γ-synuclein对结肠癌细胞SW1116体外及体内增殖潜能的影响

Effects of γ-synuclein on proliferation of colon cancer cell line SW1116 in vitro and in vivo

目的:研究γ-synuclein对人结肠癌细胞系体外及体内增殖潜能的影响。方法:利用真核表达载体p IRES2-EGFP和RNA干扰载体p GCsi-U6/neo/GFP构建针对γ-synuclein的重组质粒,并转染至人结肠癌细胞株SW1116,经G418筛选出稳定转染细胞株。应用CCK8法检测细胞增殖活性;软琼脂克隆法检测细胞克隆形成能力;裸鼠皮下成瘤实验检测细胞在裸鼠体内的成瘤能力。结果:经测序以及RT-PCR和Western blot验证,重组质粒成功构建,并能够过表达或抑制γ-synuclein基因和蛋白。结果显示,γ-synuclein受抑制后,SW1116细胞的增殖数目从48h至120h持续显著减少(P<0.05),克隆形成率亦显著降低(P<0.05)。SW1116细胞的增殖和克隆形成能力与其γ-synuclein的表达水平呈正相关。过表达γ-synuclein能够部分增强SW1116细胞的增殖和克隆形成能力,但差异无统计学意义(P>0.05)。体内实验也有类似结果,过表达γ-synuclein能够稍微加快SW1116细胞瘤体生长速度,但直至第27天,其生长速度才显著加快(P<0.05)。γ-synuclein受抑制后,SW1116细胞裸鼠皮下成瘤速度从第6天至第30天显著减慢(P<0.05)。结论:γ-synuclein与结肠癌细胞体外及体内的增殖潜能密切相关。

Objective： To study the effects of γ-synuclein on colon cancer cell line SW1116 proliferation in vitro and in vivo. Methods： The shRNA targeting γ-synuclein mRNA plasmid or γ-synuclein gene eukaryotic expression vector was constructed and transfected into the colon cancer cell line SW1116. The stable cell lines were selected with G418,and proliferation feature of these cells were examined by CCK8 assay,soft agar assay in vitro,and tumor xenograft model of tumorigenicity assay in vivo. Results： The recombinant plasmids was successfully constructed,which was stably expressed in SW1116 cells and could translate γ-synuclein protein or suppress γ-synuclein expression in vitro. γ-synuclein knockdown significantly reduced proliferation cell number by 48,72,96 and 120 h after plating（ P〈0. 05）. The colony formation rate of the p GCsi-γ-synuclein cells was also significantly reduced（ P〈0. 05）.The overexpression of γ-synuclein moderately facilitated proliferation and colony formation of SW1116 cells,but not significantly different with proliferation and colony formation of other cell groups（ P〉0. 05）,which presented a promotive effect in a γ-synuclein expression quantity-dependent manner. Similar findings were also found in the tumorigenicity assay in vivo. Tumors in mice derived from overexpressed γ-synuclein cells did not significantly exhibit faster tumor growth until day 27. γ-synuclein knockdown suppressed the tumorigenicity of SW1116 cells in mice,which presented significantly smaller tumor masses from d_6~ d_（30）,compared with empty vector cells（ P〈0. 05）. Conclusion： γ-synuclein is closely related with proliferation of colon cancer cell in vitro and in vivo.