摘要
目的通过基因沉默缺氧诱导因子-1α(HIF-1α),研究其对结直肠癌SW480细胞的生长增殖、侵袭迁移能力,及对锌指转录因子(Snail)与上皮钙黏蛋白(E-cadherin)表达的影响和作用。方法利用氯化钴(CoCl2)化学诱导建立结直肠癌SW480细胞的缺氧模型,设计合成HIF-1α的小干扰RNA(HIF-1α-siRNA)并将其转染入细胞内。通过四甲基偶氮唑兰(MTT)法检测细胞的体外生长增殖活性,体外侵袭实验(Transwell法)检测细胞的体外侵袭迁移力,逆转录聚合酶链反应(RT-PCR)及免疫蛋白印迹(Western blot)检测细胞中HIF-1α、Snail及E-cadherinmRNA与蛋白的表达情况。结果 MTT法检测结果显示,缺氧条件下,基因干扰组的OD值及细胞存活率均降低(P<0.05);体外侵袭实验结果显示,基因干扰组侵袭迁移率降低(P<0.05);RT-PCR与Western blot检测结果显示,基因干扰组中HIF-1α和Snail mRNA和蛋白的表达明显降低,E-cadherin mRNA和蛋白的表达明显增强(P<0.05),对3种因子进行相关性分析,结果显示HIF-1α和Snail的表达呈正相关,HIF-1α和Snail均与E-cadherin的表达呈负相关(P<0.05)。结论 RNA干扰沉默HIF-1α基因可抑制结直肠癌SW480细胞的体外生长增殖和体外侵袭迁移能力,其机制可能是由于基因沉默HIF-1α导致细胞中Snail低表达,E-cadherin高表达,抑制了EMT发生、发展所致。
Objective To study the roles of HIF-1α on the proliferation,invasiveness and the effects to the expressions of Snail and E-cadherin in colorectal cancer SW480 cells by HIF-1α gene silencing.Methods Chemical inducing process was used with cobalt chloride（CoCl2）to establish the hypoxia cell model of colorectal cancer SW480 cells,the small interference RNA of HIF-1α（HIF-1α-siRNA） designed and synthetized,and the cells transfection got.Methyl thiazolyl tetrazolium（MTT） method was used to detect the proliferation capability of the colorectal cancer SW480 cells,extraorgan invasion experiment（Transwell methed） was applied to evaluate the cell invasion and transference capability,the mRNA and protein expression levels of HIF-1α,Snail and E-cadherin were detected by real time PCR（RT-PCR） and Western blot,respectively.Results In the hypoxia microenvironment,MTT disclosed that both the OD value and survival rates of gene interference group were decreased（P0.05）;Transwell showed that invasion and migration rates in gene interference group were decreased（P0.05）;RT-PCR and Western blot displayed that the expressions of both mRNA and protein of HIF-1α and Snail in SW480 cells in gene interference groups were decreased,and the E-cadherin was increased obviously（P0.05）,furthermore,there were significant positive correlativity between the HIF-1α and Snail,and negative correlativity between both them and E-cadherin conspicuously（P0.05）.Conclusions The extraorgan proliferation,invasion and migration capabilities of the colorectal cancer SW480 cells are inhibited by RNA interference on HIF-1α gene silencing,and the possible mechanism is that HIF-1α gene silencing could inhibit Snail expression and promote E-cadherin expression,then restrain the incidence and development of EMT.
出处
《实用肿瘤杂志》
CAS
2012年第3期226-232,共7页
Journal of Practical Oncology
