Ueno Koji, Hiura Mikako, Suehiro Yutaka, Hazama Shoichi, Hirata Hiroshi, Oka Masaaki, Imai Kohzoh, Dahiya Rajvir, Hinoda Yuji
Department of Laboratory Medicine, Yamaguchi University Graduate School of Medicine, Ube, Japan.
Neoplasia. 2008 Jul;10(7):697-705. doi: 10.1593/neo.08320.
We investigated whether one of the Wnt receptors, frizzled-7 (FZD7), functions in the canonical Wnt signaling pathway of colorectal cancer (CRC) cells harboring an APC or CTNNB1 mutation and may be a potential therapeutic target for sporadic CRCs. The expression level of FZD gene family members in colon cancer cells and primary CRC tissues were determined by real-time PCR. Activation of the Wnt signaling pathway was evaluated by TOPflash assay. The expression level of Wnt target genes was determined by real-time polymerase chain reaction and/or Western blot analysis. Cell growth and cell invasion were assessed by MTS and matrigel assays, respectively. Among 10 FZD gene family members, FZD7 mRNA was predominantly expressed in six colon cancer cell lines with APC or CTNNB1 mutation. These six cell lines were transfected with FZD7 cDNA together with a TOPflash reporter plasmid, resulting in a 1.5- to 24.3-fold increase of Tcf transcriptional activity. The mRNA expression levels of seven known Wnt target genes were also increased by 1.5- to 3.4-fold after transfection of FZD7 cDNA into HCT-116 cells. The six cell lines were then cotransfected with FZD7-siRNA and a TOPflash reporter plasmid, which reduced Tcf transcriptional activity to 20% to 80%. FZD7-siRNA was shown to significantly decrease cell viability and in vitro invasion activity after transfection into HCT-116 cells. Our present data demonstrated that FZD7 activates the canonical Wnt pathway in colon cancer cells despite the presence of APC or CTNNB1 mutation and that FZD7-siRNA may be used as a therapeutic reagent for CRCs.
我们研究了Wnt受体之一卷曲蛋白7（FZD7）是否在携带APC或CTNNB1突变的结直肠癌（CRC）细胞的经典Wnt信号通路中发挥作用，以及它是否可能是散发性CRC的潜在治疗靶点。通过实时PCR测定结肠癌细胞和原发性CRC组织中FZD基因家族成员的表达水平。通过TOPflash试验评估Wnt信号通路的激活情况。通过实时聚合酶链反应和/或蛋白质免疫印迹分析测定Wnt靶基因的表达水平。分别通过MTS和基质胶试验评估细胞生长和细胞侵袭能力。在10个FZD基因家族成员中，FZD7 mRNA主要在6种携带APC或CTNNB1突变的结肠癌细胞系中表达。将FZD7 cDNA与TOPflash报告质粒一起转染到这6种细胞系中，导致Tcf转录活性增加了1.5至24.3倍。将FZD7 cDNA转染到HCT-116细胞后，7个已知Wnt靶基因的mRNA表达水平也增加了1.5至3.4倍。然后将FZD7-siRNA与TOPflash报告质粒共转染到这6种细胞系中，使Tcf转录活性降低至20%至80%。结果显示，将FZD7-siRNA转染到HCT-116细胞后，可显著降低细胞活力和体外侵袭活性。我们目前的数据表明，尽管存在APC或CTNNB1突变，FZD7仍能激活结肠癌细胞中的经典Wnt通路，并且FZD7-siRNA可能用作CRC的治疗试剂。
