hca-F cells compared with treated Hca-F cells. In the ECM invasion assay (in vitro ), treated cells passed through the ECMatrix gel in significantly smaller numbers than untreated cells (tunicamycin 5 μg/mL: 68 ± 8 vs 80 ± 9, P=0.0222; 10 μg/mL: 50 ± 6vs 80 ± 9,P=0.0003; 20 μg/mL: 41 ± 4 vs 80 ± 9, P=0.0001); (PNGase F 8 h: 66 ± 7 vs 82 ± 8, P=0.0098; 16 h: 49 ± 4 vs 82 ± 8, P=0.0001; 24 h: 34 ± 3 vs 82 ± 8, P=0.0001). In the tumor metastasis assay (in vivo ), average lymph node weights of the untreated Hca-F group compared with treated Hca-F groups (tunicamycin 5 μg/mL: 0.84 ± 0.21 g vs 0.72 ± 0.19 g, P=0.3237; 10 μg/mL: 0.84 ± 0.21 g vs 0.54 ± 0.11 g, P=0.0113; 20 μg/mL: 0.84 ± 0.21 g vs 0.42 ± 0.06 g, P=0.0008); (PNGase F 8 h: 0.79 ± 0.15 g vs 0.63 ± 0.13 g, P=0.0766; 16 h: 0.79 ± 0.15 g vs 0.49 ± 0.10 g, P=0.0022; 24 h: 0.79 ± 0.15 g vs 0.39 ± 0.05 g, P=0.0001). Also, average lymph node volumes of the untreated Hca-F group compared with treated Hca-F groups (tunicamycin 5 μg/mL: 815 ± 61 mm 3 vs 680 ± 59 mm 3 , P=0.0613; 10 μg/mL: 815 ± 61 mm 3 vs 580 ± 29 mm 3 , P=0.0001; 20 μg/mL: 815 ± 61 mm 3 vs 395 ± 12 mm 3 , P=0.0001); (PNGase F 8 h: 670 ± 56 mm 3 vs 581 ± 48 mm 3 , P=0.0532; 16 h: 670 ± 56 mm 3 vs 412 ± 22 mm 3 , P=0.0001; 24 h: 670 ± 56 mm 3 vs 323 ± 11 mm 3 , P=0.0001). CONCLUSION: Alteration of Axl glycosylation can at-tenuate neoplastic lymphatic metastasis. Axl N-glycans may be a universal target for chemotherapy.     

Caveolin-1和Caspase-3蛋白在肝细胞癌中的表达及意义

目的研究Caveolin．1和Caspase-3蛋白在肝细胞癌中的表达及意义。方法采用免疫组织化学检测30例肝细胞癌,相应癌旁及10例正常肝组织中Caveolin-1和Caspase-3蛋白的表达。结果（1）Caveolin-1在正常肝组织、癌旁肝组织、肝细胞癌组织中的阳性表达率分别为90％、13．33％、10％。Caspase-3在正常肝组织、癌旁肝组织、肝细胞癌组织中阳性表达率分别为90％、70％、40％。Caveolin．1和Caspase-3在不同性质组织间的表达,差异均有统计学意义（P〈0．05）;（2）Caveolin-1与Caspase-3的表迭在肝细胞癌中无明显相关性（r=0．181,P=0．337）。结论Caveolin-1与Caspase-3在肝细胞癌组织中表达均呈下降,但两者间无明显相关性,提示在肝细胞癌中,Caveolin-1的异常与Caspase-3细胞凋亡紊乱无关。