S-腺苷甲硫氨酸抑制大肠癌细胞生长的实验研究

目的探讨S-腺苷甲硫氨酸(SAM)对人大肠癌细胞生长的抑制作用及抑癌机制。方法用SAM对大肠癌细胞系HT-29进行处理,使其癌基因c-myc和H-ras启动子区域甲基化。用噻唑蓝(MTT)法检测肿瘤细胞生长状态;甲基化特异性聚合酶链反应(PCR)(MSP)法检测c-myc和H-ras启动子区域甲基化状态;细胞免疫荧光染色检测C-MYC和H-RAS蛋白的表达情况。结果大肠癌细胞系HT-29经SAM处理后,癌基因c-myc和H-ras启动子区域重新出现甲基化。经SAM处理的大肠癌细胞组与对照组相比,细胞生长受到明显抑制,差异有统计学意义(P<0.05);C-MYC和H-RAS蛋白表达明显降低,差异有统计学意义(P<0.05)。结论 SAM能使HT-29中癌基因c-myc和H-ras启动子区域重新甲基化,降低C-MYC和H-RAS蛋白的表达水平,且有效抑制了肿瘤细胞的生长,从而为肿瘤治疗提供了新的靶点。     

Fatty acids do not activate UCP2 in pancreatic beta cells: comparison with UCP1

UCP2 is expressed in pancreatic β cells where its postulated uncoupling activity will modulate glucose-induced changes in ATP/ADP ratio and insulin secretion. The consequences of UCP2 over/underexpression on β-cell function has mainly been studied in the basal state; however, a UCP has no uncoupling activity unless stimulated by fatty acids and/or reactive oxygen species. Here, UCP2 was overexpressed in INS-1 cells and parameters reflecting mitochondrial coupling measured in the basal state and after stimulation by fatty acids. For comparison, UCP1 was expressed to similar levels and the same parameters measured. Neither UCP1 expression nor UCP2 overexpression modified basal or glucose-stimulated metabolic changes. Upon addition of fatty acids, UCP1-expressing cells displayed the expected mitochondrial uncoupling effect, while UCP2 did not elicit any measurable change in mitochondrial function. Taken together, our data demonstrate that, in pancreatic β-cells, UCP2 has no uncoupling activity in the basal state or after fatty acid stimulation.     

乙型肝炎病毒X蛋白通过降低P4启动子甲基化水平上调人IGF-II基因的转录

目的:构建稳定表达乙型肝炎病毒X蛋白(HBx)的肝癌细胞株HepG2-HBx,探讨HBx对胰岛素样生长因子 II(IGF-II)基因P4启动子甲基化水平及转录表达的影响。方法:应用基因重组技术,构建含HBx基因的重组逆转录病毒载体pBABE-puro-HBx,采用磷酸钙共沉淀法将其转染293FT包装细胞产生逆转录病毒,感染HepG2肝癌细胞,采用嘌呤霉素进行阳性克隆筛选,Western blotting鉴定表达HBx蛋白的肝癌细胞株HepG2-HBx。采用亚硫酸氢盐测序法及实时荧光定量RT-PCR检测HepG2-HBx细胞中P4启动子甲基化水平及P4 mRNA表达水平变化。进一步将体外甲基化的人IGF-II基因P4启动子驱动的荧光素酶报告载体pGL3-P4及含HBx基因的pCMV-tag2B-X质粒共转染HepG2肝癌细胞,采用亚硫酸氢盐测序法及双萤光素酶实验检测pGL3-P4载体上P4启动子甲基化水平及转录调控活性变化。结果:(1)经Western blotting鉴定,成功构建了稳定表达HBx蛋白的肝癌细胞株HepG2-HBx;(2)表达HBx蛋白的HepG2-HBx细胞中P4启动子甲基化CpG位点的比例(9.0%)明显低于对照细胞HepG2-control(25.0%)(P<0.01),而其P4 mRNA表达水平则为对照细胞HepG2-control的2.8倍;(3)共转染pCMV-tag2B-X质粒的HepG2细胞中pGL3-P4载体上P4启动子甲基化CpG位点的比例(60.8%)明显低于共转染对照质粒pCMV-tag2B的HepG2细胞(84.1%)(P<0.01),而前者P4启动子相对萤光素酶活性(14.12±0.89)则明显高于后者(4.61±0.76)(P<0.01)。结论:HBx蛋白可降低IGF-II基因P4启动子甲基化水平,进而上调其转录表达。     

