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目的探讨46~50℃时抑制重组人肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-related apoptosis inducing ligand,TRAIL)蛋白促肝癌细胞凋亡相关分子机制。方法建立肝癌细胞亚致死性热温度模型,在不同温度(43、46、50℃)条件下分别处理10 min,Annexin-V/PI染色检测重组人TRAIL蛋白对肝癌细胞凋亡率的影响,以常温37℃作为对照组。根据前期肝癌细胞Agilent Human lnc RNA+mRNA Array V4.0芯片表达谱数据,选定50℃时特异性高表达的LncRNA RNU12作为研究对象。构建LncRNA RNU12-sh RNA慢病毒干扰载体,转染MHCC97-H细胞。qRT-PCR及Western blot检测感染前后不同温度组中热休克蛋白72(heat shock protein 72,HSP72)mRNA和蛋白的表达变化。利用流式细胞技术检测LncRNA RNU12慢病毒干扰载体对TRAIL蛋白诱导细胞凋亡的影响。采用荧光原位杂交(FISH)技术检测LncRNA RNU12亚细胞定位情况。结果温度在43℃时促进重组人TRAIL蛋白诱导肝癌细胞凋亡的功能,46~50℃时抑制重组人TRAIL蛋白诱导肝癌细胞凋亡的功能。HSP72 mRNA在4个温度组中的表达均较干扰前明显降低(P<0.01);在蛋白水平,37、46、50℃组较干扰前显著降低(P<0.05)。转染后,46、50℃条件下TRAIL蛋白诱导MHCC97-H细胞凋亡分别上升7.36%、8.84%(P<0.01)。利用RNA-FISH技术确定50℃时LncRNA RNU12主要在MHCC97-H细胞细胞核中表达。结论 LncRNA RNU12在转录水平上调HSP72表达可能是46~50℃时抑制重组人TRAIL蛋白促肝癌细胞凋亡分子机制之一。
OBJECTIVE: To investigate the molecular mechanism of apoptosis induced by recombinant human tumor necrosis factor-related apoptosis inducing ligand (TRAIL) in hepatocellular carcinoma at 46-50 ℃. Methods The sub-lethal thermal temperature model of hepatoma cells was established and treated with different concentrations of temperature (43, 46, 50 ℃) for 10 min respectively. The effect of recombinant human TRAIL on the apoptosis of hepatocellular carcinoma cells was evaluated by Annexin-V / PI staining. 37 ℃ room temperature as a control group. According to the data of Agilent Human lnc RNA + mRNA Array V4.0 chip, the LncRNA RNU12 with high specificity at 50 ℃ was selected as the research object. LncRNA RNU12-sh RNA lentiviral vector was constructed and transfected into MHCC97-H cells. qRT-PCR and Western blot were used to detect the expression of heat shock protein 72 (HSP72) mRNA and protein in different temperature groups before and after infection. The effect of LncRNA RNU12 lentiviral vector on apoptosis induced by TRAIL protein was detected by flow cytometry. Fluorescence in situ hybridization (FISH) was used to detect subcellular localization of LncRNA RNU12. Results The function of recombinant human TRAIL protein in inducing hepatocellular carcinoma cell apoptosis was promoted when the temperature was 43 ℃, and the function of recombinant human TRAIL protein induced apoptosis was inhibited at 46-50 ℃. The expression of HSP72 mRNA in the four temperature groups was significantly lower than that before the disturbance (P <0.01). At the protein level, the expression levels of HSP72 mRNA in the four temperature groups were significantly lower than those before the disturbance (P <0.05). After transfection, the apoptosis of MHCC97-H cells induced by TRAIL protein increased by 7.36% and 8.84% (P <0.01) at 46 and 50 ℃, respectively. The results of RNA-FISH showed that LncRNA RNU12 was mainly expressed in the nucleus of MHCC97-H cells at 50 ℃. Conclusions Upregulation of HSP72 expression by LncRNA RNU12 may be one of the molecular mechanisms that inhibit the apoptosis of human hepatoma cells induced by recombinant human TRAIL at 46-50 ℃.