CAJ | 학술논문

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。
목적：구건miR-148a/b진핵표체질립，전염이선암AsPC-1세포，병관찰기miR-148a/b적표체수평。방법근거miRBase제공적서렬합성miR-148a/b전체，PCR확증후형성쌍련，삽입선성화진핵표체질립pcDNA3．1，구건pcDNA3．1/miR-148a/b，진행매절화측서감정。장이선암 AsPC-1세포분4조진행질립전염：전염 pcDNA3．1/miR-148a（miR-148a조）、전염 pcDNA3．1/miR-148b（miR-148b조）、전염 pcDNA3．1（공질립대조조）화부가지질체（공백대조조）。전염48 h후，채용정량 PCR법검측각조세포중 miR-148a/b 적표체량。결과 pcDNA3．1/miR-148a/b적매절화측서여miRBase제공적서렬완전일치。전염48 h후，miR-148a조 AsPC-1세포중적 miR-148a표체량명현고우공질립대조조화공백대조조（6．76±0．35비1．00±0．54、1．02±0．40，균P＜0．05），miR-148b 조AsPC-1세포중적miR-148b표체량명현고우공질립대조조화공백대조조（3．28±0．08비1．02±0．35、1．01±0．28，균P＜0．05）。결론성공구건료 miR-148a/b진핵표체질립 pcDNA3．1/miR-148a/b，해질립능현저상조이선암 AsPC-1세포적miR-148a/b표체수평。
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.