Nrf2在不同肝癌细胞株中的表达及定位

目的:观察红系衍生的核因子2相关因子2(Nrf2)在肝癌细胞株(HepG2、Hep3B、SMMC-7721)中的表达及分布情况.方法:应用流式细胞术测定Nrf2阳性细胞的比例,激光共聚焦观察Nrf2在3种细胞株中的定位情况,通过Wostern blot测定Nrf2在不同细胞胞质及胞核中的表达差异.结果:流式细胞术测定Nd2在HepG2、Hep3B和SMMC-77213种细胞中表达率分别为99.39%、99.94%和99.98%,激光共聚焦观察到Nd2在3种肝癌细胞株胞质胞核中均有分布,但不同细胞的分布规律各不相同,Western blot结果显示:Nrf2在HepG2中以胞质分布为主,在Hep3B中胞质、胞核分布相当,在SMMC-7721中以胞核分布为主.结论:Nrf2在3种肝癌细胞株(HeqG2、Hep3B、SMMC-7721)中均有表达,且在胞质胞核中均有分布,但在不同细胞中的分布规律各不相同,为下一步探讨肝癌细胞耐药机制的相关研究奠定了基础.     

LGR5在肝癌细胞及肝癌组织中的表达及意义

目的 探讨LGR5在4个肝癌细胞系、癌旁组织及肝癌组织中的表达,及在肝癌中的作用.方法 免疫印迹法及实时定量RT-PCR检测正常细胞和4种肝癌细胞中LGR5的表达;采用免疫组织化学检测36例肝癌及癌旁组织中LGR5及β-catenin的表达;利用免疫细胞化学检测HepG2细胞中LGR5的表达.结果 免疫印迹法及实时定量...     

Calpain 2-dependent IκBα degradation mediates CPT-11 secondary resistance in colorectal cancer xenografts

CPT-11 (irinotecan), the first-line chemotherapy for advanced stage colorectal cancer, remains inactive in about half of patients (primary chemoresistance) and almost all initial responders develop secondary resistance after several courses of treatment (8 months on average). Nude mice bearing HT-29 colon cancer xenografts were treated with CPT-11 and/or an NF-κB inhibitor for two courses. We confirm that NF-κB inhibition potentiated CPT-11 anti-tumoural effect after the first course of treatment. However, tumours grew again at the end of the second course of treatment, generating resistant tumours. We observed an increase in the basal NF-κB activation in resistant tumours and in two resistant sublines, either obtained from resistant HT-29 tumours (HT-29R cells) or generated in vitro (RSN cells). The decrease of NF-κB activation in HT-29R and RSN cells by stable transfections with the super-repressor form of IκBα augmented their sensitivity to CPT-11. Comparing gene expression profiles of HT-29 and HT-29R cells, we identified the S100A10/Annexin A2 complex and calpain 2 as over-expressed potential NF-κB inducers. SiRNA silencing of calpain 2 but not of S100A10 and/or annexin A2, resulted in a decrease in NF-κB activation, an increase in cellular levels of IκBα and a partial restoration of the CPT-11 sensitivity in both HT-29R and RSN cells, suggesting that calpain 2-dependent IκBα degradation mediates CPT-11 secondary resistance. Thus, targeted therapies directed against calpain 2 may represent a novel strategy to enhance the anti-cancer efficacy of CPT-11.     

