TREATMENT OF HEPATOCELLULAR CARCINOMA BY THE REGULATION OF GLYCOSYLTRANSFERASE

通过糖基转移酶的调节治疗肝细胞癌

• 批准号: 13670500
• 负责人: AOYAGI Yutaka
• 金额: $ 2.3万
• 依托单位: NIIGATA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670500/
• 关键词: Alpha 1-6 fucosyltransferase; hepatocellular carcinoma; lipofection; antisense oligonucleotide; Lens culinaris agglutinin; treatment; アルファフェトプロテイン

In Japan, hepatocellular carcinoma (HCC) is one of the most prevalent human cancers. The diagnosis and treatment for HCC are still difficult although several therapeutic modalities and serological tumor markers, such as alpha-fetoprotein (AFP), have been developed. We have already shown that the relative amount of the Lens culinaris agglutinin (LCA)-reactive species of AFP is significantly greater in HCC than in non-neoplastic liver diseases and that the higher proportion of (LCA)-reactive species of AFP is significantly associated with the tumor invasiveness. The molecular basis of the LCA-reactive species of AFP is the fucosylation at the innermost N-acetylglucosamine residue of the biantennary sugar chain of AFP. Thus, it is supposed that these phenotypic changes must be provided by GDP-L-Fuc: N-acethyl-β-D-glucosaminide: α1-6 fucosyltransferase (αFT), which catalyzes the addition of fucose from GDP-fucose through an α1-6 linkage to the reducing end of N-acetylglucosamine residue of … More N-linked oligosaccharides of glycoproteins. Accordingly, we attempt to perform the treatment of HCC by the regulation of αFT in the present project.For this issue, we evaluated whether the proportion of LCA-reactive species of AFP could be diminished by the transfer of antisense oligonucleotide, which was deduced from the sequence reported as αFT : GenBank D89289. Because the conformation of αFT mRNA estimated by RNA structure software (David H Mathews, et al) indicated that the vicinity at the nucleotide position 861 would be available for antisense access, 2'-methoxyethyl phosphorothioate RNA oligonucleotide, 5'-CACTCATCTTGGA-3', was transfected to HepG2 cells by lipofection. The AFP concentrations at 24 hours after lipofection were 3.2 ± 0.4 ng/ml, 3.3 ± 0.3 ng/ml, 3.8 ± 0.2 ng/ml, and 2.4 ± 0.1 ng/ml in the medium of the cells without lipofection, with RNA(-)-lipofection, sense-lipofection, and antisense-lipofection, respectively. The concentration in antisense-lipofection was significantly lower than that in sense-lipofection (Dunn's test P<0.05). The proportions of LCA-reactive species of AFP at 24 hours after lipofection were 76.2 ± 2.8 %, 82.8 ± 0.8 %, 58.7 ± 2.9 %, and 48.0 ± 9.6 % in the medium of the cells without lipofection, with RNA(-)-lipofection, sense-lipofection, and antisense-lipofection, respectively. The proportion in antisense lipofection was significantly lower than that in RNA(-)-lipofection (P<0.01). These results suggested that αFT (D89289) is responsible for the fucosylation of AFP and that the depletion of αFT activity may reduce AFP production affecting the cell viability.In the next step, we are engaged in an establishment of αFT knock-down cells by inducing small interfering RNA against αFT. The 19 nucleotides from the nucleotide position 1002 were cloned into pSUPER.retro vector (OligoEngine, USA), which expresses a target RNA connected with the complementary sequence forming double-stranded RNA in an intrastranded fashion. Although the clones carrying empty and mutated sequences have been established, none of carrying the target sequence has been established yet. There are some possibilities that αFT activity is critical for cell viability. Less

