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目的构建miR-26b过表达载体,观察其对MKN-45人胃癌细胞增殖的影响。方法合成miR-26b DNA寡聚核苷酸单链,经退火形成双链;用限制性内切酶EcoRⅠ和HindⅢ对pc DNATM6.2-GW-miR真核表达载体进行双酶切(大片段),纯化后与之前退火形成的DNA双链连接,转化至大肠杆菌感受态细胞DH5α,扩增后提取质粒,经DNA测序和BLAST比对,验证其是否构建成功;将成功构建的pc DNATM6.2-GW-miR-26b载体瞬时转染MKN-45人胃癌细胞,采用实时定量PCR检测miR-26b的表达水平,Western blot法检测细胞分裂周期蛋白6(CDC6)蛋白,MTT法检测细胞增殖的变化。结果通过DNA测序BLAST比对结果显示成功构建了miR-26b的真核表达载体;瞬时转染MKN-45细胞后,实时定量PCR结果显示miR-26b的表达水平显著增加;Western blot结果表明过表达miR-26b明显抑制CDC6蛋白的表达,MTT法显示过表达miR-26b能显著抑制MKN-45细胞的增殖。结论过表达miR-26b可抑制MKN-45人胃癌细胞的增殖。
Objective To construct miR-26b overexpression vector and observe its effect on the proliferation of MKN-45 human gastric cancer cells. Methods Single-stranded RNA was synthesized and annealed to form double-stranded DNA. Double enzyme digestion (large fragment) of pcDNA3.1-GW-miR eukaryotic expression vector was performed with restriction endonucleases EcoRI and HindIII. After purification, the DNA was ligated with DNA previously annealed and transformed into E. coli competent cells DH5α. After amplification, the plasmid was extracted and verified by DNA sequencing and BLAST. The constructed recombinant plasmid pcDNA3.1 The expression of miR-26b was detected by real-time quantitative PCR. The expression of CDC6 protein was detected by Western blot. The proliferation of MKN-45 cells was detected by MTT assay . Results The DNA sequencing BLAST results showed that miR-26b was successfully constructed and the expression of miR-26b was significantly increased after transient transfection of MKN-45 cells. Western blot results showed that overexpression of miR-26b miR-26b significantly inhibited CDC6 protein expression, MTT assay showed that over-expression of miR-26b can significantly inhibit the proliferation of MKN-45 cells. Conclusion Overexpression of miR-26b can inhibit the proliferation of MKN-45 human gastric cancer cells.