A polymorphism in the promoter of UCP2 gene modulates lipid levels in patients with type 2 diabetes

A G/A single nucleotide polymorphism (SNP) in the position -866 of the UCP2 promoter modulates UCP2 expression in adipose tissue and pancreatic beta-cell, and is associated with variations of body mass index (BMI) and insulin secretion in nondiabetic subjects. We investigated associations of this SNP with traits related to obesity, dyslipidemia, and hyperglycemia in patients with type 2 diabetes. The -866 G/A SNP in the UCP2 promoter was genotyped by PCR/RFLP in 681 type 2 diabetic patients. Increased triglyceride (> or = 1.70 mM), total cholesterol (> or = 6.0 mM) and LDL-cholesterol (> or = 3.35 mM) levels were significantly less frequent in homozygous carriers of the G-allele than in homozygous carriers of the A-allele. Odds ratios for the risk of dyslipidemia in GG vs AA carriers were 0.45, 0.57, and 0.50, for triglyceride, total cholesterol and LDL-cholesterol, respectively (all p<0.007). No genetic effects of this polymorphism on the BMI or on traits related to the severity of hyperglycemia were observed. In conclusion, a common polymorphism in the promoter region of the UCP2 gene modulates triglycerides and cholesterol levels in French Caucasian subjects with type 2 diabetes. The implications of this effect in the evolution of type 2 diabetes and its macrovascular complications deserve to be investigated.     

DNA methylation dominates transcriptional silencing of Pax5 in terminally differentiated B cell lines

Pax5 plays a key role in the progression of B cell development. Its expression is observed in a wide range of cell types from early lineage-committed precursors up to mature B cells, but is silenced in terminal differentiated plasma cells. In this report, we show that DNA methylation is involved in the silencing of Pax5. In the Pax5-expressing cell lines 38B9 (pre-B) and 2PK-3 (mature B), all CpG sites in TATA-containing upstream promoter were unmethylated, whereas these sites were completely methylated in myeloma cell lines FO and Sp-2/0, which do not express Pax5. Demethylation of FO and Sp-2/0 with 5-aza-2'-deoxycytidine (5-aza-dC) resulted in Pax5 re-expression with the concomitant expression of CD19 and mb-1 genes, which are known to be the target genes of Pax5. Re-expression of Pax5 was also induced by trichostatin A (TSA), which was a specific inhibitor of histone deacetylase. This re-expression was, however, transcribed only from the TATA-less downstream promoter. Taken together, we concluded that the upstream promoter was predominantly inactivated by DNA methylation, while the downstream promoter was repressed by the histone deacetylation. This synergetic inactivation of two promoters results in the final silencing of Pax5 expression in terminally differentiated B cell lines.     

Aberrant Cosmc genes result in Tn antigen expression in human colorectal carcinoma cell line HT-29

The Tn antigen, which arises from mutation in the Cosmc gene is one of the most common tumor associated carbohydrate antigens. Cosmc resides in X24 encoded by a single gene and functions as a specific molecular chaperone for T-synthase. While the Tn antigen cannot be detected in normal cells, Cosmc mutations inactivate T-synthase and consequently result in Tn antigen expression within certain cancers. In addition to this Cosmc mutation-induced expression, the Tn antigen is also expressed in such cell lines as Jurkat T, LSC and LS174T. Whether the Cosmc mutation is present in the colon cancer cell line HT-29 is still unclear. Here, we isolate HT-29-Tn⁺ cells from HT-29 cells derived from a female colon cancer patient. These HT-29-Tn⁺ cells show a loss of the Cosmc gene coding sequence (CDS) leading to an absence of T-synthase activity and Tn antigen expression. Additionally, almost no methylation of Cosmc CpG islands was detected in HT-29-Tn⁺ as well as in HT-29-Tn^- and Tn^- tumor cells from male patients. In contrast, the methylation frequency of CpG island of Cosmc in normal female cells was ~50%. Only one active allele of Cosmc existed in HT-29-Tn⁺ and HT-29-Tn^- cells as based upon detection of SNP sites. These results indicate that Tn antigens expression and T-synthase inactivity in HT-29-Tn⁺ cells can be related to the absence of CDS in Cosmc active alleles, while an inactive allele deletion of Cosmc in HT-29 cells has no influence on Cosmc function.     

