体外转染KISS-1基因对SW480细胞系侵袭和迁移力的影响

Effects of in vitro tranfection of KISS-1 gene on invasion and migration of SW 480 cell line

目的探讨人肿瘤转移抑制基因KiSS-1对人结肠癌SW480细胞增殖、侵袭、迁移能力的影响。方法将携有KISS-1基因的重组真核表达质粒pRc/CMV2-KISS-1转化大肠杆菌DH5et并扩增,抽提纯化质粒并进行酶切鉴定,pRc/CMV2-KISS-1体外转染SW480细胞(SW480-FH1T),筛选稳定转染的细胞并扩增培养,以转染了空质粒pRc/CMV2的SW480细胞(SW480-pRc/CMV2)和正常SW480细胞为对照,用RT-PCR检测KISS-1的表达情况,并用四甲基偶氮唑蓝比色法(MTT法)和细胞侵袭实验和迁移实验分别检测KISS-1基因转染前、后SW480细胞增殖、侵袭和迁移活性的变化。结果 pRc/CMV2-KISS-1的酶切鉴定证实含有目的基因KISS-1;SW480-KISS-1细胞的RT-PCR产物经凝胶电泳有明显的阳性条带,而对照组则无;MTT实验结果显示重组质粒转染组SW480细胞的增殖明显受到抑制;Transwell体外侵袭实验显示转染pRc/CMV2-KISS-1能明显抑制SW480细胞的体外侵袭力(P<0.01);体外迁移实验显示转染pRc/CMV2-KISS-1能明显抑制SW480细胞的体外迁移力(P<0.01)。结论 KiSS-1基因能显著抑制人结肠癌SW480细胞的侵袭和迁移能力,在结肠肿瘤的转移中起重要作用。

Objective To investigate the effects of tumormetastasis suppressor gene KiSS-1 on the invasionand migration abilities of colon cancer SW480 cells. Methods Eukaryotic expression plasmid containing KISS-1,pRc/CMV2-KISS-1,was introduced into E.coli DH5α and amplified.The plasmid was purified and identified by restrictive enzyme digestion. Plasmid pRc/CMV2-KISS-1 was transfected into SW480 cells,and positive cell clones（SW480-KISS-1）were selected and amplified.plasmid-transfected SW480 cells（SW480-pRc/CMV2）and normal SW480 cells were set as control.Reverse transcription-polymerase chain reaction（RT-PCR）was applied to assess the expression of KISS-1.Proliferation、invasion and migration ability of SW480 cells were measured before and after transfection by MTT assay and cell invasion assay, respectively. Results SW 480 cell transfected with pRc/CWV2-KISS-1 Was approved to express KISS-1.while KISS-1 expression was completety absent in the control group. The expression of KISS-1 significantly inhibited invasion; migration of SW480 cells in the experimental group（P0.01）. Conclusion KiSS-1 gene can significantly inhibit the invasion and migration abilities of colon cancer SW480 cells, which may play a key role in metastasis of colon cancer.