Xu Jue, Ren Gang, Zhao De-An, Li Bo-An, Cai Cheng-Fu, Zhou Yi, Luo Xian-Yang
Department of Otolaryngology, The First Hospital Affiliated to Huzhou University Medical College, Huzhou, Zhejiang 313000, P.R. China.
Department of Otolaryngology, The First Hospital Affiliated to Huzhou University Medical College, Huzhou, Zhejiang 313000, P.R. China.
Oncol Rep. 2014 Nov;32(5):1947-56. doi: 10.3892/or.2014.3451. Epub 2014 Aug 29.
Chibby (Cby) inhibits Wnt/β-catenin-mediated transcriptional activation by competing with Lef-1 (the transcription factor and target of β-catenin) to bind to β-catenin. This suggests that Cby could be a tumor suppressor protein. In the present study, we examined Cby expression in laryngeal squamous cell carcinoma (LSCC) and its function and mechanism in laryngeal carcinoma cell lines. Cby expression levels were investigated by immunohistochemistry in a panel of 36 LSCC patient cases. The expression of β-catenin, c-myc and cyclin D1 in Hep-2 were determined through RT-PCR and western blot analysis. Activity of Wnt/β-catenin signaling pathway after overexpression of Cby was measured by TCF/LEF luciferase reporter gene assay. Proliferation, clone forming ability, cell cycle distribution and cell apoptosis of Hep-2 cells were detected by MTT assay, plate colony forming assay, flow cytometry and TUNEL assay, respectively. This study showed that expression of Cby protein was strongly downregulated in LSCC tumor tissues in comparison to normal laryngeal mucosa samples. No significant correlation was found between the expression of Cby in tumor tissue and gender, age, clinical stage and tumor differentiation of laryngeal cancer patients. When Cby was overexpressed in Hep-2 cells, the expression of cyclin D1 was reduced and β-catenin activity was inhibited. Proliferation and plate colony forming assays revealed a significant inhibitory effect of Cby on growth and colony formation ability of Hep-2 cells after Cby overexpression in comparison to control and mock-infected cells. In addition, we also found that upregulated expression of Cby resulted in accumulation of numbers of cells in G0/G1 phase with concomitant decrease in S phase by cell cycle assay. TUNEL staining demonstrated that, compared with the control group, the rate of apoptosis in the plv-cs2.0-Cby group was significantly increased. Taken together, downregulation of Cby was observed in LSCC, but with no significant correlation to the clinicopathological features of LSCC patients. Overexpression of Cby effectively suppressed laryngeal carcinoma cell growth and promoted its apoptosis. A better understanding of the mechanisms of Cby gene activation in LSCC could provide potential novel therapeutic targets for human laryngeal carcinoma.
奇比（Cby）通过与淋巴样增强因子1（Lef-1，β-连环蛋白的转录因子及作用靶点）竞争结合β-连环蛋白，从而抑制Wnt/β-连环蛋白介导的转录激活。这表明Cby可能是一种肿瘤抑制蛋白。在本研究中，我们检测了喉鳞状细胞癌（LSCC）中Cby的表达情况及其在喉癌细胞系中的功能和机制。通过免疫组织化学方法对36例LSCC患者病例进行检测，以研究Cby的表达水平。通过逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹分析确定Hep-2细胞中β-连环蛋白、c-myc和细胞周期蛋白D1的表达。通过TCF/LEF荧光素酶报告基因检测法检测Cby过表达后Wnt/β-连环蛋白信号通路的活性。分别采用MTT法、平板克隆形成试验、流式细胞术和TUNEL法检测Hep-2细胞的增殖、克隆形成能力、细胞周期分布和细胞凋亡情况。本研究表明，与正常喉黏膜样本相比，LSCC肿瘤组织中Cby蛋白的表达明显下调。肿瘤组织中Cby的表达与喉癌患者的性别、年龄、临床分期和肿瘤分化之间未发现显著相关性。当Cby在Hep-2细胞中过表达时，细胞周期蛋白D1的表达降低，β-连环蛋白的活性受到抑制。增殖试验和平板克隆形成试验显示，与对照细胞和模拟感染细胞相比，Cby过表达后对Hep-2细胞的生长和克隆形成能力有显著抑制作用。此外，通过细胞周期检测我们还发现，Cby表达上调导致G0/G1期细胞数量增加，同时S期细胞数量减少。TUNEL染色显示，与对照组相比，plv-cs2.0-Cby组的细胞凋亡率显著增加。综上所述，LSCC中观察到Cby表达下调，但与LSCC患者的临床病理特征无显著相关性。Cby过表达有效抑制了喉癌细胞的生长并促进其凋亡。深入了解LSCC中Cby基因激活的机制可为人类喉癌提供潜在的新型治疗靶点。
