CARMA3基因敲减对结肠癌细胞HCT116生长和侵袭转移的抑制
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摘要
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 protein 3( CARMA3) knockdown on the growth,migration and invasion of human colonic carcinoma HCT116 cells and to analyze 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 stablytransfected HCT116-sh CARMA3 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 system. 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 observed 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-sh CARMA3 cells with stably-silenced CARMA3 gene were successfully established. Among them,HCT116-sh CARMA3-93 cells showed the greatest inhibition of CARMA3 at mRNA and protein levels. Therefore,HCT116-sh CARMA3-93 cells were chosen as the cell model. Compared with control group,the morphological changes of the HCT116-sh CARMA3-93 cells had epithelial-mesenchymal transition( EMT) reversion. The abilities of proliferation,colony formation,migration and invasion in the HCT116-sh CARMA3-93 cells were obviously suppressed( P < 0. 01). G_0/G_1 phase proportion was increased and S phase proportion was correspondingly decreased( P <0. 05). Bcl10 and NF-κB 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 markers 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-κB signaling pathway to change cell cycle and metastasis-related markers and to regulate EMT.
AIM: To study the effcts of caspase recruitment domain membrane-associated guanylate kinase protein 3( CARMA3) knockdown on the growth,migration and invasion of human colonic carcinoma HCT116 cells and to analyze 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 stablytransfected HCT116-sh CARMA3 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 system. 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 observed 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-sh CARMA3 cells with stably-silenced CARMA3 gene were successfully established. Among them,HCT116-sh CARMA3-93 cells showed the greatest inhibition of CARMA3 at mRNA and protein levels. Therefore,HCT116-sh CARMA3-93 cells were chosen as the cell model. Compared with control group,the morphological changes of the HCT116-sh CARMA3-93 cells had epithelial-mesenchymal transition( EMT) reversion. The abilities of proliferation,colony formation,migration and invasion in the HCT116-sh CARMA3-93 cells were obviously suppressed( P < 0. 01). G_0/G_1 phase proportion was increased and S phase proportion was correspondingly decreased( P <0. 05). Bcl10 and NF-κB 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 markers 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-κB signaling pathway to change cell cycle and metastasis-related markers and to regulate EMT.
引文
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[17]Qin M,Zhang J,Xu C,et al.Knockdown of NIK and IKKβ-binding protein(NIBP)reduces colorectal cancer metastasis through down-regulation of the canonical NF-κΒsignaling pathway and suppression of MAPK signaling mediated through ERK and JNK[J].PLo S One,2017,12(1):e0170595.
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[20]刘宝玉,黄杰安,刘诗权,等.NF-κB对人结肠癌细胞上皮间质转化及侵袭转移的影响[J].世界华人消化杂志,2014,22(23):3403-3409.
[2]Jiang C,Zhou Z,Quan Y,et al.CARMA3 is a host factor regulating the balance of inflammatory and antiviral responses against viral infection[J].Cell Rep,2016,14(10):2389-2401.
[3]Jiang C,Lin X.Analysis of epidermal growth factor-induced NF-κB signaling[J].Methods Mol Biol,2015,1280:75-102.
[4]赵婷婷,刑鹏,李继光.新型接头蛋白CARD10在结直肠癌中的表达与意义[J].中华结肠疾病电子杂志,2015,4(1):58-61.
[5]Xia ZX,Li ZX,Zhang M,et al.CARMA3 regulates the invasion,migration,and apoptosis of non-small cell lung cancer cells by activating NF-кB and suppressing the P38MAPK signaling pathway[J].Exp Mol Pathol,2016,100(2):353-360.
[6]Zhao T,Miao Z,Wang Z,et al.CARMA3 overexpression accelerates cell proliferation and inhibits paclitaxel-induced apoptosis through NF-κB regulation in breast cancer cells[J].Tumor Biol,2013,34(5):3041-3047.
[7]Xie C,Han Y,Fu L,et al.Overexpression of CARMA3is associated with advanced tumor stage,cell cycle progression,and cisplatin resistance in human epithelial ovarian cancer[J].Tumor Biol,2014,35(8):7957-7964.
[8]Wu GL,Yuan JL,Huang XD,et al.Evaluating the expression of CARMA3 as a prognostic tumor marker in renal cell carcinoma[J].Tumor Biol,2013,34(6):3431-3435.
[9]Man X,He J,Kong C,et al.Clinical significance and biological roles of CARMA3 in human bladder carcinoma[J].Tumor Biol,2014,35(5):4131-4136.
[10]Miao Z,Zhao T,Wang Z,et al.CARMA3 is overexpressed in colon cancer and regulates NF-κB activity and cyclin D1 expression[J].Biochem Biophys Res Commun,2012,425(4):781-787.
[11]Wang L,Qian L,Li X,et al.MicroRNA-195 inhibits colorectal cancer cell proliferation,colony-formation and invasion through targeting CARMA3[J].Mol Med Rep,2014,10(1):473-478.
[12]Du S,Jia L,Zhang Y,et al.CARMA3 is upregulated in human pancreatic carcinoma,and its depletion inhibits tumor proliferation,migration,and invasion[J].Tumor Biol,2014,35(6):5965-5970.
[13]贾陌杨,王要军.结直肠癌转移相关信号通路[J].国际消化病杂志,2015,35(3):183-185.
[14]阮妙华,王凯,王丹,等.病毒性心肌炎小鼠心肌中MMP-2和NF-κB的表达[J].中国病理生理杂志,2016,32(9):1704-1707.
[15]李中信,贾漪涛,马顺茂,等.结直肠癌组织NF-κB和MMP-9表达与腹腔微转移关系的初步探讨[J].中华肿瘤防治杂志,2008,5(13):1014-1017.
[16]Feng X,Miao G,Han Y,et al.CARMA3 is overexpressed in human glioma and promotes cell invasion through MMP9 regulation in A172 cell line[J].Tumour Biol,2014,35(1):149-154.
[17]Qin M,Zhang J,Xu C,et al.Knockdown of NIK and IKKβ-binding protein(NIBP)reduces colorectal cancer metastasis through down-regulation of the canonical NF-κΒsignaling pathway and suppression of MAPK signaling mediated through ERK and JNK[J].PLo S One,2017,12(1):e0170595.
[18]Pires BR,Mencalha AL,Ferreira GM,et al.NF-kappa B is involved in the regulation of EMT genes in breast cancer cells[J].PLo S One,2017,12(1):e0169622.
[19]Zhang S,Zhang C,Liu W,et al.MicroRNA-24 upregulation inhibits proliferation,metastasis and induces apoptosis in bladder cancer cells by targeting CARMA3[J].Int J Oncol,2015,47(4):1351-1360.
[20]刘宝玉,黄杰安,刘诗权,等.NF-κB对人结肠癌细胞上皮间质转化及侵袭转移的影响[J].世界华人消化杂志,2014,22(23):3403-3409.