Xu Mei-fang, Zang Sheng-bing, Liu Jing-feng, Gao Ling-yun, Gao Mei-qin, Yang Ying-hong, Huang Ai-min
Department of Pathology, the Affiliated Union Hospital, Fujian Medical University, Fuzhou, China.
Zhonghua Gan Zang Bing Za Zhi. 2012 Dec;20(12):925-9. doi: 10.3760/cma.j.issn.1007-3418.2012.12.011.
To investigate the impact of expression of kisspeptin-1 (KiSS-1) metastasis-suppressor gene on the proliferative, adhesive and invasive abilities of human hepatocellular carcinoma (HCC) using an in vitro cell system.
The highly metastatic human hepatoma cell line MHCC97-H was transiently transfected with the pcDNA3.1/HisC vector expressing the KiSS-1 gene (experimental group) or the vector without the KisS-1 gene (blank control group). Untransfected cells served as the negative control group. Proliferative abilities of the three groups were assessed by flow cytometry and MTT assay. Adhesive abilities were assessed by MTT assays using matrigel and fibronectin. Invasive abilities and cell motility were assessed by chemoinvasion chamber assay using reconstituted matrigel and migration chamber assay using polycarbonate filters, respectively.
The experimental group showed significantly lower adhesion capacity to matrigel (0.257+/-0.029) than either the blank control group (0.374+/-0.016; t=-7.90345, P less than 0.01) or the negative control group (0.394+/-0.031; t=-7.22752, P less than 0.01). Similarly, the experimental group showed significantly lower adhesion capacity to fibronectin (0.292+/-0.004) than either the blank control group (0.394+/-0.010; t=-20.93138, P less than 0.01) or the negative control group (0.412+/-0.023; t=-11.31371, P less than 0.01). The experimental group also showed significantly lower numbers of cells with invasive capacity (42.40+/-1.14) than either the blank control group (66+/-1.58; t=-27.0711, P less than 0.01) or the negative control group (67.80 +/- 1.92; t=-25.4, P less than 0.01). Similarly, the experimental group showed significantly lower numbers of cells with chemotactic movement (65.80+/-1.92) than either the blank control group (93.80+/-2.28; t=-30.11750, P less than 0.01) or the negative control group (96.40+/-2.07; t=-24.19142, P less than 0.01). The experimental group showed slightly, but not significantly, lower cell proliferation (0.644+/-0.027) than either the blank control group (0.669+/-0.022; t=-1.60371, P?>?0.05) or the negative control group (0.678+/-0.027; t=-1.97828, P?>?0.05). In addition, there were no obvious differences between the three groups in the amounts of cells arrested in either the G1 phase or the S phase.
KiSS-1 overexpression suppresses the adhesion, invasion and motility, but not the proliferation, of hepatoma carcinoma cells in vitro. These findings imply that KiSS-1 might represent a promising new candidate for gene therapy against human hepatocellular carcinoma.
利用体外细胞系统研究转移抑制基因kisspeptin-1(KiSS-1)的表达对人肝癌(HCC)细胞增殖、黏附及侵袭能力的影响。
将高转移人肝癌细胞系MHCC97-H分别用表达KiSS-1基因的pcDNA3.1/HisC载体(实验组)或不含KiSS-1基因的载体(空白对照组)进行瞬时转染。未转染的细胞作为阴性对照组。采用流式细胞术和MTT法评估三组细胞的增殖能力。使用基质胶和纤连蛋白,通过MTT法评估黏附能力。分别使用重组基质胶通过化学侵袭小室试验和使用聚碳酸酯滤膜通过迁移小室试验评估侵袭能力和细胞运动能力。
实验组对基质胶的黏附能力(0.257±0.029)显著低于空白对照组(0.374±0.016;t=-7.90345,P<0.01)和阴性对照组(0.394±0.031;t=-7.22752,P<0.01)。同样,实验组对纤连蛋白的黏附能力(0.292±0.004)显著低于空白对照组(0.394±0.010;t=-20.93138,P<0.01)和阴性对照组(0.412±0.023;t=-11.31371,P<0.01)。实验组具有侵袭能力的细胞数量(42.40±1.14)也显著低于空白对照组(66±1.58;t=-27.0711,P<0.01)和阴性对照组(67.80±1.92;t=-25.4,P<0.并对其进行了分析。同样,实验组具有趋化运动能力的细胞数量(65.80±1.92)显著低于空白对照组(93.80±2.28;t=-30.11750,P<0.01)和阴性对照组(96.40±2.07;t=-24.19142,P<0.01)。实验组细胞增殖能力(0.644±0.027)略低于空白对照组(0.669±0.022;t=-1.60371,P>0.05)和阴性对照组(0.678±0.027;t=-1.97828,P>0.05),但差异不显著。此外,三组细胞在G1期或S期的停滞细胞数量无明显差异。
KiSS-1基因过表达可抑制肝癌细胞的体外黏附、侵袭及运动能力,但不影响其增殖能力。这些发现提示KiSS-1可能是一种有前景的抗人肝癌基因治疗新候选基因。