在日本，肝细胞癌（HCC）是最普遍的人类癌症之一。尽管已经开发了几种治疗方法和血清学肿瘤标志物，例如α-毒素（AFP），但HCC的诊断和治疗仍然很困难。我们已经表明，HCC的AFP晶状体凝集蛋白（LCA）反应性物种的相对量明显高于非肿瘤肝病，并且AFP的（LCA）反应性较高的AFP比例与肿瘤的侵入性相关。 AFP的LCA反应性物种的分子基础是AFP双胞质糖链的最内向的N-乙酰葡萄糖住所。这是可以预期的，GDP-L-FUC必须提供这些表型变化：N-乙基-β-D-D-葡萄糖酰胺：α1-6诱导糖基转移酶（αFT），催化通过α1-6与α1-6链接的N-烯基烯酸化液相链接的GDP-ucose从GDP-ucose中添加岩藻糖的添加……糖蛋白。根据这个问题，我们试图通过在本项目中对αFT的调节进行HCC处理。对于此问题，我们评估了AFP的LCA反应性物种的比例是否可以通过反义寡核苷酸的转移来减少，而反义寡核苷酸的转移是根据序列报道为αFT：GenBank D89289的序列。因为通过RNA结构软件估算的αFTmRNA的构象（David H Mathews等）表明，核苷酸位置的附近861可用于反义访问，2'-甲氧基甲氧基磷酸胸酯RNA RNA RNA RNA RNA寡核苷酸，5'- cactcatcatcatcatctcattgga-3'，由5'- cactcatcattgga-3'，由lipfg2 to lipff2细胞。在没有脂肪触发的细胞中，脂肪产后24小时的AFP浓度为3.2±0.4 ng/ml，3.3±0.3 ng/ml，3.8±0.2 ng/ml，和2.4±0.1 ng/ml的浓度在没有脂肪的培养基中，RNA（ - ） - 脂肪触发，理性 - 透镜，理性 - 透镜，理性，与静态质量分析。反义 - 脂肪触发的浓度明显低于感官脂肪的浓度（Dunn的测试p <0.05）。脂化后24小时，AFP的LCA反应性种类的比例为76.2±2.8％，82.8±0.8％，58.7±2.9％，在没有脂肪的培养基的培养基中，带有RNA（ - ） - 唇部 - 唇形，理性，感觉，静脉曲张和反式渗透，分别为rna（ - ） - 含量为48.0±9.6％。反义lipofection的比例明显低于RNA（ - ） - 脂肪触发（p <0.01）的比例。这些结果表明，αFT（D89289）负责AFP的脉糖基化，并且αFT活性的耗竭可以减少影响细胞活力的AFP产生。在下一步中，我们通过诱导的小型干扰RNA诱导的小型干扰RNA来建立αft敲击细胞。将1002个核苷酸位置的19个核苷酸克隆到psuper.Retro矢量（美国寡旋转）中，该载体表达了与形成双链RNA的完整序列相连的靶RNA。尽管已经建立了携带空和突变序列的克隆，但尚未建立任何携带目标序列的克隆。 αFT活性对于细胞活力至关重要。较少的

项目成果

Waguri N, Suda T, Kamura K, Aoyagi Y.: "Heterogeneous hepatic enhancement on CT angiography in idiopathic portal hypertension"Liver. 22. 276-280 (2002)

Waguri N、Suda T、Kamura K、Aoyagi Y.：“特发性门静脉高压 CT 血管造影的异质肝脏增强”肝脏。

Shoji Y, Mita T, Isemura M, Mega T, Hase S, Isemura S, Aoyagi Y: "A fibronectin-binding protein from rice bran with cell adhesion activity for animal tumor cells"Biosci. Biotechnol. Biochem.. 65. 1181-1186 (2001)

Shoji Y、Mita T、Isemura M、Mega T、Hase S、Isemura S、Aoyagi Y：“来自米糠的纤连蛋白结合蛋白，对动物肿瘤细胞具有细胞粘附活性”Biosci。

Suda T, Fujiyama A, Takimoto M, Igarashi M, Kuroiwa T, Waguri N, Kawai H, Mita Y, Aoyagi Y: "Interchromosomal telomere length variation"Biochem Bioph Res Comm. 291. 210-214 (2002)

Suda T、Fujiyama A、Takimoto M、Igarashi M、Kuroiwa T、Waguri N、Kawai H、Mita Y、Aoyagi Y：“染色体间端粒长度变异”Biochem Bioph Res Comm。

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Furukawa K、Hrada T、Aoaygi Y：“使用顶端带有气球的乳头刀成功进行内镜下乳头括约肌切开术，治疗胆总管结石并发十二指肠憩室的病例”Am J Gastroenterol。

Shoji Y, Mita T, Isemura M, et al.: "A fibronectin-binding protein from rice bran with cell adhesion activity for animal tumor cells"Biosci. Biotechnol. Biochem.. 65. 1181-1186 (2001)

Shoji Y、Mita T、Isemura M 等人：“来自米糠的纤连蛋白结合蛋白，对动物肿瘤细胞具有细胞粘附活性”Biosci。