摘要
目的探讨肝癌缺失基因-1（DLC-1）基因主要结构域在调控人结肠癌HT29细胞增殖中的作用。方法分别构建DLC-1基因Rho蛋白GTP酶活化蛋白（RhoGAP）、SAM、START结构域敲除的亚克隆重组质粒，并转染至人结肠癌HT29细胞，另设野生型HT29细胞组（对照组）、pcDNA3．1-HT29细胞组（空载体组）和pcDNA3．1-HT29-DLC-1细胞组（全长转染组）作为对照。应用四甲基偶氮唑盐（MTT）实验、平板克隆实验检测细胞增殖的改变；流式细胞术检测细胞凋亡；pull-down方法分析RhoA蛋白活性。组间差异采用方差分析。结果转染成功48h后，与对照组及空载体组相比，全长转染组细胞增殖（F=146．36）及RhoA蛋白活性（F=698．08）明显抑制（P均〈0．05），细胞早期凋亡增加（F=294．08，P〈O．05）。敲除RhoGAP转染组细胞的增殖能力（F=0．99）、细胞凋亡（F=0．049）及RhoA蛋白活性（F=5．769）与对照组及空载体组相比差异均无统计学意义（P均〉0．05）；敲除SAM转染组细胞比DLC-1基因全长转染组更显著地抑制了细胞增殖（F=31．00，P〈0．05），使Rh0A蛋白活性下调（F=92．57，P〈0．05），凋亡增加（F=130．44，P〈0．05）；敲除START转染组相对于DLC-1基因全长转染组，细胞增殖能力增强（F=15．47，P〈0．05），凋亡细胞减少（F=110．23，P〈0．06），RhoA蛋白活性上调（F=199．39，P〈0．05）。结论DLC-1基因抑制HT29细胞增殖具有RhoGAP依赖性，SAM结构域可能是内源性RhoGAP活性的自身抑制区域，START结构域可能协助RhoGAP结构域而起作用。
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 48hours 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. a6, 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.
出处
《中华消化杂志》
CAS
CSCD
北大核心
2012年第11期744-749,共6页
Chinese Journal of Digestion
基金
国家自然科学基金（30471937）
高等学校博士点基金（200802860040）
