CARMA3 gene knockdown in HCT116 cells inhibits cell growth, migration and invasion / 中国病理生理杂志

ABSTRACT

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 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 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-shCARMA3 cells with stably-silenced CARMA3 gene were successfully 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 suppressed (P<0.01).G0 /G1 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 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-κB signaling pathway to change cell cycle and metastasis-related markers and to regulate EMT.