乙型肝炎病毒上调的lnc-HUR1抑制肝癌细胞凋亡

HBV-upregulated Lnc-HUR1 inhibits the apoptosis of liver cancer cells

摘要:

Lnc-HUR1是乙型肝炎病毒(hepatitis B virus,HBV)上调的长链非编码RNA，具有促进肝癌细胞增殖和促进肝癌发生发展的功能。为探究lnc-HUR1对肝癌细胞凋亡的影响，通过免疫印迹、实时荧光定量PCR、双荧光素酶报告基因、免疫共沉淀、流式细胞术等实验方法，检测lnc-HUR1对肝癌细胞凋亡的影响。转化生长因子-β(tansforming growth factor-β,TGF-β)、五氟尿嘧啶和星孢菌素诱导肝癌细胞凋亡的实验结果显示，过表达lnc-HUR1能够显著降低caspase3/7的活性以及PARP-1的剪切，而敲低lnc-HUR1则能够显著增加caspase3/7的活性，促进PARP-1的剪切；Annexin-Ⅴ和PI联合染色实验也表明过表达lnc-HUR1能够抑制细胞凋亡，敲低lnc-HUR1能够促进细胞的凋亡。同时过表达lnc-HUR1能够在RNA水平和蛋白水平上调凋亡抑制因子Bcl-2(B cell lymphoma-2)、下调促凋亡因子BAX (B cell lymphoma-2-associated x)，从而抑制细胞的凋亡。在CCL4诱导的小鼠急性肝损伤模型中，lnc-HUR1转基因小鼠肝组织中Bcl-2的表达高于对照小鼠。染色质免疫共沉淀(chromatin immunoprecipitation,ChIP)实验也证实lnc-HUR1能够降低p53在Bcl-2和BAX启动子区的富集。以上结果说明，lnc-HUR1通过促进凋亡抑制因子Bcl-2及抑制凋亡促进因子BAX的表达抑制肝癌细胞的凋亡。进一步的实验表明，在HCT116细胞中lnc-HUR1能够调控Bcl-2和BAX的转录，而在HCT116 p53^−/−细胞中，lnc-HUR1对Bcl-2和BAX的表达没有影响，说明lnc-HUR1对肝癌细胞凋亡的抑制作用依赖于p53的活性。综上所述，HBV上调的lnc-HUR1具有抑制肝癌细胞凋亡的作用，lnc-HUR1通过抑制p53转录活性，上调凋亡抑制因子Bcl-2、抑制凋亡促进因子BAX的转录，从而抑制细胞凋亡。上述结果提示Lnc-HUR1在HBV相关肝癌发生发展中发挥重要作用。

Abstract:

Lnc-HUR1 is an HBV-related long non-coding RNA, which can promote the proliferation of hepatoma cells and the occurrence and development of liver cancer. In this study we explored the effect of lnc-HUR1 on the apoptosis of hepatocellular carcinoma cells by taking the approach of immunoblotting, quantitative real time PCR, luciferase reporter assay, chromatin immunoprecipitation (ChIP) and flow cytometry. We found that overexpression of lnc-HUR1 significantly reduced the activity of caspase3/7 and the cleavage of PARP-1, while knocking down of lnc-HUR1 significantly increased the activity of caspase3/7 and promoted the cleavage of PARP-1 in HepG2 cells treated with TGF-β, pentafluorouracil or staurosporine. Consistently, the data from Annexin-V/PI staining showed that overexpression of lnc-HUR1 inhibited apoptosis, while knockdown of lnc-HUR1 promoted apoptosis. Moreover, overexpression of lnc-HUR1 up-regulated the apoptosis inhibitor Bcl-2 and down-regulated the pro-apoptotic factor BAX at both RNA and protein levels. In the CCL4-induced acute liver injury mice model, the expression of Bcl-2 in the liver tissue of lnc-HUR1 transgenic mice was higher than that of the control mice. The data from ChIP assay indicated that lnc-HUR1 reduced the enrichment of p53 on Bcl-2 and BAX promoters. All these results indicated that lnc-HUR1 inhibited the apoptosis by promoting the expression of apoptosis inhibitor Bcl-2 and inhibiting the expression of apoptosis promoting factor BAX. Further studies showed that lnc-HUR1 regulated the transcription of Bcl-2 and BAX in HCT116 cells, but had no effect on the expression of Bcl-2 and BAX in HCT116 p53^−/− cells, indicating that lnc-HUR1 regulates the transcription of Bcl-2 and BAX dependent upon the activity of p53. In conclusion, HBV upregulated lnc-HUR1 can inhibit the apoptosis of hepatoma cells. Lnc-HUR1 inhibits apoptosis by inhibiting the transcriptional activity of p53. These results suggest that lnc-HUR1 plays an important role in the occurrence and development of HBV-related hepatocellular carcinoma.