降低S100A6基因表达对胃癌细胞生物学特性影响的研究

The study on the changes of biologic characteristics after knockdown S100A6 in gastric cancer cell lines

目的:初步探讨S100A6基因对于胃癌细胞生长、增殖状态,细胞周期,凋亡状态等生物学特性及成瘤、浸润转移能力等恶性表型的影响。方法:S100A6基因的RNAi载体通过脂质体介导法转染S100A6基因高表达SGC7901细胞,后经G418压力筛选法获得稳定转染的细胞系,经过RT-PCR法、免疫细胞化学法及Western-bloting法证实。对稳定表达株进行鉴定,而后使用流式细胞仪、生长曲线法、平板克隆形成实验法、细胞迁徙实验等方法分析稳定表达株相关生物学特性与恶性表型的变化,每种检测实验均设立RNAi载体稳定转染细胞组、IMG800空载体稳定转染细胞组和空白SGC7901细胞组。结果:稳定的S100A6基因RNAi细胞系经过RT-PCR法、免疫细胞化学法及Western-bloting法证实,mRNA抑制率可达75%左右,蛋白产物的抑制率达85%左右。与转染IMG800空载体及空白SGC7901胃癌细胞相比转染S100A6 RNAi载体的稳定表达细胞株生长减慢,各时间点细胞计数显著低于对照组(P<0.05);后两组之间亦无显著差异,细胞周期检测显示S100A6 RNA干扰组G0-G1期比例显著高于对照组,而G2-M及S期比例显著低于对照组(P<0.05),其它各期细胞比例均无显著差异。平板克隆形成实验结果显示S100A6 RNA干扰组克隆形成率显著低于对照组(P<0.05);,细胞迁徙实验结果提示S100A6 RNA干扰组穿膜率显著低于对照组(P<0.05)。结论:S100A6基因可能具有促进细胞生长增殖作用,同时可影响细胞周期,增加处于分裂期细胞的比例可能具有促进细胞分裂作用,并可促进胃癌细胞侵袭转移。降低S100A6基因表达可能抑制胃癌细胞的恶性生物学行为。

Objective：To investigate the influence of SIOOA6 on the growth,praliferation,apepatosis,infiltration and cell cycle of the gastric cancer line SGC7901. Methods： As a critical member of S100 gene family ,the effective silengcing sequence to S100A6 was selected,designed and synthesized based on the sequence of S100A6 mRNA. They were separately subcloned into the plasmid of IMG800 containing U6 promoter. The RNA interference eukaryotic expression vector specific to SIOOA6 gene were constructed,followed by transfection in SGC7901 by using liposome. Then stable expression clones were selected and appraised. The apeptosis and cell cycles were detected using flow cytometry. The growth and proliferation were analyzed by making cell growth curves and colony formation assay respectively. The ability of infiltration were tested using cancer cell migration assay. The experimental group and two control groups were detected. Results： The stable cell lines whose expression of S100A6 gene were suppressed by 75% in mRNA level or 85% in protein level appraised by immunohistochemistry, RT - PCR and Westen - bloting methods. The stable RNAi for SIOOA6 cell lines grow slower significantly than SGC7901 and SGC - IMG group. The cell counts in the fifth, sixth and seventh days were significantly different with that of others （P 〈 0. 05 ）. Cell cycle analysis showed that the stable RNAi for S100A6 cell lines proliferated slower,proportions of cells in G0 - G1 were higher and proportions in G2 - M and S were lower significantly than that of control groups （ P 〈 0.05 ）. Cell apoptosis analysis showed that proportions of apoptosis cells were higher than that of control groups（P 〈 0.05）. Results of colony formation assay showed that the colony formation rate of S100A6 RNAi cell lines was lower than that of control groups （P 〈 0.05）. Further more,the cell migration rate of S100A6 RNAi cell lines was much lower than that of control groups （P 〈0.05）. Conclusion： S100A6 also can promote the growth and proliferation and restrain apoptosis of gastric cancer cells. It can help tumor cell maintain malignant phenotype and promote infiltration and metastasis of gastric cancer cells. Knockdown S100A6 in gastric cancer cell lines could ameliorate malignant phenotype.