沉默PHF8重组慢病毒的制备及其对食管鳞癌细胞增生的影响

目的 制备锌指蛋白8（plant homeodomain finger protein 8,PHF8）RNA干扰（RNA interference,RNAi）重组慢病毒（lentivirus）颗粒,建立PHF8表达沉默的人食管鳞癌（esophageal squamous-cell carcinoma,ESCC）稳定细胞系并观察PHF8对细胞增生的影响.方法将目的 基因或阴性对照短发卡RNA（short hairpin RNA,shRNA）载体与包装载体共转染病毒包装细胞293T,收集并浓缩上清液获得重组慢病毒;测定病毒滴度后用于感染食管鳞癌细胞;通过荧光显微镜及流式细胞仪观察感染效率,嘌呤霉素筛选法建立稳定细胞系;定量PCR及Western Blotting检测PHF8的表达沉默;MTS法检测细胞的增生特性.结果成功制备了沉默PHF8的重组慢病毒;应用重组病毒感染食管鳞癌细胞后,PHF8 mRNA 和蛋白的表达水平显著降低,细胞的增生明显下降.结论慢病毒介导的shRNA干扰能有效抑制食管鳞癌细胞的PHF8表达及细胞增生.

Preparation of lentivirus silencing PHF8 and its effects on human esophageal squamous-cell carcinoma cells

Objective To construct a recombinant lentivirus for RNA interference of PHF8 gene, establish a human esophageal squamous-cell carcinoma cell lines with PHF8 gene knockdown and observe its effect on cell proliferation. Methods The PHF8 or Nonsilencing shRNA and packaging vectors were cotransfected to 293T cells to produce the recombinant lentivirus, which were further ultra-centrifuged, tittered and used to infect the human esophageal squamous-cell carcinoma cell lines. The infection efficiency was evaluated with fluorescence microscope and flow cytometry and stable cell lines were constructed using puromycin selection assay. Real-time PCR and Western Blotting were used to confirm the silencing of PHF8. Research on the effect of PHF8 on cell proliferation was performed using MTS asssy. Results We successfully prepared the recombinant lentivirus for RNA interference of PHF8 gene. The expression of PHF8 mRNA and protein in expremental group were lower than that of the control group (P<0.05). The proliferation of expremental group cells were reduced than that of control group (P<0.05). Conclusion Lentivirus mediated shRNA effectively inhibited the expression of PHF8 in human esophageal squamous-cell carcinoma cell lines and the cell proliferation ability.