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目的:构建携microRNA-155(miR-155)的真核表达载体并观察其转染高转移性人巨细胞肺癌95D细胞后细胞的生物学行为变化。方法:以95D细胞基因组RNA为模板,经PCR法扩增miR-155的前体序列,由BamHⅠ和HindⅢ双酶切后将其亚克隆入真核表达载体pcDNA 3.1(-),并进行双酶切及测序鉴定;将构建成功的pcDNA3.1(-)-pri-miR-155载体(命名为p-miR-155)体外瞬时转染人肺癌95D细胞,利用Real-time PCR探针法检测miR-155成熟体的表达水平,并利用CCK-8法、克隆形成实验和划痕法检测95D细胞的增殖、克隆形成以及体外迁移能力。结果:成功构建携miR-155的真核表达载体;与空白(Mock)和对照组(p-Ctrl)相比,转染后的95D细胞过表达miR-155[(2.04±0.62)vs(0.76±0.62)、(1.00±0.45),均P<0.01]、p-miR-155载体转染组95D细胞的增殖抑制明显增加[(46.70±6.89)%vs(3.70±1.40)%、(1.11±0.75)%,P<0.01]、克隆形成能力(在100和1 000个细胞/孔接种条件下)明显下降[(12±3)vs(34±3)、(35±3)个,P<0.01;(78±4)vs(159±4)、(165±4)个,P<0.01)],此外,细胞的迁移细胞数也明显减少[(110±5)vs(295±5)、(325±5)个,P<0.01]。结论:通过miR-155真核表达载体转染产生的过表达miR-155可显著抑制人肺癌95D细胞的增殖和迁移能力。
OBJECTIVE: To construct an eukaryotic expression vector carrying microRNA-155 (miR-155) and observe the changes of its biological behavior after transfection into 95D cells with highly metastatic human giant cell lung cancer. Methods: The precursor sequence of miR-155 was amplified by PCR using genomic RNA of 95D as a template. The precursor of miR-155 was amplified by PCR and subcloned into the eukaryotic expression vector pcDNA 3.1 (-) by BamHⅠand HindⅢ. (PcDNA3.1 (-) - pri-miR-155 vector (named as p-miR-155) was transiently transfected into human lung cancer 95D cells in vitro and Real-time PCR was used to detect the expression of miR -155 mature body, and the proliferation, colony formation and migration in vitro of 95D cells were detected by CCK-8 assay, clone formation assay and scratch assay. Results: The eukaryotic expression vector carrying miR-155 was constructed successfully. Compared with Mock group and control group (p-Ctrl), 95D cells overexpressing miR-155 [(2.04 ± 0.62) vs (0.76 (P <0.01). The proliferation inhibition of 95D cells in the group transfected with p-miR-155 was significantly higher than that in the control group [(46.70 ± 6.89)% vs (3.70 ± 1.40)% vs (12 ± 3) vs (34 ± 3), (35 ± 3), P <0.01), P <0.01] 0.01; 78 ± 4 vs 159 ± 4, 165 ± 4, P <0.01). In addition, the number of migrating cells was significantly decreased (110 ± 5 vs 295 ± 5, (325 ± 5), P <0.01]. Conclusion: The overexpression of miR-155 by miR-155 eukaryotic expression vector significantly inhibits the proliferation and migration of human lung cancer 95D cells.