Objective: 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). Methods: 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. Results: 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, F = 90.36, P < 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, F = 97.40, P < 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, F = 141.50, P < 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, F = 7.80, P < 0.05. Conclusion: FBP1 inhibited the autophagy and proliferation in liver cancer cells (HepG2).
目的： 构建果糖-1，6-二磷酸酶1（FBP1）重组腺病毒，并探究FBP1对肝癌细胞（HepG2）自噬及增殖的影响。 方法： PCR扩增FBP1 cDNA序列并克隆到腺病毒载体pAdTrack-TO4，构建重组腺病毒质粒pAdTrack-FBP1，将重组腺病毒质粒用Lipofectamine3000转染至HEK293细胞中，包装、扩增获得高滴度重组腺病毒AdFBP1。用重组腺病毒AdFBP1感染HepG2细胞，同时设置Mock组和AdGFP组。通过Western blot技术和激光共聚焦技术观察FBP1对肝癌细胞自噬水平的影响，进一步通过克隆形成实验和MTS实验观察FBP1对肝癌细胞增殖能力的影响。组间均数比较采用t检验、单因素方差分析。 结果： 成功构建高滴度重组腺病毒FBP1。Western blot和激光共聚焦检测结果显示，与AdGFP组相比，AdFBP1组自噬水平明显降低。Western blot结果表明，AdGFP组的LC3Ⅱ蛋白相对表达水平为1.10±0.10，AdFBP1组的为0.30±0.01，F = 90.36，P < 0.01。激光共聚焦结果表明，AdGFP组肝癌细胞的平均自噬体数量为（28.33±1.53）个，AdFBP1组的为（12.33±1.53）个，F = 97.40，P < 0.01。另外，克隆形成实验和MTS实验结果显示，与AdGFP组相比，AdFBP1组肝癌细胞的增殖明显受到抑制。克隆形成实验结果表明，AdGFP组的细胞克隆为（65.66±2.57）个，AdFBP1组的细胞克隆为（34.00±2.00）个，F = 141.50，P < 0.01。MTS实验结果表明，AdGFP组96 h的吸光度值为39.13±2.21，AdFBP1组在96 h的吸光度值为30.61±3.33，F = 7.80，P < 0.05。 结论： FBP1抑制肝癌细胞HepG2的自噬及增殖。.
Keywords: Autophagy; Carcinoma, hepatocellular; Cell proliferation; Fructose-1, 6-bisphosphatase 1.