miR-29c过表达载体的构建及其对95D肺癌细胞增殖的影响

Construction of miR-29c expression vector and its effect on proliferation of lung cancer 95D cells

关键词:  miR-29c  肺肿瘤  95D细胞  细胞增殖

Key words:  miR-29c  lung cancer  95D cell  cell proliferation

摘要:

目的构建miR-29c真核表达载体并转染95D肺癌细胞，观察miR-29c过表达对95D细胞增殖能力的影响。方法克隆miR-29c片段插入质粒pcDNA3．1，构建miR-29c表达质粒pcDNA3．1-miR-29c；通过脂质体瞬时转染人肺癌细胞株95D，荧光PCR检测细胞miR-29c表达水平，MTT法检测过表达miR-29c对细胞增殖能力的影响。结果重组质粒pcDNA3．1-miR-29c经酶切及测序证明构建成功，荧光PCR证实转染后95D细胞miR-29c表达水平显著升高，是对照组的36倍。MTT检测结果表明，过表达miR-29c的95D细胞增殖受到显著抑制，至72h抑制率最高，达38．63％。结论成功构建miR-29c真核表达质粒pcDNA3．1-miR-29c，初步观察显示过表达miR-29c显著抑制95D肺癌细胞增殖效应。为进-步深入研究miR-29c对肺癌的作用打下良好基础。

Abstract:

Objective To construct eukaryotic expression vector of miR-29c and to investigate its effect on proliferation of human lung cancer 95D ceils. Methods pcDNA3. 1-miR-29c was constructed by inserting cloned gene miR-29c to eukaryotic expression vector pcDNA3.1. Sequencing and restriction endonucleases were used to verify the recombinant vector. Human lung cancer 95D cells were transfected by pcDNA3. 1-miR-29c. The expression level of miR-29c in 95D cells after transfection was identified by Real-Time PCR. The cell proliferative levels were assessed by MTT test. Results The expression vector of recombined pcDNA3.1-miR-29c was successfully constructed. The miR-29c expression levels in 95D cells after transfection significantly increased and were 36 times as that of control group. The proliferative activity was suppressed in pcDNA3. 1-miR-29c cells and the inhibition rate reached the highest 38.63% at 72 hour. Conclusion MiR-29c eukaryotic expression plasmid pcDNA3. 1-miR-29c was successfully constructed. Preliminary study indicated that over- expression of miR-29c can inhibit the proliferation of human lung cancer 95D cells.