miRNA let-7a1真核表达载体的构建及对肺癌细胞增殖的影响

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

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miRNApre-let-7a1基因序列,将miRNApre-let-7a1基因克隆到真核表达载体pSilencerTM4.1-CMVneo中,构建成pSilencerTM4.1-let-7a1重组体。将pSilencerTM4.1-let-7a1表达载体瞬时转染肺癌A549细胞,RT-PCR法检测miRNAlet-7a1在转录水平的表达。根据miRBaseTargets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pSilencerTM4.1-let-7a1表达载体共转染A549细胞,通过荧光素酶活性检测pSilencerTM4.1-let-7a1质粒对其靶序列的作用。MTT法检测pSilencerTM4.1-let-7a1转染A549细胞后,对细胞增殖的影响。结果:pSilencerTM4.1-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。pSilen-cerTM4.1-let-7a1转染A549细胞后,经RT-PCR证明能有效表达miRNAlet-7a1。pSilencerTM4.1-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pSilencerTM4.1-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:pSilencerTM4.1-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pSilencerTM4.1-let-7a1,转染肺腺癌A549细胞后能有效表达,miRNAlet-7a1基因过表达抑制A549细胞的增殖。

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 ceils 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- 7al 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 - trans- fected with pSilencer 4. 1 -let-Ta1 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.