Protective effect of RIP and c-FLIP in preventing liver cancer cell apoptosis induced by TRAIL

TRAIL (TNF-related apoptosis-inducing ligand) is a member of the tumor necrosis factor superfamily that can induce tumor selective death by up-regulating death receptor 4 (DR4) and DR5 expression. The study aimed to explore the role of RIP and c-FLIP genes in TRAIL induced liver cancer cell HepG2 and Hep3B apoptosis and related mechanism. RIP and c-FLIP silenced HepG2 and Hep3B cell model were established through siRNA. Western blot was applied to test c-FLIP, RIP, DR4, DR5, FADD, Caspase-3/8/9, ERK1/2, and DFF45 protein expression. Caspase-8 kit was used to detect Caspase-8 expression. Flow cytometry was performed to measure cell apoptosis rate. Acid phosphatase method was applied to determine cell cycle. TRAIL had no significant effect on Caspase-3/8/9, DR4, DR5, ERK1/2, and DFF45 protein expression, but up-regulated c-FLIP and RIP protein expression and reduced FADD expression level. After treated by the chemotherapy drug mitomycin and adriamycin, c-FLIP and RIP expression decreased significantly, while FADD increased. After knockout c-FLIP and RIP gene, HepG2 and Hep3B cell apoptosis rate induced by TRAIL increased obviously. Meanwhile, cell subG1 percentage increased markedly and exhibited G1 phase growth retardation. In addition, after two kinds of gene knockout, Caspase-8 was activated and produce Caspase-3 P20 and P24, leading DFF45 appeared DNA fragment P17 and P25. c-FLIP and RIP can inhibit Caspase-8 activation and prompting HepG2 and Hep3B resistant to cell apoptosis induced by TRAIL.     

miR-483-5p通过上调IGF2基因转录促进肝癌细胞生长、迁移与侵袭

目的:探讨miR-483-5p对人胰岛素样生长因子2(IGF2)基因P3启动子驱动的m RNA(P3 m RNA)表达的影响及其在肝细胞癌发生发展中的作用。方法:(1)采用real-time PCR检测人肝癌细胞株Huh7、Hep3B、Bel-7402、Hep G2和SMMC-7721,人正常肝细胞株HL-7702,83例人肝细胞癌组织和配对的癌旁组织,22例正常肝组织中miR-483-5p和P3 m RNA的表达水平,并应用Pearson相关分析评估P3 m RNA与miR-483-5p表达水平之间的关系。(2)将IGF2基因P3 m RNA的5’端非翻译区(5’UTR)克隆入p GL3启动子载体,构建P3 m RNA 5’UTR野生型(p GL3-P3-5’UTR-WT)及P3 m RNA 5’UTR突变型(p GL3-P3-5’UTR-MUT)重组萤光素酶报告质粒,将其分别与miR-483-5p mimic、miR-483-5p inhibitor及scrambled control共转染He La、293T及Huh7细胞,采用双萤光素酶报告系统检测萤光素酶活性。(3)分别将miR-483-5p mimic、miR-483-5p inhibitor及scrambled control转染Huh7及Hep3B肝癌细胞,应用real-time PCR检测这2种肝癌细胞P3 m RNA表达水平的变化。(4)应用real-time PCR检测肝癌细胞Huh7及Hep3B的细胞核和细胞质中miR-483-5p的表达水平;应用核连缀实验(nuclear run-on assay)分析miR-483-5p对P3 m RNA转录的影响;应用RNA稳定性实验分析miR-483-5p对P3 m RNA稳定性影响。(5)应用体外细胞功能实验研究miR-483-5p对Huh7肝癌细胞生长、凋亡、迁移与侵袭能力的影响。结果:(1)5种肝癌细胞株miR-483-5p及P3 m RNA表达水平均明显高于正常肝细胞株HL-7702(P0.01),肝细胞癌组织中miR-483-5p及P3 m RNA表达水平均明显高于配对的癌旁组织及正常肝组织(P0.01);线性相关分析显示,在5种肝癌细胞株及肝细胞癌组织中,P3 m RNA表达水平均与miR-483-5p水平呈正相关。(2)萤光素酶实验显示,miR-483-5p与P3m RNA 5’UTR的同源位点互补结合可促进P3 m RNA的表达。(3)瞬时转染实验显示,过表达miR-483-5p呈剂量依赖性促进Hep3B和Huh7肝癌细胞P3 m RNA表达水平的增高。(4)miR-483-5p表达实验显示,成熟miR-483-5p存在于肝癌细胞Hep3B和Huh7的细胞质和细胞核中;核连缀实验显示,miR-483-5p诱导Huh7肝癌细胞核中新生P3 m RNA转录;RNA稳定性实验表明,miR-483-5p不改变Huh7肝癌细胞P3 m RNA稳定性。(5)体外细胞功能实验显示,miR-483-5p促进Huh7肝癌细胞增殖,抑制其凋亡,并增强迁移与侵袭能力。结论:miR-483-5p高表达可部分通过上调IGF2基因P3 m RNA转录促进肝癌细胞生长、迁移与侵袭,进而参与肝细胞癌发生。     

