Regulation of deleted in liver cancer-1 gene domains on the proliferation of human colon cancer HT29 cell
10.3760/cma.j.issn.0254-1432.2012.11.007
- VernacularTitle:肝癌缺失基因-1基因结构域调控人结肠癌HT29细胞增殖的实验研究
- Author:
Pingping WU
;
Peng WU
;
Qiqiang LONG
;
Nan LI
;
Zhi JIN
;
Xiaoqiang TIAN
;
Peilin HUANG
- Publication Type:Journal Article
- Keywords:
Colonic neoplasms;
Plasmids;
Codon,initiator;
Rho GTPase-activating protein;
Cell proliferation
- From:
Chinese Journal of Digestion
2012;32(11):744-749
- CountryChina
- Language:Chinese
-
Abstract:
Objective To study the role of deleted in liver cancer-1 (DLC-1) gene main domains on the regulation of human colon cancer HT29 cell proliferation.Methods Subcloning recombinant plasmid vectors with Rho GTPase activating protein (RhoGAP),sterile alpha motif (SAM) or steroidogenic acute regulatory-related lipid-transfer (START) domains of DLC-1 gene knockout were constructed and transfected into human colon cancer cell HT29.Wild HT29 cell group (control group),pcDNA3.1-HT29 cell group (vector group) and pcDNA3.1-HT29-DLC-1 cell group (whole DLC-1 gene transfected group) were set as control.The change of cell proliferation was detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and colony formation test.The cell apoptosis was analyzed by flow cytometry.The activity of RhoA protein was detected by pull-down assay.The differences between the groups were analyzed by the analysis of variance.Results At 48 hours after the successful transfection,compared with control group and vector group,cells proliferation and the activity of RhoA protein were significantly suppressed in whole DLC-1 gene transfected group (F=146.36,698.08,both P＜0.05) and early cell apoptosis increased (F=294.08,P＜0.05).Compared with control group and vector group,there was no significant difference in cell proliferation ability,cell apoptosis and the activity of RhoA protein activity in RhoGAP knockout transfected cells (F=0.99,0.049,5.769,all P＞0.05).Compared with whole DLC-1 gene transfected group,the suppression of cell proliferation was more significant in SAM knockout transfected cells (F=31.00,P＜0.05),the activity of RhoA protein down regulated (F=92.57,P＜0.05) and apoptosis increased (F=130.44,P＜0.05).Compared with whole DLC-1 gene transfected group,the ability of cell proliferation increased (F=15.47,P＜0.05),apoptosis cell decreased (F＝110.23,P＜0.05) and the activity of RhoA protein up regulated (F=199.39,P＜0.05) in START knockout transfected cells.Conclusions The role of DLC-1 gene in the suppression of cell proliferation in HT29 cells was RhoGAP-dependent.SAM domain may be the self suppression domain for endogenous RhoGAP activity.START domain may take effect through enhancing RhoGAP domain.
