人支气管上皮hsa-miR-148a-3p低表达细胞株的建立

Construction of a human bronchial epithelial hsa-miR-148a-3p knockdown cell line

目的:建立稳定的hsa-miR-148a-3p低表达人支气管上皮细胞株(16HBE)。方法:根据miRBase中提供的序列信息设计hsa-miR-148a-3p tough decoy RNA(TuD RNA),并将其连接到慢病毒载体pLKO.1-puro上。将重组慢病毒载体转染至293FT细胞并包装为慢病毒后,收集病毒上清,感染正常16HBE细胞。用嘌呤霉素筛选出has-miR-148a-3p低表达的16HBE细胞株,通过荧光定量PCR对其进行鉴定,然后对筛选出的细胞分别采用荧光定量PCR和Western blot检测DNA甲基转移酶1(DNMT1)mRNA和蛋白的表达水平。结果:测序结果表明含hsa-miR-148a-3p TuD RNA的重组慢病毒载体构建成功;荧光定量PCR检测显示has-miR-148a-3p低表达的16HBE细胞株has-miR-148a-3p的表达量比正常16HBE细胞低44%(P<0.01),其作用靶基因DNMT1的mRNA和蛋白表达水平分别为正常细胞的3.4倍和2.0倍(P均<0.01)。结论:成功建立hsa-miR-148a-3p低表达的16HBE细胞,hsa-miR-148a-3p的低表达能提高DNMT1 mRNA和蛋白的表达水平。

OBJECTIVE： To construct a 16HBE cell line with low expression of hsa-miR-148a-3p. METHODS：A pair of TuD RNA sequences of hsa-miR-148a-3p was designed according to information provided in miRBase, which was ligated to lentiviral vector pLKO.1-puro afterwards. The recombinant vector was transfected into 293FT cells for lentivirus packaging. The viruses were collected and used to infect normal 16HBE cells. The target cells were selected with Puromycin and was identified by quantitative PCR. The mRNA expression and the protein expression level of DNMT1 were then tested. RESULTS：Sequencing results indicated the successful construction of recombinant lentiviral vector with hsa-miR-148a-3p TuD RNA,quantitative PCR showed that the expression level of hsa-miR-148a-3p in the target cells was 44%lower than that of normal 16HBE cells（P〈0.01）,whereas a 3.4-fold increase of the mRNA expression level and a two-fold increase of protein expression level of DNMT1 were observed in the cell line（P〈0.01）. CONCLUSION：Low expression of hsa-miR-148a-3p of 16HBE cell line was successfully constructed,and inhibition of hsa-miR-148a-3p could lead to higher expression and higher protein levels of DNMT1.