miR-485-5p靶向作用于SOX5抑制肝癌细胞Hep3B的活力及侵袭和迁移能力

目的:研究微小RNA-485-5p(miR-485-5p)对肝癌细胞的活力及迁移和侵袭能力的影响及其潜在机制。方法:以正常肝细胞THLE-3为对照,采用RT-qPCR检测肝癌细胞中性别决定区Y框蛋白5(SOX5)mRNA和miR-485-5p的表达,Western blot检测SOX5、增殖细胞核抗原(PCNA)、Ki67、细胞周期蛋白D1(cyclin D1)及基质金属蛋白酶2(MMP-2)的表达水平,MTT法检测Hep3B细胞的活力,Transwell实验检测细胞的迁移和侵袭能力,双萤光素酶报告基因系统验证细胞中miR-485-5p与SOX5的调控关系。结果:与对照组相比,在肝癌细胞Hep3B、Huh7和HCCLM3中miR-485-5p的表达水平显著降低(P<0.05),SOX5的mRNA和蛋白表达水平均显著升高(P<0.05);过表达miR-485-5p可抑制Hep3B细胞的活力及迁移和侵袭能力(P<0.05);miR-485-5p靶向调控SOX5的表达,敲减SOX5的表达也可抑制Hep3B细胞的活力及迁移和侵袭能力;过表达SOX5可部分逆转过表达miR-485-5p对Hep3B细胞活力及迁移和侵袭能力的抑制作用(P<0.05)。结论:miR-485-5p通过靶向调控SOX5基因抑制Hep3B细胞的活力及迁移和侵袭能力,有望成为肝癌诊断和治疗的潜在分子靶点。     

Nuclear factor kappa B plays a pivotal role in polyinosinic-polycytidylic acid-induced expression of human β-defensin 2 in intestinal epithelial cells

Intestinal epithelial cells (IECs) play an important role in protecting the intestinal surface from invading pathogens by producing effector molecules. IECs are one of the major sources of human beta-defensin 2 (hBD-2), and can produce it in response to a variety of stimuli. Although IECs express Toll-like receptor 3 (TLR-3) and can respond to its ligand, double-stranded RNA (dsRNA), hBD-2 expression in response to dsRNA has not been elucidated. In the present study, using an artificial analogue of dsRNA, polyinosinic-polycytidylic acid (poly I:C), we investigated whether the human IEC line, HT-29, can produce hBD-2 in response to poly I:C. HT-29 cells can express hBD-2 mRNA only when stimulated with poly I:C. The induction of hBD-2 mRNA expression was observed at 3 h after stimulation and peaked at 12 h of post-stimulation. Pre-incubation of the cells with nuclear factor kappa B (NF-κB)-specific inhibitor, l-1-4'-tosylamino-phenylethyl-chloromethyl ketone (TPCK) and isohelenine abolished the expression of hBD-2. Detection of the poly I:C signal by TLR-3 on the surface of HT-29 cells was revealed by pre-incubating the cells with anti-TLR-3 antibody. The 5'-regulatory region of the hBD-2 gene contains two NF-κB binding sites. A luciferase assay revealed the importance of the proximal NF-κB binding site for poly I:C-induced expression of hBD-2. Among NF-κB subunits, p65 and p50 were activated by poly I:C stimulation and accumulated in the nucleus. Activation of the p65 subunit was investigated further by determining its phosphorylation status, which revealed that poly I:C stimulation resulted in prolonged phosphorylation of p65. These results indicate clearly that NF-κB plays an indispensable role in poly I:C induced hBD-2 expression in HT-29 cells.     

