pcDNA3-HIF-1α真核表达质粒的构建及其在MCF-7细胞中的表达

Construction of pcDNA3-HIF-1α eukaryotic expression vector and its expression in MCF-7 cells

目的构建低氧诱导因子1α(H IF-1α)真核表达质粒,并在MCF-7细胞中进行功能验证。方法利用RT-PCR扩增带有XbaⅠ和H indⅢ酶切位点的H IF-1α编码基因,插入T载体测序正确后,克隆入pcDNA3真核表达载体,然后将其转染MCF-7细胞。W estern b lot和双荧光素酶报告基因法分别检测H IF-1α表达及其DNA结合活性;实时定量PCR检测H IF-1α对低氧诱导基因C-METmRNA表达的影响;Caspase3/7活性检测来初步判断H IF-1α对低氧MCF-7细胞凋亡的影响。结果酶切及测序结果证明pcDNA3-H IF-1α表达质粒的DNA序列完全正确。将此质粒转染MCF-7细胞后,H IF-1α蛋白表达明显增加,它不仅增强了pGL3-EPO-HRE报告基因活性而且促进了C-METmRNA的表达,同时还降低了低氧MCF-7细胞中Caspase3/7的水平。结论pcDNA3-H IF-1α真核表达质粒构建成功,它不但具有很强的DNA结合和诱导活性,而且在细胞低氧过程中具有一定的抗凋亡作用。

Objective To construct pcDNA3-HIF-1α eukaryotic expression vector and to investigate its function in breast cancer MCF-7 ceils. Methods RT-PCR was applied to amplify human HIF-1α cDNA from MCF-7 ceils with a pair of sequence specific primers carrying a restriction enzyme site Xba Ⅰ or Hind Ⅲ on each 5＇end. HIF-1α cDNA was inserted into pMD18-T after sequencing, and then inserted into pcDNA3 vector. The recombinant vector was transfected into MCF-7 ceils to observe its expression and function by Western blot and dual-luciferase reporter gene assay. Realtime-PCR was performed to detect the inducible expression of C-MET mRNA by HIF-1 α the antiapoptotic effect of HIF-1 under hypoxia was analysed by detecting the Caspase3/7 activity. Results The sequence of pcDNA3-HIF-1α is identical with the gene bank. It enhances the expression of HRE reporter gene and C-MET mRNA, but decreases the Caspase3/7 activity in MCF-7 ceils under hypoxia. Conclusion The pcDNA3-HIF-1α eukaryotic expression vector was successfully constructed, it not only has strong DNA binding and inducing activity, but also has anti-apoptotic effect in hypoxia MCF-7 ceils.