胱天蛋白酶募集域蛋白9在胃癌中的表达及作用机制研究

Expression of caspase recruitment domain protein 9 in gastric cancer and its mechanism

目的研究作为参与调节细胞凋亡的关键蛋白之一的胱天蛋白酶募集域蛋白9(CARD9)在胃癌中的表达及作用机制。方法收集2012年3月至2014年3月深圳市龙岗区第二人民医院及第四人民医院收治的90例中晚期胃癌患者的胃癌组织和癌旁组织,采用Western blot法和免疫组织化学法检测其胃癌组织和癌旁组织中CARD9表达情况;在37℃,5%CO2条件下常规培养胃癌细胞株BGC-823,分别转染无意义干扰RNA[绿色荧光蛋白(GFP)]作对照(C GFP)和含于Sh Card 9 pool中的短发夹RNA(ShRNA)后,通过三种方法作如下检测:用四氮唑盐比色法(MTT比色法)检测抑制CARD9作用后对胃癌细胞增殖的影响;用流式细胞术分析抑制CARD9作用后细胞周期变化及细胞凋亡情况;用Western blot法检测抑制CARD9对抑制性κB激酶α(IKK-α)及磷酸化IKK-α(p-IKK-α)表达的影响。结果胃癌组织中CARD9表达水平高于癌旁组织,同时阳性率高于癌旁组织(70.0%vs36.0%,χ2=20.58,P<0.01)。胃癌组织中IKKα蛋白水平低于癌旁组织,p-IKKα水平高于癌旁组织。抑制CARD9后,胃癌细胞中p-IKKα水平降低,而IKKα水平升高;转染ShRNA的胃癌细胞增殖能力高于转染C GFP的细胞,并且抑制CARD9后细胞凋亡程度低于转染C GFP的胃癌细胞。结论 CARD9可能通过促进IKKα的磷酸化激活抑制胃癌细胞的增殖,继而促进胃癌细胞的凋亡。

Objective To study the expression of caspase recruitment domain protein 9（CARD9） as one of the key proteins participating in regulating apoptosis in gastric cancer and its mechanism.Methods The gastric cancer tissues and paracancerous tissues from 90 patients with mid-and late-stage gastric cancer who were treated in Longgang District Second People＇s Hospital and Longgang District Fourth People＇s Hospital of Shenzhen City from March 2012 to March 2014 were collected.Western blot method and immunohistochemistry were used to detect the expressions of CARD9 in gastric cancer tissues and paracancerous tissues.Gastric cancer cell line BGC-823 were cultured routinely at 37℃ and 5%CO_2 and transferred with nonsense interference RNA（green fluorescent protein,GFP） as control（C GFP） and short hairpin RNA（ShRNA） containing in ShCard 9 pool.The tetrazolium salts colorimetric method（MTT colorimetric method） was used to detect the influence on proliferation of gastric cells after inhibiting the action of CARD9.The flow cytometry was used to analyze the changes of cell cycle and the condition of cells apoptosis after inhibiting the action of CARD9.Western blot method was used to detect the influence of inhibiting CARD9 on expressions of inhibitory κB kinase alpha（IKK-α） and phosphorylated IKK-α（p-IKKα）.Results The expression level of CARD9 in gastric cancer tissues was significantly higher than that in paracancerous tissues,and its positive expression rate was also significantly higher than that in paracancerous tissues（70.0%vs 36.0%,χ~2=20.58,P 0.01）.After inhibiting CARD9,the IKK-α protein level in gastric cancer tissues was lower than that in paracancerous tissues,and the p-IKKα level in gastric cancer tissues was higher than that in paracancerous tissues.Compared with the cells transferred with C GFP,the proliferation ability of gastric cancer cells transferred with ShRNA increased,and the apoptosis degree of cells transferred with ShRNA decreased.Conclusion CARD9 inhibits the proliferation of gastric cancer cells probably through promoting the activation of IKKα phosphorylation,and then promoting the apoptosis of gastric cancer cells.