Analysis of the HNF4A isoform-regulated transcriptome identifies CCL15 as a downstream target in gastric carcinogenesis

Objective:Hepatocyte nuclear factor 4α (HNF4A) has been demonstrated to be an oncogene in gastric cancer (GC). However, the roles of different HNF4A isoforms derived from the 2 different promoters (P1 and P2) and the underlying mechanisms remain obscure.Methods:The expression and prognostic values of P1- and P2-HNF4A were evaluated in The Cancer Genome Atlas (TCGA) databases and GC tissues. Then, functional assays of P1- and P2-HNF4A were conducted both in vivo and in vitro. High-throughput RNA-seq was employed to profile downstream pathways in P1- and P2-HNF4A-overexpressing GC cells. The expression and gene regulation network of the candidate target genes identified by RNA-seq were characterized based on data mining and functional assays.Results:HNF4A amplification was a key characteristic of GC in TCGA databases, especially for the intestinal type and early stage. Moreover, P1-HNF4A expression was significantly higher in tumor tissues than in adjacent non-tumor tissues (P < 0.05), but no significant differences were found in P2-HNF4A expression (P > 0.05). High P1-HNF4A expression indicated poor prognoses in GC patients (P < 0.01). Furthermore, P1-HNF4A overexpression significantly promoted SGC7901 and BGC823 cell proliferation, invasion and migration in vitro (P < 0.01). Murine xenograft experiments showed that P1-HNF4A overexpression promoted tumor growth (P < 0.05). Mechanistically, RNA-seq showed that the cytokine-cytokine receptor interactions pathway was mostly enriched in P1-HNF4A-overexpressing GC cells. Finally, chemokine (C-C motif) ligand 15 was identified as a direct target of P1-HNF4A in GC tissues.Conclusions:P1-HNF4A was the main oncogene during GC progression. The cytokine-cytokine receptor interaction pathway played a pivotal role and may be a promising therapeutic target.     

Hirschsprung-associated enterocolitis develops independently of NOD2 variants

Backgroud/Purpose

Hirschsprung-associated enterocolitis (HAEC) represents a cause for significant pre- and postoperative morbidity and mortality in Hirschsprung disease (HD). Although multiple studies on HAEC have been performed and several mechanisms have been presumed, the pathogenesis of this condition remains unclear. As changes in colonic mucosal defense are key factors suggested in both Crohn's disease (CD) and HAEC pathogenesis, the aim of the current study was to investigate genetic alterations in the most important susceptibility gene for Crohn's enterocolitis (NOD2) to see whether carriers of polymorphisms within the NOD2 gene are predisposed to the development of HAEC.

Methods

Genotyping for the NOD2 variants in exon 4 (p.Arg702Trp [rs2066844]), exon 8 (p.Gly908Arg [rs2066845]), and exon 11 (p.1007fs [rs2066847]) was performed in 52 white children with HD (41 boys, 11 girls), 152 healthy controls, and 152 children with CD (onset of disease <17 years; mean, 11.8 years). Seventeen patients with HD (32.7%) were carriers of a RET germline mutation, 35 children (67.3%) had short segment disease, and 17 (32.7%) had long segment disease.

Results

Ten children (19.2%) with HD were heterozygous carriers of at least one NOD2 variant vs 17 (11.2%) in the healthy control group and 69 (45.4%) in the CD cohort. Hirschsprung-associated enterocolitis was observed in 7 children (13.5%), with 4 having short segment HD and 3 with long segment HD; but none of them were carriers of NOD2 variants.

Conclusion

Our study shows that NOD2 variants described to be causatively associated with CD do not predispose to the development of HAEC. As data on the molecular basis of HAEC are limited, the distinct mechanisms involved in the pathogenesis of this complication remain unclear.     

