RNAi抑制H157细胞Wnt5a表达

Inhibited Expression of Wnt5a in H157 Cell by RNA Interference

目的构建抑制大鼠Wnt5a基因表达的重组质粒,达到抑制H157细胞Wnt5a表达的目的。方法根据大鼠Wnt5a的基因序列,设计合成含有发夹结构的2条寡核苷酸片段,经退火形成双链后克隆至PAVU6+27载体质粒中,对阳性重组子进行酶切和测序鉴定后转染正常H157细胞,并采用West-ern blot检测Wnt5a蛋白水平表达的变化。结果重组质粒经双酶切后,有目的条带出现,提示Wnt5a干扰片断已克隆至PAVU6+27载体质粒中,DNA测序结果显示插入序列与预先设计完全一致。重组质粒转染H157细胞后经G418筛选,成功获得阳性克隆,Western blot结果显示Wnt5a的蛋白水平表达显著下降。结论成功构建Wnt5a基因RNA干扰表达质粒,明显抑制H157细胞中Wnt5a表达,这为深入研究该基因在非小细胞肺癌发生及侵袭转移中的作用奠定了基础。

Objective To construct PAVU6-Wnt5a/siRNA expression plasmid and inhibit its function in H157 cells. Methods Human siwnt5a was subcloned into PAVU6 ＋ 27, DH10B E. col. i was transformed with the recombinant plasmid, and the ampicillin-resistant clones were identified by double digestion with HindⅢ and BamH Ⅰ and DNA sequencing. H157 cells were transfected with the identified PAVU6-siWnt5a and PAVU6 ＋ 27 by Lipofectamine 2000. Results The DNA fragment obtained by double digestion and DNA sequencing showed that siWnt5a was inserted into the PAVU6 ＋ 27 in correct orientation and reading frame. G418-resistant colonies of H157 cells were obtained. The expression of Wnt5a protein decreased by Western blotting. Conclusion PAVU6-siWnt5a expression plasmid was constructed and inhibited obviously the expression of Wnt5a in H157 cells, which help to further study the carcinogenesis and metastasis role in non-small cell lung cancer of this protein.