Interferon-inducible IFI16, a negative regulator of cell growth, down-regulates expression of human telomerase reverse transcriptase (hTERT) gene.
Interferon-inducible IFI16, a negative regulator of cell growth, down-regulates expression of human telomerase reverse transcriptase (hTERT) gene.
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DOI：
10.1371/journal.pone.0008569
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发表时间：
2010-01-05
期刊：
影响因子：
3.7
通讯作者：
Choubey D
中科院分区：
文献类型：
--
作者：
Song LL;Ponomareva L;Shen H;Duan X;Alimirah F;Choubey D
人正常前列腺上皮细胞和二倍体成纤维细胞（HDF）中干扰素（IFN）诱导的IFI 16蛋白（由位于1 q22的IFI 16基因编码）水平升高与细胞衰老的发生相关。然而，IFI 16蛋白促进细胞衰老相关细胞生长停滞的分子机制仍有待阐明。在这里，我们报告说，增加IFI 16蛋白在正常的HDFs和HeLa细胞的水平负调节人端粒酶逆转录酶（hTERT）基因的表达。我们优化了实时PCR，免疫印迹和端粒重复扩增协议（TRAP）测定的条件，以检测相对较低水平的hTERT mRNA，蛋白质和端粒酶活性，发现在HDFs。使用优化的条件下，我们报告说，治疗HDFs与细胞周期进程的抑制剂，如阿非迪霉素或CGK 1026，这导致稳态水平降低IFI 16 mRNA和蛋白，与hTERTmRNA和蛋白水平和端粒酶活性的增加。相反，细胞中IFI 16表达的敲低增加了c-Myc的表达，c-Myc是hTERT表达的正调节因子。此外，IFI 16蛋白在细胞中的过表达抑制了c-Myc介导的对hTERT-luc-reporter活性的刺激，并降低了c-Myc和hTERT的稳态水平。这些数据表明，在HDFs中IFI 16蛋白水平的增加下调hTERT基因的表达。我们的观察将为理解IFI 16蛋白的细胞水平增加如何导致某些衰老依赖性疾病奠定基础。
Increased levels of interferon (IFN)-inducible IFI16 protein (encoded by the IFI16 gene located at 1q22) in human normal prostate epithelial cells and diploid fibroblasts (HDFs) are associated with the onset of cellular senescence. However, the molecular mechanisms by which the IFI16 protein contributes to cellular senescence-associated cell growth arrest remain to be elucidated. Here, we report that increased levels of IFI16 protein in normal HDFs and in HeLa cells negatively regulate the expression of human telomerase reverse transcriptase (hTERT) gene. We optimized conditions for real-time PCR, immunoblotting, and telomere repeat amplification protocol (TRAP) assays to detect relatively low levels of hTERT mRNA, protein, and telomerase activity that are found in HDFs. Using the optimized conditions, we report that treatment of HDFs with inhibitors of cell cycle progression, such as aphidicolin or CGK1026, which resulted in reduced steady-state levels of IFI16 mRNA and protein, was associated with increases in hTERT mRNA and protein levels and telomerase activity. In contrast, knockdown of IFI16 expression in cells increased the expression of c-Myc, a positive regulator of hTERT expression. Additionally, over-expression of IFI16 protein in cells inhibited the c-Myc-mediated stimulation of the activity of hTERT-luc-reporter and reduced the steady-state levels of c-Myc and hTERT. These data demonstrated that increased levels of IFI16 protein in HDFs down-regulate the expression of hTERT gene. Our observations will serve basis to understand how increased cellular levels of the IFI16 protein may contribute to certain aging-dependent diseases.