Two opposite effects of desmoglein 3 on the growth of oral squamous cell carcinoma between anchorage -dependent and -independent conditions

Desmoglein 3 (Dsg3) is one of the desmosomal cadherins. Recent studies have reported that Dsg3 is abnormally expressed in oral squamous cell carcinoma (OSCC) and associated with tumor growth and poor survival. A role of Dsg3 in the progression of OSCC remains controversial, since both positive and negative effect on the cancer progression have been reported. Previously we have demonstrated that OSCC cells from the metastatic lymph nodes showed significantly higher DSG3 expression compared to those from the primary tumor of the same patients. Here we investigated the effect of Dsg3 expression on the growth of OSCC between anchorage-dependent (AD) and -independent (AID) conditions. Cell lines established from the primary tumor (P) and metastatic lymph nodes (LY) of three OSCC patients and two commercial OSCC cell lines were used for the experiments. Under AD conditions, Dsg3-low cell lines (Dsg3-low) had higher cell proliferation and migration ability than Dsg3-high cell lines (Dsg3-high) of the same patients. After transferred into AID conditions, all the cell lines except Dsg3-negative 7P showed increased Dsg3 expression by up to 50-fold, compared with AD conditions. Under AID conditions, all the patients showed higher cell proliferation ability in the Dsg3-high compared with Dsg3-low, which was marked contrast to AD conditions. Dsg3 knockdown under AD conditions increased the cell proliferation rate of all the cell lines except Dsg3-negative line. On the other hand, cell growth was not affected under AID conditions. These results suggested that Dsg3 may have two opposite effects on the growth of OSCC cells between AD and AID conditions, and that the involvement of molecules other than Dsg3 may also be important. Therefore, we investigated DEGs by Dsg3 knockdown under AD and AID conditions and their functions. To investigate the differences in DSG3 function and DEGs on anchorage conditions, OSCC cell lines were cultured under AD and AID conditions, and DSG3 was knocked down by siRNA DEGs were extracted when Dsg3-positive cells were knocked down. Gene otology (GO) analysis was performed to identify the functional annotations of the extracted DEGs.

桥粒芯糖蛋白3（Desmoglein 3, Dsg3）属于桥粒钙粘蛋白家族成员之一。近期研究表明，Dsg3在口腔鳞状细胞癌（oral squamous cell carcinoma, OSCC）中存在异常表达，且与肿瘤生长及不良预后密切相关。但Dsg3在OSCC进展中的作用仍存在争议，已有研究报道其对癌症进展可产生双向影响。既往本团队已证实，相较于同一患者的原发肿瘤来源OSCC细胞，转移性淋巴结来源的OSCC细胞中DSG3的表达水平显著更高。本研究旨在探究Dsg3表达在贴壁依赖型（anchorage-dependent, AD）与非贴壁依赖型（anchorage-independent, AID）培养条件下对OSCC细胞生长的影响。实验纳入3例OSCC患者原发肿瘤（P）及转移性淋巴结（LY）来源的细胞系，以及2株商业化OSCC细胞系开展相关实验。在贴壁依赖培养条件下，同一患者来源的低表达Dsg3（Dsg3-low）细胞系的增殖与迁移能力均高于高表达Dsg3（Dsg3-high）细胞系。当细胞切换至非贴壁依赖培养条件后，除Dsg3阴性的7P细胞系外，其余所有细胞系的Dsg3表达水平较贴壁依赖条件下最高可上调50倍。在非贴壁依赖培养条件下，所有患者来源的细胞系中，高表达Dsg3的细胞增殖能力均高于低表达Dsg3的细胞系，这与贴壁依赖条件下的结果形成显著反差。在贴壁依赖条件下，敲低Dsg3可提升除Dsg3阴性细胞系外所有细胞系的增殖速率；而在非贴壁依赖条件下，细胞生长未受明显影响。上述结果提示，Dsg3在贴壁依赖与非贴壁依赖两种培养条件下对OSCC细胞生长可能存在双向调控作用，且除Dsg3之外的其他分子的参与或许同样至关重要。因此，本研究通过在贴壁依赖与非贴壁依赖条件下对Dsg3进行敲低，筛选得到差异表达基因（differentially expressed genes, DEGs）并分析其功能。为探究不同贴壁培养条件下Dsg3的功能及差异表达基因的调控差异，本研究将OSCC细胞系分别置于贴壁依赖与非贴壁依赖条件下培养，通过小干扰RNA（small interfering RNA, siRNA）敲低Dsg3，并提取Dsg3阳性细胞敲低后的差异表达基因。随后采用基因本体（Gene Ontology, GO）分析对提取得到的差异表达基因进行功能注释鉴定。