miRNA let-7a1真核表达载体的构建及对肺癌细胞增殖的影响

目的： 构建miRNA let-7a1真核表达载体，研究其在肺癌A549细胞株的表达及对A549细胞增殖的影响。方法：以人肺癌细胞A549的总RNA为模板，RT-PCR扩增miRNA pre-let-7a1基因序列, 将miRNA pre-let-7a1基因克隆到真核表达载体pSilencerTM4.1-CMV neo中，构建成pSilencerTM 4.1-let-7a1重组体。将pSilencerTM 4.1-let-7a1表达载体瞬时转染肺癌A549细胞，RT-PCR法检测miRNA let-7a1在转录水平的表达。根据miRBase Targets数据库，查hsa-let-7a1靶序列，构建 let-7a1靶序列-报道基因融合质粒pMIR-report-let-7a1T，与pSilencerTM 4.1-let-7a1表达载体共转染A549细胞，通过荧光素酶活性检测pSilencerTM 4.1-let-7a1质粒对其靶序列的作用。MTT法检测pSilencerTM 4.1-let-7a1转染A549细胞后，对细胞增殖的影响。结果：pSilencerTM 4.1-let-7a1真核表达栽体和let-7a1靶序列-报道基因融合质粒经酶切及测序鉴定正确。pSilencerTM 4.1-let-7a1转染 A549细胞后，经RT-PCR证明能有效表达miRNA let-7a1。 pSilencerTM 4.1-let-7a1 质粒和pMIR-report-let-7a1T质粒共转染A549细胞后，通过报告基因检测，相对荧光素酶活性明显降低，表明pSilencerTM 4.1-let-7a1转染A549细胞后，可表达let-7a1并具有生物学活性。MTT检测结果显示：pSilencerTM 4.1-let-7a1 转染后的A549活细胞数目明显减少。结论：成功构建了真核表达载体pSilencerTM 4.1-let-7a1，转染肺腺癌A549细胞后能有效表达，miRNA let-7a1基因过表达抑制A549细胞的增殖。

Construction of eukaryotic expression vector miRNA let-7a1 and its effect on the proliferation of lung cancer A549 cells

AIM: To construct a recombinant eukaryotic expression vector, pSilencer 4.1-let-7a1 and to express it in lung cancer A549 cells for detecting its effect on the proliferation of A549 cells. METHODS: The pre-let-7a1 sequence was amplified by RT-PCR using RNA from human lung cancer A549 cells, and then inserted into pSilencer 4.1-CMV neo vector to generate pSilencer 4.1-let-7a1 which was transfected into lung cancer A549 cells. The expression of miRNA let-7a1 was verified by RT-PCR. Its activity in A549 cells was determined by luciferase reporter assay after cotransfection of let-7a1 target sequence-reporter gene plasmid with pMIR-report let-7a1T, which was constructed by inserting let-7a1 target sequence into the luciferase reporter 3’UTR of pMIR-report luciferase vector. The effect of pSilencer 4.1-let-7a1 transfection on A549 cell proliferation was detected by MTT method. RESULTS: The sequences of cloned pre-let-7a1 were correct. RT-PCR results indicated that pSilencer 4.1-let-7a1 was effectively expressed in the transfected A549 cells. The relative luciferase activity was decreased significantly after A549 cells were co-transfected with pSilencer 4.1-let-7a1 and pMIR-report let-7a1T, indicating that let-7a1 was expressed effectively and had biologic activity in A549 cells that were transfected with pSilencer 4.1-let-7a1. MTT results showed that miRNA let-7a1 gene overexpression in A549 inhibited cell proliferation. CONCLUSION: The eukaryotic expression vector pSilencer 4.1-let-7a1 is successfully constructed and effectively expresses in A549 cell. The overexpression of miRNAlet-7a1 gene inhibits lung cancer A549 cell proliferation.