CARMA3基因敲减对结肠癌细胞HCT116生长和侵袭转移的抑制

CARMA3 gene knockdown in HCT116 cells inhibits cell growth,migration and invasion

目的:探讨CARMA3基因在人结肠癌细胞HCT116生长和侵袭转移中的作用及其机制。方法:选取高表达CARMA3的人结肠癌细胞株。应用慢病毒技术敲减CARMA3基因,puromycin筛选后构建稳定转染的HCT116-sh CARMA3细胞株。Real-time PCR和Western blot鉴定mRNA和蛋白表达的抑制情况。WST-1法和RTCA S16系统分析细胞增殖情况。集落形成实验观察集落形成。流式细胞术检测细胞周期。显微镜下观察上皮-间充质转化(EMT)形态变化。划痕实验和Transwell实验检测细胞迁移与侵袭能力的改变。Western blot分析相关分子变化,探讨可能机制。结果:4株人结肠癌细胞株中HCT116细胞的CARMA3 mRNA和蛋白表达量最高,构建稳定沉默CARMA3的HCT116-sh CARMA3细胞株,其中HCT116-sh CARMA3-93细胞中CARMA3的mRNA和蛋白受抑制最明显,将其作为细胞模型。相比于对照组,HCT116-sh CARMA3-93细胞形态发生EMT逆转,其增殖、集落形成、迁移和侵袭能力明显下降(P<0.01)。HCT116-sh CARMA3-93的G_0/G_1细胞所占比例明显升高,S期细胞比例相应下降(P<0.05)。信号通路分子Bcl10和NF-κB表达明显下调,MALT-1变化不明显;细胞周期相关蛋白cyclin D1显著下调,cyclin A表达略有下降;侵袭转移相关分子MMP-2和MMP-9的表达下调,MMP-7未见改变,TIMP-1和TIMP-2的表达上调;EMT相关分子E-cadherin的表达水平升高,N-cadherin、Snail、Slug和Twist的表达水平呈不同程度降低。结论:CARMA3可通过改变细胞周期和侵袭转移分子的表达、调控EMT来影响结肠癌细胞HCT116的生长和侵袭转移。这可能与NF-κB信号通路发生改变有关。

AIM ： To study the effcts of caspase recruitment domain membrane-associated guanylate kinase pro-tein 3 （ CARMA3） knockdown on the growth, migration and invasion of human colonic carcinoma HCT116 cells and to ana-lyze the mechanism. METHODS ： A colonic carcinoma cell line with CARMA3 over-expression was selected. The CARMA3 gene in the HCT116 cells was knocked down by lentivirus technique. After screening by puromycin, the stably- transfected HCT116-shCARMA3 cell line was constructed. CARMA3 expression at mRNA and protein levels was detected by real-time PCR and Western blot, respectively. The cell proliferation was analyzed by WST-1 assay and RTCA S16 sys-tem. The colony formation ability was measured by colony-forming assay. The cell cycle was analyzed by flow cytometry.The cell morphological changes were observed under microscope. The abilities of migration and invasion in vitro were ob-served by wound healing assay and Transwell assay. The changes of related molecules were determined by Western blot to explore the mechanism. RESULTS： The expression of CARMA3 at mRNA and protein levels in the HCT116 cells was the highest in the 4 colonic carcinoma cell lines. HCT116-shCARMA3 cells with stably-silenced CARMA3 gene were success-fully established. Among them, HCT116-shCARMA3-93 cells showed the greatest inhibition of CARMA3 at mRNA and protein levels. Therefore, HCT116-shCARMA3-93 cells were chosen as the cell model. Compared with control group, the morphological changes of the HCT116-shCARMA3-93 cells had epithelial-mesenchymal transition （EMT） reversion. The abilities of proliferation, colony formation, migration and invasion in the HCT116-shCARMA3-93 cells were obviously sup-pressed （P 〈 0. 01 ）. G0 /G1 phase proportion was increased and S phase proportion was correspondingly decreased （ P 〈 0.05）. BcllO and NF-kB were down-regulated, and mucosa-associated lymphoid tissue lymphoma translocation protein 1 （MALT-1） showed no change. Cyclin D1 was decreased obviously and cyclin A declined slightly. Metastasis-related mar-kers matrix metalloproteinase （MMP）-2 and MMP-9 were reduced, MMP-7 remained unchanged, while tissue inhibitor of metalloproteinase （TIMP） -1 and TIMP-2 were up-regulated. Furthermore, EMT-associated molecule E-cadherin was increased, while N-cadherin, Snail, Slug and Twist were decreased to some extent. CONCLUSION： CARMA3 has an impact on the growth, migration and invasion of colonic carcinoma cell line, which is possibly related to NF-kB signaling pathway to change cell cycle and metastasis-related markers and to regulate EMT.