LncRNA RNU12上调热休克蛋白72抑制重组人TRAIL蛋白诱导肝癌细胞凋亡

LncRNA RNU12 inhibits apoptosis of hepatocellular carcinoma cells induced by recombinant human TRAIL protein through up-regulating heat shock protein 72

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 inhibiting the apoptosis of hepatocellular carcinoma （HCC） ceils induced by recombinant human tumor necrosis factor-related apoptosis inducing ligand （TRAIL） protein at 46 -50 ℃. Methods A sub-lethal heat treatment model of HCC cells was established. The Annexin-V/PI staining was used to detect the effect of treatment of recombinant human TRAIL on the apoptotic rate of HCC cells under different temperatures （43, 46 and 50℃, and 37℃ as control temperature） for 10 min. According to the expression profile from the Agilent human lncRNA ＋ mRNA Array V4.0 microarray data in HCC cells in our previous study, 50 ℃ specific highly expressed LncRNA RNU12 was selected as the research object. Lentiviral vector LncRNA RNU12 was constructed, and then transfected into MHCC97-H cells. The expression of heat shock protein 72 （HSP72） at mRNA and protein levels were measured in different temperature treatment groups before and after transfection by qRT-PCR and Western blotting. Meanwhile, flow cytometry was used to detect the effect of lentiviral vector LncRNA RNU12 on the apoptosis of MHCC97-H cells induced by TRAIL protein. The fluorescence in situ hybridization （FISH） was used to detect the sub-cellular localization of LncRNA RNU12 in HCC cells. Results Temperature at 43 ℃ promoted recombinant human TRAIL protein to induce the apoptosis in HCC cells, but it exerted opposite effect at 46 - 50℃. The mRNA level of HSP72 was significantly decreased in all 4 treatment groups after lentiviral vector infection （P 〈 0.01 ）. And TRAIL protein levels were obviously lower at the 37, 46 and 50 ℃ groups than before lentivirus-mediated interference （P 〈 0.05 ）. After the lentiviral vector infection, the apoptosis of MHCC97-H cells induced by TRAIL protein were increased by 7.36% （P 〈 0.01 ） and 8.84% （P 〈 0.01）, respectively at the 46 ℃and 50℃ groups. RNA-FISH showed that LncRNA RNU12 was mainly expressed in the nucleus of MHCC97-H cells at 50℃. Conclusion LncRNA RNU12 up- regulating HSP72 at transcriptional level may be one of the molecular mechanisms of inhibiting the apoptosis of HCC cells induced by recombinant human TRAIL protein at 46 -50℃.