FAM83B对肝癌细胞的作用及机制研究

Effect of FAM83B on hepatocellular carcinoma cells and its mechanism

摘要:

目的 探讨具有序列相似性的家族83成员B（FAM83B）对肝癌细胞增殖和侵袭能力的影响及其机制。方法 采用实时荧光定量聚合酶链反应（qRT-PCR）、Western blotting、免疫组织化学法检测肝癌组织、癌旁正常组织、人类肝癌细胞系（HepG2、Hep3B）、正常人类肝细胞系（LO2）中FAM83B mRNA和蛋白的表达。采用靶向siRNA敲低HepG2细胞系中FAM83B作为si-FAM83B组，HepG2细胞转染si-NC慢病毒载体作为si-NC组。分别用PBS、40 ng/mL胰岛素样生长因子1（IGF-1）处理si-FAM83B组细胞48 h，并将细胞分为si-FAM83B+PBS组和si-FAM83B+IGF-1组，分析敲低FAM83B对肝癌细胞增殖、侵袭、细胞周期和凋亡的影响，并研究敲低FAM83B对磷脂酰肌醇3-激酶/蛋白激酶B/雷帕霉素哺乳动物靶蛋白（PI3K/Akt/mTOR）信号通路的影响。结果 肝癌组织FAM83B mRNA和蛋白相对表达量高于癌旁正常组织（P <0.05）。HepG2、Hep3B细胞FAM83B mRNA和蛋白相对表达量高于LO2细胞（P <0.05）。si-FAM83B组FAM83B mRNA和蛋白相对表达量低于si-NC组（P <0.05）。si-NC组与si-FAM83B组24 h、48 h、72 h、96 h的OD值比较，采用重复测量设计的方差分析，结果 ①不同时间点OD值有差异（F =773.510，P =0.001）；②si-NC组与si-FAM83B组的OD值有差异（F =516.980，P =0.000），si-FAM83B组OD值较低；③两组OD值变化趋势有差异（F =820.782，P =0.000）。si-FAM83B组细胞凋亡率高于si-NC组，侵袭细胞数低于si-NC组（P <0.05）。si-FAM83B组PI3K、p-Akt、p-mTOR蛋白相对表达量低于si-NC组（P <0.05），LC3-Ⅱ高于si-NC组（P <0.05）。si-NC组与si-FAM83B组Akt、mTOR蛋白相对表达量比较，差异均无统计学意义（P >0.05）。si-FAM83B+IGF-1组PI3K、p-Akt和p-mTOR蛋白相对表达量高于si-FAM83B+PBS组（P <0.05），LC3-Ⅱ低于si-FAM83B+PBS组（P <0.05）。si-FAM83B+PBS组与si-FAM83B+IGF-1组Akt、mTOR蛋白相对表达量比较，差异均无统计学意义（P >0.05）。si-FAM83B+PBS组与si-FAM83B+IGF-1组24 h、48 h、72 h、96 h的OD值比较，采用重复测量设计的方差分析，结果 ①不同时间点OD值有差异（F =5211.626，P =0.000）；②si-FAM83B+PBS组与si-FAM83B+IGF-1组OD值有差异（F =453.499，P =0.000），si-FAM83B+IGF-1组OD值较高；③两组OD值变化趋势有差异（F =384.347，P =0.000）。si-FAM83B+IGF-1组细胞凋亡率低于si-FAM83B+PBS组（P <0.05），侵袭细胞数高于si-FAM83B+PBS组（P <0.05）。结论 敲低FAM83B可通过沉默PI3K/Akt/mTOR通路抑制肝癌细胞生长，并促进肿瘤细胞自噬。

Abstract:

Objective To explore the effect of family with sequence similarity 83 member B (FAM83B) on the proliferation and invasion of liver cancer cells and its underlying mechanisms.Methods The quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting and immunohistochemistry were used to detect the mRNA and protein expressions of FAM83B in liver cancerous tissues and adjacent tissues, as well as in liver cancer cell lines (HepG2, Hep3B) and normal human hepatocyte cell line (LO2). The targeted knockdown of the expression of FAM83B in liver cancer cell line HepG2 was achieved via small interfering RNA (siRNA), which was set as the si-FAM83B group. The HepG2 cells transfected with control lentiviral vectors were set as the si-NC group. The cells in the si-FAM83B group were treated with PBS and insulin-like growth factor 1 (IGF-1) at a concentration of 40 ng/mL for 48 h, and were set as si-FAM83B + PBS group and si-FAM83B + IGF-1 group, respectively. The effects of the knockdown of the expression of FAM83B on the proliferation, invasion, cell cycle and apoptosis of liver cancer cells, and on the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/ Akt/ mTOR) signaling pathway were analyzed.Results The relative mRNA and protein expressions of FAM83B in liver cancer tissues were significantly higher than those in the adjacent tissues (P < 0.05). Compared with those in the HepG2 and Hep3B cells, the relative mRNA and protein expressions of FAM83B were lower in the LO2 cells (P < 0.05). Besides, the relative mRNA and protein expressions of FAM83B were lower in the si-FAM83B group than those in the si-NC group (P < 0.05). The optical density (OD) values at 24 h, 48 h, 72 h, and 96 h in the si-FAM83B and si-NC groups were compared via repeated measures ANOVA, and the results demonstrated that the OD values were different among the time points (F = 773.510, P = 0.001) and between the groups (F = 516.980, P = 0.000). Specifically, the OD values were lower in the si-FAM83B group. Besides, there were differences in the change trends of OD values between the two groups (F = 820.782, P = 0.000). The apoptosis rate was higher but the number of invasive cells was lower in the si-FAM83B group than in the si-NC group (P < 0.05). Compared with the si-NC group, the protein expressions of PI3K, p-Akt and p-mTOR were lower, while the protein expression of LC3-Ⅱ was higher in the si-FAM83B group (P < 0.05). There was no difference in the relative protein expressions of Akt and mTOR between the si-FAM83B + IGF-1 group and the si-FAM83B + PBS group (P > 0.05). The OD values at 24 h, 48 h, 72 h, and 96 h in the si-FAM83B + IGF-1 and si-FAM83B + PBS groups were compared via repeated measures ANOVA, and the results demonstrated that the OD values were different among the time points (F = 5211.626, P = 0.000) and between the groups (F = 453.499, P = 0.000), where the OD values in the si-FAM83B + IGF-1 group were higher. The change trends of the OD values were also different between the two groups (F = 384.347, P = 0.000). The apoptosis rate in the si-FAM83B + IGF-1 group was lower than that in the si-FAM83B + PBS group (P < 0.05), whereas the number of invasive cells in the si-FAM83B + IGF-1 group was higher than that in the si-FAM83B+PBS group (P < 0.05).Conclusions Knockdown of FAM83B can suppress the growth of liver cancer cells and promote their autophagy by silencing the PI3K/Akt/mTOR signaling pathway.