Li Kui, Zhou Zhong-Yin, Ji Pan-Pan, Luo He-Sheng
Department of Gastroenterology, Renmin Hospital, Wuhan University, Wuhan, Hubei 430060, P.R. China.
Oncol Lett. 2016 Jun;11(6):3896-3900. doi: 10.3892/ol.2016.4481. Epub 2016 Apr 20.
The aim of the present study was to explore the effect of knocking down the expression of β-catenin by small interference (si)RNA on the activity of the Wnt/β-catenin signaling pathway, and the proliferation, apoptosis and invasion abilities of the human colon cancer cell line SW480. For that purpose, double-stranded siRNA targeting β-catenin (β-catenin-siRNA) was synthesized and transfected into SW480 cells. Reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were used to detect the messenger (m)RNA and protein levels of β-catenin in SW480 cells. To detect cell proliferation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed, while cell apoptosis and caspase-3 activity were detected by flow cytometry and caspase-3 activity assay, respectively. Matrigel invasion assay was performed to detect the influence of siRNA-mediated gene silencing on the invasion and metastasis of SW480 cells . The results of RT-PCR and western blot analysis demonstrated that, compared with the blank control, negative control and liposome groups, β-catenin-siRNA transfected SW480 cells had significantly decreased mRNA and protein levels of β-catenin. In addition, following β-catenin-siRNA transfection, the proliferation of SW480 cells was significantly lower than that of the blank control, negative control and liposome groups, while the apoptosis rate increased in β-catenin-siRNA transfected cells, compared with the aforementioned groups. Invasion assay showed that, following β-catenin-siRNA transfection, the number of SW480 cells infiltrating through the Matrigel membrane was significantly lower than that of the blank control, negative control and liposome groups. Following β-catenin-siRNA transfection, the caspase-3 activity in SW480 cells was lower than that in the blank control, negative control and liposome groups. These results indicate that siRNA-mediated silencing of β-catenin could inhibit the proliferation and invasion of SW480 cells and induce apoptosis, thus providing novel potential strategies for the clinical treatment of colon cancer, and may serve as a novel target for cancer therapy.
本研究的目的是探讨通过小干扰(si)RNA敲低β-连环蛋白表达对Wnt/β-连环蛋白信号通路活性以及人结肠癌细胞系SW480增殖、凋亡和侵袭能力的影响。为此,合成了靶向β-连环蛋白的双链siRNA(β-连环蛋白-siRNA)并转染到SW480细胞中。采用逆转录-聚合酶链反应(RT-PCR)和蛋白质印迹法检测SW480细胞中β-连环蛋白的信使(m)RNA和蛋白质水平。为检测细胞增殖,进行了3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐检测,而细胞凋亡和半胱天冬酶-3活性分别通过流式细胞术和半胱天冬酶-3活性检测进行检测。进行基质胶侵袭试验以检测siRNA介导的基因沉默对SW480细胞侵袭和转移的影响。RT-PCR和蛋白质印迹分析结果表明,与空白对照、阴性对照和脂质体组相比,转染β-连环蛋白-siRNA的SW480细胞中β-连环蛋白的mRNA和蛋白质水平显著降低。此外,转染β-连环蛋白-siRNA后,SW480细胞的增殖明显低于空白对照、阴性对照和脂质体组,而与上述组相比,转染β-连环蛋白-siRNA的细胞凋亡率增加。侵袭试验表明,转染β-连环蛋白-siRNA后,穿过基质胶膜浸润的SW480细胞数量明显低于空白对照、阴性对照和脂质体组。转染β-连环蛋白-siRNA后,SW480细胞中的半胱天冬酶-3活性低于空白对照、阴性对照和脂质体组。这些结果表明,siRNA介导的β-连环蛋白沉默可抑制SW480细胞的增殖和侵袭并诱导凋亡,从而为结肠癌的临床治疗提供新的潜在策略,并可能作为癌症治疗的新靶点。