A novel recessive 15-hydroxyprostaglandin dehydrogenase mutation in a family with primary hypertrophic osteoarthropathy

Abstract

We present two PHO siblings having a novel homozygous truncating mutation in HPGD. The purpose of the study was to attempt medical treatment, and to find the HPGD mutation causing the disease, in a 22-year old Turkish male and his 23-year old sister afflicted with primary hypertrophic osteoarthropathy (PHO). In combination with NSAIDs and colchicine, treatment with sulfasalazine was started in both cases, and methotrexate was added to the treatment regimen of the female patient at the end of the first year. The patients were found to be typical PHO. Ultrasonographic examination of the joints revealed synovitis and inflammation by B mode and power Doppler ultrasonography. Joint symptoms responded to sulfasalazine treatment in both patients. However, after the addition of methotrexate, the female patient had better remission. All exons of HPGD, the known disease gene, were analyzed by Sanger sequencing. A homozygous 2-bp deletion (c.310_311delCT or p.L104AfsX3) was identified. Seven relatives carrying the mutation in the heterozygous state were examined and none was found affected. Although not specific for this disease, skin, soft tissue and joint ultrasonography can be helpful for evaluation of the musculoskeletal findings in the patients.     

Leukotriene B4 12-hydroxydehydrogenase/15-ketoprostaglandinΔ13-reductase (LTB4 12-HD/PGR) responsible for the reduction of a double-bond of the α,β-unsaturated ketone of an aryl propionic acid non-steroidal anti-inflammatory agent CS-670

CS-670 is a non-steroidal anti-inflammatory agent with an α,β-unsaturated ketone structure. It exerts its pharmacological activity after being transformed to the active metabolite (2S,1′R,2′S)-trans-alcohol. Two consecutive reductions are needed for the formation of the active metabolite, reduction of the double-bond of the α,β-unsaturated ketone moiety, followed by reduction of the resulting saturated ketone. The objective of the current study was to identify the enzyme responsible for reduction of the double-bond. An enzyme purified from rat liver cytosol as a single band on sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was analysed by a Mascot database search of nano-LC tandem mass spectrometry (MS/MS) data and the enzyme was identified as 2-alkenal reductase (EC 1.3.1.74), which is known as an β-nicotinamide adenine dinucleotide phosphate (NADPH)-dependent alkenal/one oxidoreductase and has a role for leukotriene B₄ 12-hydroxydehydrogenase/15-ketoprostaglandinΔ13-reductase (LTB₄ 12-HD/PGR). The identification was confirmed by cloning LTB₄ 12-HD/PGR cDNA from rat liver, expressing it in Escherichia coli, and characterizing the properties of the enzyme. The identity was further supported by the subcellular localization in cytosol, a cofactor requirement for NADPH, substrate specificity, and substantial inhibition by 15-ketoPGF_2α, benzylideneacetophenone, indomethacin, and quercitrin. In addition to catalysing the biological reduction of eicosanoids, including prostaglandins, leukotrienes, and lipoxins, LTB₄ 12-HD/PGR was also determined to function as a xenobiotic-metabolizing enzyme.     

Relationship between lipogenesis,ketogenesis, and malonyl-CoA content in isolated hepatocytes from the obese Zucker rat adapted to a high-fat diet

