lincRNA ULK4P2对肝癌细胞生物学行为的影响

Role of lincRNA ULK4P2 in biological behavior of hepatocellular carcinoma cells

目的通过干扰肝癌细胞株中长链基因间非编码RNA ULK4P2(lincRNA ULK4P2的表达,初步观察lincRNA ULK4P2对肝癌细胞生物学行为的影响。方法运用实时荧光定量聚合酶链反应(RT-qPCR)检测lincRNA ULK4P2在不同肝癌细胞株和正常肝细胞株中的表达,根据RT-qPCR检测结果,选择肝癌细胞株HepG2进行后续干扰实验。将针对lincRNA ULK4P2的小干扰RNA(siRNA)序列,包括siRNA-1、siRNA-2、siRNA-3、siRNA-mock(空白对照)、siRNA-Scramble(将目的 siRNA序列打乱后重新组合所得的阴性对照)转染HepG2细胞,根据转染序列siRNA后对HepG2细胞lincRNA ULK4P2的干扰效果,选择siRNA-2进行之后的功能检测。应用siRNA下调HepG2细胞中lincRNA ULK4P2的表达,运用CCK-8技术研究lincRNA ULK4P2表达下降对肝癌细胞增殖情况的影响,运用流式细胞仪检测lincRNA ULK4P2表达下降对肝癌细胞凋亡和细胞周期分布的影响。对CCK-8实验中siRNA-2组、siRNA-mock组和siRNA-Scramble组HepG2细胞增殖活性(吸光度)的比较采用单因素方差分析。对分别转染siRNA-2、siRNA-mock、siRNA-Scramble的HepG2细胞通过流式细胞仪检测所得细胞周期分布情况,采用单因素方差分析。结果 CCK-8实验显示,与siRNA-mock组和siRNA-Scramble组比较,siRNA-2组HepG2细胞在转染后72 h增殖活性明显下降,差异具有统计学意义(P <0.05)。下调HepG2细胞lincRNA ULK4P2的表达后,细胞各个周期时相中细胞数目无明显变化;siRNA-2组HepG2细胞中G2/M期细胞的比例(43.92%)相对于siRNA-mock组(7.81%)和siRNA-Scramble组(8.21%)明显上升,差异具有统计学意义(P<0.05)。结论 lincRNA ULK4P2可能参与了肝癌细胞增殖和细胞周期的分子调控过程。

Objective To investigate the role of the long-strand intergenic non-coding RNA(lincRNA)ULK4P2 in the biological behavior of hepatocellular carcinoma cells by interfering with the expression of lincRNA ULK4P2 in hepatocellular carcinoma cell lines.Methods Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of lincRNA ULK4P2 in different hepatoma cell lines and normal cells.According to the results of RT-qPCR,hepatocellular carcinoma cell line HepG2 was selected for subsequent interference experiments.Small interfering RNA(siRNA)targeting lincRNA ULK4P2,including siRNA-1,siRNA-2,and siRNA-3,as well as siRNA-mock(blank control)and siRNA-Scramble(reconstructed after shuffling the siRNA sequence of interest;negative control)were transfected into HepG2 cells,and siRNA-2 was selected for subsequent experiments according to the interference effect on lincRNA ULK4P2.The expression of lincRNA ULK4P2 in HepG2 cells was downregulated by siRNA-mediated interference.The role of lincRNA ULK4P2 expression in the proliferation of hepatoma cells was studied by CCK-8 assay.Flow cytometry was used to detect the expression of lincRNA ULK4P2 and the influence on apoptosis and cell cycle distribution of liver cancer cells.One-way analysis of variance was used to compare the proliferation activity(absorbance)and cell cycle distribution of HepG2 cells transfected with siRNA-2,siRNA-mock,or siRNA-Scramble.Results Compared with the siRNA-mock group and siRNA-Scramble group,the proliferation of hepatocellular carcinoma cells was significantly inhibited after the down-regulation of lincRNA ULK4P2 in the siRNA-2 group(P<0.05).After downregulating the expression of lincRNA ULK4P2 in HepG2 cells,there was no significant change in the number of cells in each phase.The proportion of G2/M phase cells in the siRNA-2 group(43.92%)was significantly higher than that in the siRNA-mock group(7.81%)and siRNA-Scramble group(8.21%)(P<0.05).Conclusion Our findings indicate that lincRNA ULK4P2 may be involved in the proliferation of hepatocellular carcinoma cells.