Pan X M, Zhang G J, Chen X M, Liang L, Tang N, Wang K
The Key Laboratory of Molecular Biology for Infectious Diseases (Ministry of Education), Chongqing Medical University, Chongqing 400016, China.
Zhonghua Gan Zang Bing Za Zhi. 2019 Sep 20;27(9):687-692. doi: 10.3760/cma.j.issn.1007-3418.2019.09.006.
To construct the recombinant adenoviral containing fructose 1, 6-biphosphatase 1 (FBP1), and to investigate whether FBP1 has effect on autophagy and proliferation in liver cancer cells (HepG2). FBP1 cDNA sequence was amplified by PCR and cloned in adenovirus vector pAdTrack-TO4, and then recombinant adenovirus plasmid pAdTrack-FBP1 was constructed. The recombinant adenovirus plasmid was transfected into HEK293 cells by Lipofectamine 3000. High-titer of recombinant adenovirus AdFBP1 was obtained by packaging and amplification. HepG2 cells were infected with recombinant adenovirus AdFBP1, and the Mock and AdGFP group were set at the same time. Western blot and confocal laser scanning microscopy were used to observe the effect of FBP1 on the level of autophagy in hepatocellular carcinoma cells, and the effect of FBP1on the proliferation was observed by MTS and colony formation assay. A t-test and one-way ANOVA were used to compare the mean between group. A high-titer recombinant adenovirus FBP1 was successfully constructed. Western blot and confocal laser scanning microscopy showed that the level of autophagy in AdFBP1 group was significantly lower than that in AdGFP group. Western blot results showed that LC3-II protein expression level in AdGFP was 1.10 ± 0.10 and 0.30 ± 0.01 in AdFBP1 group, = 90.36, < 0.01. Confocal laser scanning microscopy analysis showed that the average number of autophages in AdGFP was 28.33 ± 1.53 and 12.33 ± 1.53 in AdFBP1group, = 97.40, < 0.01. In addition, the results of colony formation assay and MTS assay showed that the proliferation of liver cancer cells in the AdFBP1 group was significantly inhibited compared with the AdGFP group. The results of colony formation showed that the cell clones in the AdGFP group was 65.66 ± 2.57 and 34.00 ± 2.00 in AdFBP1 group, = 141.50, < 0.01. MTS results showed that the absorbance of AdGFP group at 96h was 39.13 ± 2.21 and 30.61 ± 3.33 in AdFBP1 group, = 7.80, < 0.05. FBP1 inhibited the autophagy and proliferation in liver cancer cells (HepG2).
构建含果糖1,6 -二磷酸酶1（FBP1）的重组腺病毒，并研究FBP1对肝癌细胞（HepG2）自噬和增殖的影响。通过PCR扩增FBP1 cDNA序列并克隆至腺病毒载体pAdTrack - TO4，构建重组腺病毒质粒pAdTrack - FBP1。用Lipofectamine 3000将重组腺病毒质粒转染至HEK293细胞。通过包装和扩增获得高滴度的重组腺病毒AdFBP1。用重组腺病毒AdFBP1感染HepG2细胞，同时设置Mock和AdGFP组。采用蛋白质免疫印迹法和共聚焦激光扫描显微镜观察FBP1对肝癌细胞自噬水平的影响，通过MTS法和集落形成试验观察FBP1对增殖的影响。采用t检验和单因素方差分析比较组间均值。成功构建了高滴度的重组腺病毒FBP1。蛋白质免疫印迹法和共聚焦激光扫描显微镜显示，AdFBP1组的自噬水平显著低于AdGFP组。蛋白质免疫印迹结果显示，AdGFP组LC3 - II蛋白表达水平为1.10±0.10，AdFBP1组为0.30±0.01，F = 90.36，P < 0.01。共聚焦激光扫描显微镜分析显示，AdGFP组自噬体平均数量为[28.33±1.53]，AdFBP1组为12.33±1.53，F = 97.40，P < 0.01。此外，集落形成试验和MTS试验结果显示，与AdGFP组相比，AdFBP1组肝癌细胞的增殖受到显著抑制。集落形成结果显示，AdGFP组细胞克隆数为65.66±2.57，AdFBP1组为34.00±2.00，F = 141.50，P < 0.01。MTS结果显示，AdGFP组96小时吸光度为39.13±2.21，AdFBP1组为30.61±3.33，F = 7.80，P < 0.05。FBP1抑制肝癌细胞（HepG2）的自噬和增殖。 （注：原文中部分数据未给出完整的统计量符号，翻译时补充了常见的统计量符号如F、P等，以符合医学统计表达习惯）
