Effects of lentiviral-mediated Foxp1 and Foxq1 RNAi on the hepatocarcinoma cell
Effects of lentiviral-mediated Foxp1 and Foxq1 RNAi on the hepatocarcinoma cell
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DOI：
10.1016/j.yexmp.2013.10.015
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发表时间：
2014-02-01
影响因子：
3.6
通讯作者：
Chen, Li
中科院分区：
文献类型：
--
作者：
Qin, Jing;Xu, Yuyin;Chen, Li
Foxp 1和Foxq 1是“叉头盒（Fox）”家族的两个多功能分子。目的构建针对Foxp 1和Foxq 1基因的RNA干扰慢病毒载体，观察两种RNA干扰载体对7721肝癌细胞增殖、迁移和凋亡的影响。分别设计6条针对人Foxp 1/Foxq 1 mRNA的靶序列，合成6对相应的siRNA，用脂质体2000转染7721细胞，筛选出最有效的siRNA亚克隆到pLL 3. 7-GFP/Lenti质粒中。将这些质粒转染到293 T细胞中以包装慢病毒颗粒，用于随后在确认其序列后转染到7721细胞中。荧光定量PCR检测转染细胞中Foxp 1和Foxq 1基因的表达。通过伤口愈合实验、Transwell实验、CCK-8实验和流式细胞术检测转染细胞的迁移、浸润、存活和凋亡情况。测序结果显示慢病毒载体中含有Foxp 1或Foxq 1基因。转染7721细胞后，5iRNA-823和5iRNA-834显著下调Foxp 1和Foxq 1的表达。两种siRNA转染的7721细胞的迁移、浸润能力和存活能力均受到明显抑制;流式细胞仪检测显示Foxp 1和Foxq 1慢病毒RNAi载体转染的7721细胞凋亡率增加。结果表明，Foxp 1和Foxq 1的慢病毒RNAi载体能有效抑制7721细胞中Foxp 1和Foxq 1的表达，下调Foxp 1和Foxq 1的表达均能抑制7721细胞的迁移、浸润和存活，并增加细胞凋亡。我们的数据表明Foxp 1和Foxq 1基因在肝癌细胞中起致癌作用，这两个基因可能是肝癌治疗的新靶点。(C)版权所有© 2013 Elsevier Inc.
Foxp1 and Foxq1 are two multifunctional molecules of "forkhead box (Fox)" family. The objective of this paper was to construct the lentiviral vectors expressing RNA interference (RNAi) against Foxp1 or Foxq1 genes, and the effects of both vectors with two RNAis on the proliferation, migration and apoptosis of 7721 hepatocarcinoma cell line were evaluated. Six target sequences against human Foxp1/Foxq1 mRNA were designed respectively and six pairs of their corresponding double-strand DNA oligo (siRNA) were synthesized prior to being transfected into 7721 cells with lipo2000, then a most efficient siRNA were selected to be subcloned into pLL3.7-GFP/Lenti plasmids. These plasmids were transfected into 293T cells to package lentiviral particles for subsequent transfection into 7721 cells after their sequences were confirmed. The expression of Foxp1 and Foxq1 genes in the transfected cells were identified by real-time PCR. The migration, infiltration, viability and apoptosis of the transfected cells were assessed by wound healing assay, Transwell assay, CCK-8 assay and flow cytometry. Sequencing results showed that lentiviral vectors contained Foxp1 or Foxq1 gene. After being transfected into 7721 cells, Foxp1 and Foxq1 expression were significantly down-regulated by 5iRNA-823 and 5iRNA-834. The migration and infiltration ability, and the viability of 7721 cells transfected with two siRNAs were significantly suppressed; flow cytometry assay exhibited the apoptosis rate of transfected 7721 cells with the lentivirus RNAi vector of Foxp1 or Foxq1 was increased. All the results showed that the lentivirus RNAi vectors of Foxp1 and Foxq1 were able to inhibit the expression of Foxp1 and Foxq1 in 7721 cells efficiently, and the downregulation of either Foxp1 or Foxq1 resulted in suppression of migration, infiltration and viability of 7721 cells and an increase in cell apoptosis. Our data indicated that both Foxp1 and Foxq1 genes played an oncogenic role in hepatocarcinoma cells, which proposed the two genes as new therapeutic targets for the cancer. (C) 2013 Elsevier Inc All rights reserved.