EXTL3 promoter methylation down-regulates EXTL3 and heparan sulphate expression in mucinous colorectal cancers

Exostoses like-3 (EXTL3) is a putative tumour suppressor gene but its involvement in colorectal cancer (CRC) is unclear. We have investigated the role of methylation of the EXTL3 promoter as a mechanism for EXTL3 regulation and tested the hypothesis that loss of EXTL3 expression is associated with mucinous differentiation and alteration of glycoprotein expression in CRC cells. The methylation status of the EXTL3 gene promoter was analysed by methylation-specific PCR following bisulphite modification in CRC cell lines and microdissected primary CRC tissues and their corresponding adjacent normal colorectal mucosa. EXTL3 promoter methylation was detected in seven of 11 mucinous CRCs (63.6%) but in none of 26 non-mucinous CRCs examined. EXTL3 promoter methylation was also detected in the normal colonic mucosa of three patients with mucinous CRC but not in the normal colonic mucosa of any patients with non-mucinous CRC. The presence of EXTL3 methylation was significantly associated with the partial loss of HS expression in mucinous CRC lesions. The mucinous CRC cell lines, Colo201 and Colo205, showed EXTL3 promoter methylation and loss of EXTL3 mRNA expression. However 5-aza-2'-deoxycytidine treatment demethylated the EXTL3 gene promoter and restored its mRNA expression. Furthermore, the basal expression of HS in CRC cells was abolished by treatment with EXTL3-siRNA. We conclude that EXTL3 promoter methylation and its related loss of EXTL3 expression are involved in the loss of HS expression in mucinous CRCs.     

HAPLN1通过IKK/p65信号通路诱导大肠癌HT-29细胞产生MTX耐药

目的:研究甲氨蝶呤(methotrexate,MTX)耐药前后人大肠癌(结直肠癌)HT-29细胞中透明质酸蛋白聚糖连结蛋白1(hyaluronan and proteoglycan link protein 1,HAPLN1)表达的变化及其对MTX耐药性的影响,并探究可能的分子机制。方法:采用浓度梯度递增法构建耐药细胞株HT-29/MTX;RT-PCR检测HAPLN1和多药耐药相关蛋白2(multidrug resistance-associated protein 2,MRP2)的mRNA表达水平;采用脂质体介导法将人HAPLN1和MRP2基因的干扰质粒转染HT-29/MTX细胞并筛选出稳定表达的细胞系;采用CCK-8法检测细胞活力;流式细胞术检测细胞凋亡;采用Western blot检测HAPLN1、MRP2、IκB激酶(IκB kinase,IKK)α/β、p-IKKα/β(Ser176/Ser177)、p65和p-p65(Ser536)的蛋白水平。结果:HT-29/MTX细胞中HAPLN1和MRP2的mRNA和蛋白表达水平都显著高于其亲代HT-29细胞(P0.05),耐药倍数高达463.756。抑制HAPLN1和MRP2基因表达使MTX对HT-29/MTX细胞的IC_(50)从15.304μmol/L分别降至6.119μmol/L和7.801μmol/L,逆转倍数分别为2.501和1.962,并增强MTX诱导的细胞凋亡(P0.05)。敲减HAPLN1基因表达和使用IKK抑制剂IKK16均可下调IKKα/β和p65蛋白磷酸化水平以及MRP2蛋白表达水平(P0.05),但IKK16未对HAPLN1蛋白表达产生影响(P0.05)。结论:敲减HAPLN1基因表达可在体外增强人大肠癌耐药细胞株HT-29/MTX对MTX的敏感性,这可能与其抑制IKK/p65信号通路活化,继而下调MRP2基因表达有关。     

Jmjd3和Ezh2酶活性抑制剂对人间充质干细胞成脂分化的影响及机制研究

目的 研究EZH2和JMJD3对人脂肪间充质干细胞成脂分化的影响及可能机制。方法 体外培养人脂肪来源间充质干细胞并进行诱导分化。分化过程中分别加入EZH2酶活性抑制剂GSK126（6 μM）和JMJD3酶活性抑制剂GSKJ4（20 μM）,对照组加入DMSO。采用Realtime PCR方法检测脂肪细胞分化基因PPARγ、FABP4和ADIPOQ;棕色化标志基因UCP1、PRDM16和CIDEA,以及米色脂肪独有的标志基因CD137、TMEM26和TBX1,并计算各组与对照组的相对表达量。采用West