靶向抑制存活素基因对大肠癌细胞SW80侵袭和转移的实验研究

目的 构建靶向存活素(survivin)基因的短发夹干扰RNA(shRNA)表达载体,导入人大肠癌细胞SW480中,研究靶向抑制survivin基因对SW480细胞侵袭和转移的影响.方法 根据siRNA设计原则,在survivin序列中选取设计含19个核苷酸(19 nt)的靶序列,间以9个核苷酸的茎环序列,两端分别加上对应的酶切位点,形成shRNA的DNA模板并克隆到shRNA表达载体pRNAT-U6.1/Neo中,获得靶向抑制survivin基因的sihNA表达载体pRNAT-U6.1/Neo-survivin;采用Lipofectamine 2000TM转染试剂将干扰质粒pRNAT-U6.1/Neo-survivin导入到大肠癌细胞SW480中;用Western blotting从蛋白水平检测干扰效果;分别采用肿瘤侵袭粘附实验和明胶酶谱分析法检测pRNAT-U6.1/Neo-survivin对SW480细胞的侵袭和转移潜能的影响.结果 Survivin基因的蛋白表达均得到显著抑制;沉默SW480的survivin基因可以显著抑制SW480细胞的侵袭和转移潜能,而且survivin基因表达被抑制后,SW480细胞分泌基质金属蛋白酶明显减少.结论 Survivin基因可能在SW480的侵袭和转移潜能中起重要作用,沉默SW480的survivin基因可以显著抑制其侵袭、转移和基质金属蛋白酶分泌,因而survivin影响SW480的侵袭和转移可能与调控基质金属蛋白酶分泌密切相关.

Short hairpin RNA-mediated suvivin gene silencing inhibits invasion and metastasis of human colon carcinoma cell line SW480 in vitro

Objective To investigate the effect of short hairpin RNA(shRNA) targeting survivin on adhesion and invasion of human colon carcinoma cell line SW480 in vitro.Methods According to the sequence of the coding region of survivin gene,two strings of 19 nucleotides of inverted sequence flanking the loop sequence of two complementary 9-base oligonucleotides were designed and synthesized to prepare the hairpin construct as the DNA templates for the target shRNA.The shRNA templates were cloned into shRNA expression vector pRNAT-U6.1/Neo,and the resulted vector pRNAT-U6.1/Neo-survivin was transfected into SW480 cells using Lipofectamine 2000TM.Western blotting was performed to evaluate survivin gene silencing induced by shRNA transfection at the protein level,and the biological behaviors of the SW480 cells were investigated by cell-matrix adhesion,invasion and gelatin-zymography assays.Results Western blotting revealed significantly lowered survivin protein expression in transfected SW480 cells,and survivin gene silencing induced by shRNA significantly suppressed the metastatic potential of SW480 cells in association with suppressed MMPs activity.Conclusions Survivin may play an important role in modulating human colorectal carcinoma cell invasion and metastasis,and survivin gene silencing can inhibit human colorectal cancer cell invasion and the production of MMP-2 and MMP-9.Survivin may affect invasion and metastasis of human colorectal carcinoma cells via regulating the production of MMPs.