沉默表达miR-26a A549细胞株的构建

Construction of A549 cell lines silence expressing miR-26a

目的构建稳定沉默表达miR-26a的人肺腺癌细胞株（A549）。方法体外合成成熟的人miR．26a-5p（Hsa—miR-26a-5p）特异性互补序列，在片段两侧添加AgeI、EcoRI酶切位点，与经双酶切后的GV280载体连接，构建GV280-miR一26a—down慢病毒载体。应用DNA测序对此重组载体进行鉴定。将GV280-miR-26a．down慢病毒载体与病毒包装质粒pGC—LV载体、pHelper1．0载体和pHelper2．0载体共转染293T细胞，包装产生慢病毒。依据293T细胞绿色荧光蛋白的表达量确定病毒滴度。将沉默表达miR-26a的慢病毒转染A549细胞，嘌呤霉素筛选后获得稳定细胞株。应用Westernblot法检测miR-26a一个靶基因SMADl的表达情况，验证此细胞株的功能。结果经DNA测序鉴定，成功构建了GV280-miR-26a—down慢病毒载体。在293T细胞中将该载体包装成慢病毒，测定病毒滴度为2×10^12TU／L。重组慢病毒转染A549细胞的效率高达95％，用2．5mg／L嘌呤霉素筛选2周后几乎100％的A549细胞表达GFP。Westernblot结果显示miR-26a的靶基因SMADl的表达量在miR-26a沉默组比空载病毒组和空白对照组明显上调。结论成功构建了GV280．miR．26a—down慢病毒载体，并构建稳定沉默表达miR-26a的A549细胞株，为后续研究奠定了基础。

Objective To construct a lentiviral expression vector stably down-regulating has-miR-26a-Sp in human lung adenocarcinoma cell line（A549）. Methods Specific complementary sequence of mature hsa-miR-26a-Sp was synthesized in vitro. Restriction enzyme cutting sites of Age I and EcoRI was added to this sequence, which was then cloned into the plasmid of GV280 by double digestion to obtain the ]entiviral vector named GV280-miR-26a-down. The recombinant plasmid was confirmed by DNA sequencing, subsequently cotransfected into 293T cell with viral pac- kaging plasmid pGC-LV vector, pHelper 1.0 vector,and pHclper 2.0 vector. Virus titer was measured according to the expression level of green fluorescent protein. The lentivirus down-regulating miR-26a was transfected into A549 cells which was screened by puromycin subsequently to obtain stable cell line. One of miR-26a＇s target gene SMAD1 was de- tected by Western blot to ensure the function of this A549 cell line. Results The lentivirus vector of GV280-miR-26a- down was successfully constructed confirmed by DNA sequencing, and then was packaged in 293T cells to produce lentiviral particles and the virus titer reached to 2 ×10^12 TU/L. The efficiency of recombinant lentivirus transfecting A549 cells was up to 95%. Nearly 100% A549 cells expressed GFP after being screened by 2.5 mg/L puromycin for a- bout 2 weeks. Western blot results showed that the expression of miR-26a＇s target gene SMAD1 significantly upregulated in miR-26a-down group compared with viruses in the control group and the blank control group. Conclusion The lenti- virus vector of GV280-miR-26a-down and the 3.549 cell line stably down-regulating has-miR-26a-Sp can be successful- ly constructed,which provides the basis for further study of miR-26a.