目的：构建miR-148a/b真核表达质粒，转染胰腺癌AsPC-1细胞，并观察其miR-148a/b的表达水平。方法根据miRBase提供的序列合成miR-148a/b前体，PCR扩增后形成双链，插入线性化真核表达质粒pcDNA3．1，构建pcDNA3．1/miR-148a/b，进行酶切和测序鉴定。将胰腺癌 AsPC-1细胞分4组进行质粒转染：转染 pcDNA3．1/miR-148a（miR-148a组）、转染 pcDNA3．1/miR-148b（miR-148b组）、转染 pcDNA3．1（空质粒对照组）和仅加脂质体（空白对照组）。转染48 h后，采用定量 PCR法检测各组细胞中 miR-148a/b 的表达量。结果 pcDNA3．1/miR-148a/b的酶切和测序与miRBase提供的序列完全一致。转染48 h后，miR-148a组 AsPC-1细胞中的 miR-148a表达量明显高于空质粒对照组和空白对照组（6．76±0．35比1．00±0．54、1．02±0．40，均P＜0．05），miR-148b 组AsPC-1细胞中的miR-148b表达量明显高于空质粒对照组和空白对照组（3．28±0．08比1．02±0．35、1．01±0．28，均P＜0．05）。结论成功构建了 miR-148a/b真核表达质粒 pcDNA3．1/miR-148a/b，该质粒能显著上调胰腺癌 AsPC-1细胞的miR-148a/b表达水平。%Objective To construct miR-148a/b eukaryotic expression vector,and to explore its expression in pancreatic cancer AsPC-1 cells.Methods The pri-miR-148a/b was synthesized ac-cording to the miRBase and double-stranded oligonucleotides were generated by PCR.The miR-148a/b were cloned into pcDNA3.1 vector and identified by enzyme digestion and sequence analy-sis.AsPC-1 cells were divided into four groups:pcDNA3.1/miR-148a transfection group(miR-148a group),pcDNA3.1/miR-148b transfection group(miR-148b group),pcDNA3.1 transfection group(empty vector control group),lipofectamine transfection group(blank control group).The expression of miR-148a/b was detected by real-time quantitative PCR at 48 hours after transfec-tion.Results The pcDNA3.1/miR-148a/b identified by enzyme digestion and sequence analysis was completely consistent with the sequence from miRBase database.After of transfection 48 hours,the AsPC-1 cells expression of miR-148a in miR-148a group(6.76±0.35)was significantly higher than that in empty vector control group(1.00±0.54)and blank control group(1.02± 0.40)(P<0.05).In addition,the expression of miR-148b in miR-148b group(3.28±0.08)was significantly higher than in empty vector control group(1.02±0.35)and blank control group(1. 01±0.28)(P<0.05).Conclusion The miR-148a/b eukaryotic expression vector has been suc-cessfully constructed.The pcDNA3.1/miR-148a/b can significantly increase the expression of miR-148a/b in pancreatic cancer AsPC-1 cells.
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