The relationship between lipogenesis and ketogenesis and the concentration of malonyl coenzyme A (CoA) was investigated in hepatocytes from adult obese Zucker rats and their lean littermates fed either a control low-fat diet or a high-fat diet (30% lard in weight). With the control diet, lipogenesis—although strongly inhibited in the presence of either 1 mmol/L oleate, 10^?6 mol/L glucagon or 0.1 mmol/L TOFA (a hypolipidemic drug)—remained about fifteen-fold higher in the obese rats than in the lean rats. In contrast, ketogenesis under some conditions (oleate + TOFA) was not significantly lower (30%) as compared with the lean rats. After adaptation to the high-fat diet, lipogenesis was depressed fourfold in the lean rats and ninefold in the obese ones; however its magnitude remained significantly higher in the latter, namely at a value close to that measured in control-fed lean rats. Ketogenesis was comparable in lean and obese rats and much higher in the presence of 1 mmol/L oleate than of 0.3 mmol/L oleate, whereas lipogenesis did not vary with increasing oleate concentration in the medium. Acetyl-CoA carboxylase activity measured in liver homogenates was higher in the obese group, but was stepwise inhibited by increasing concentrations of oleyl-CoA regardless of the diet for both lean and obese rats, thus showing no abnormality of in vitro responsiveness to this inhibitor. With the control diet, hepatocyte malonyl-CoA levels were significantly higher in the obese rats, both in the basal state and after inhibition of lipogenesis by oleate and TOFA. However, after the high-fat diet, there was no longer a significant difference between the genotypes. These results show that in the obese Zucker rats, ketogenesis is dependent on hepatocyte malonyl-CoA content in the sense that their ketogenic capacity becomes “normalized” when malonyl-CoA is decreased to the levels found in the lean littermates, as it is the case after fat-feeding. This normalization of malonyl-CoA levels in spite of higher lipogenesis in the obese rats may result from the activities of enzymes of its formation and utilization.     

乳腺癌组织中细胞粘附分子CD15的定量研究

为了探讨细胞粘附分子ＣＤ１５表达及含量与乳腺癌分化程度和淋巴结转移状况的关系,应用微波－ＳＰ法和图像分析技术,对９４例乳腺癌和１０例正常乳腺组织中ＣＤ１５的表达及含量进行检测。结果：ＣＤ１５表达阳性物质在正常乳腺中有很极性地位于腺上皮游高面,而在乳腺癌中主要分布于浆膜;ＣＤ１５表达阳性率和图像定量分析平均光密度值在乳腺癌组织中均显著高于正常乳腺组织,并均随乳腺癌分化程度减低和淋巴结转移而     

三氧化二砷对人脐静脉内皮细胞中蛋白激酶C活性和白细胞介素-15分泌、表达的影响及其临床意义

目的 通过研究三氧化二砷(As2O3)对人脐静脉内皮细胞(HUVEC)中蛋白激酶C(PKC)活性和白细胞介素(IL)-15表达和分泌的影响,探讨内皮细胞在As2O3抑制白血病或肿瘤中的作用。方法用不同浓度的As2O3作用于培养的HUVEC细胞,测定其生长情况、胞膜和胞浆PKC活性以及对IL-15基因表达和分泌的影响。结果0.25 μmol／L~50 μmol／L的.As2O3均抑制HUVEC细胞的增殖(P<0.01),随着药物浓度或作用时间的增加,细胞逐渐死亡。在As2O3作用细胞4 h测定胞浆和胞膜的PKC活性,两者在不同浓度作用下活性水平均得到提升(P<0.01),但胞膜的活性上升幅度更大。在As2O3的测定浓度范围内,PKC的活性水平与As2O3的浓度呈正相关。1、5、16 μmol／L的As2O3导致IL-15基因表达和分泌水平的增加(P<0.01),并呈浓度依赖性关系。结论As2O3可能是通过激活内皮细胞的PKC活性,促进IL-15或其他细胞因子的分泌,来抑制白血病或肿瘤发展的。     

CA19-9及其同分异构体CD15s在大肠癌表达的对比研究

目的:探讨CA19-9及其同分异构体CD15s在大肠癌中的表达及其与肿瘤发生、分化、转移及预后的关系.方法:应用催化信号放大法(CSA)检测150例大肠癌、84例转移淋巴结和50例远离癌组织的正常大肠黏膜CA19-9及其同分异构体CD15s的表达状况.结果:CA19-9和CD15s阳性物质在远离癌组织的正常大肠黏膜中阳性表达率分别为88.0%(44/50)和18.0%(9/50),在大肠癌组织中阳性率分别为81.3%(122/150)和94.0%(141/150),转移淋巴结标本中阳性率分别为66.7%(56/84)和95.2%(80/84).结论:作为预测大肠癌侵袭、转移及评估患者预后的肿瘤相关抗原,CD15s比CA19-9具有更重要的临床意义.