Cell death by inhibition of Wnt signal pathway in cancer of the esophagus

食管癌Wnt信号通路抑制导致细胞死亡

• 批准号: 16591337
• 负责人: KIMURA Masahiro
• 金额: $ 2.3万
• 依托单位: Nagoya City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16591337/
• 关键词: esophageal cancer; Wnt signal pathway; Wntシグナル; TCF4

Transfection did siRNA into cell lines TE1 and 2 which TCF expression is high, from among esophageal cancer cell lines TE series, using Oligofectamine. After transfection, we examined the RNA expression after 48 hours because the apparant inhibition was seen most 48 hours later. Moreover, it was 30-40% though the efficiency of transfection was different according to the cell. The expression of TCF4 was measured by using Light Cycler RT-PCR, measured, and decreased in 90% or more at the mRNA level of TCF4 was confirmed compared with the control.Oligofectamine was used for esophageal cancer cell lines (TE1,2) and colon cancer cell line (SW480,LoVo), siRNA was compared with the transfection doing and the control, and the cell death was detectable in all the cell lines.A decrease of the number of cells of the inhibition group was confirmed with Giemsa stain by present. However, a significant difference has not been obtained at that time when MTTassay was performed. It was not possible to perform it to the FACS analysis. SiRNA is made again again, and being analyzed now.RNA was extracted by first using cell line HET1A and 15 kinds of the TE series, and cDNA was made. The microarray of about 30000 genes was done by using the cDNA. It interrupts now though the cluster is analyzed in cell line group from which the cell death is not derivable with cell line group from which the cell death is derivable by siRNA, and the key gene that induced the cell death was scheduled to be identified. As for the result of these microarray, it is scheduled to apply it to the identification of the anti-cancer drug receptivity gene and the development research into new anti-cancer drug.

使用Oligofectamine将siRNA转染到食管癌细胞系TE系列中TCF表达高的细胞系TE 1和2中。转染后，我们在48小时后检测RNA表达，因为在48小时后观察到最明显的抑制。此外，尽管转染效率因细胞而异，但转染率为30-40%。通过使用Light Cycler RT-PCR检测TCF 4的表达，检测结果证实，与对照相比，TCF 4的mRNA水平降低了90%或更多。（SW 480，LoVo），将siRNA与转染组和对照组进行比较，结果显示，细胞凋亡率明显降低，Giemsa染色显示细胞凋亡率明显降低。然而，当时进行MTTassay时尚未获得显着差异。无法进行FACS分析。首先利用HET 1A细胞系和15种TE系提取RNA，并制备cDNA。利用该cDNA芯片制作了约30000个基因的微阵列。现在中断，尽管在不能通过siRNA导致细胞死亡的细胞系组中分析簇，并且计划鉴定诱导细胞死亡的关键基因。对于这些微阵列的结果，计划将其应用于抗癌药物受体基因的鉴定和新抗癌药物的开发研究。