Yang Jing-Hua, Li Xiao-Yan, Wang Xin, Hou Wei-Jian, Qiu Xue-Shan, Wang En-Hua, Wu Guang-Ping
Department of Pathology, The First Affiliated Hospital and College of Basic Medical Sciences, China Medical University, Shenyang, China.
Department of Pathology, Cancer Hospital of China Medical University, Shenyang, China.
PLoS One. 2017 Aug 16;12(8):e0182775. doi: 10.1371/journal.pone.0182775. eCollection 2017.
HPV 16 E6 upregulates hTERT expression in lung cancer cells. However, the underlying molecular mechanism is unclear. In this paper, E6, LKB1, SP1, and hTERT mRNA expression levels were detected in brushing cells of patients with lung cancer (n = 106) and with benign lung disease (n = 68) by qRT-PCR. The mRNA expression levels of E6, SP1, and hTERT were significantly increased in the malignant group compared with the benign group (P < 0.01). Conversely, the mRNA expression level of LKB1 was significantly decreased in the malignant group (P < 0.01). Furthermore, the correlation between E6, Sp1, hTERT, and LKB1 was performed, our results indicated that E6, Sp1, and hTERT with positive, but LKB1 with negative correlation (P < 0.01). To investigate the potential relationship between these genes, using double directional genetic manipulation, we showed that overexpression of E6 in H1299 cells down-regulated LKB1 mRNA and protein expression but up-regulated SP1 and hTERT as well as the transcriptional activity of Sp1. In contrast, knockdown of E6 in A549 cells by short-interference RNAs (siRNAs) up-regulated LKB1 expression, but down-regulated SP1 and hTERT expression as well as Sp1 activity. LKB1 loss upregulated both SP1 and hTERT at the protein and mRNA level as well as SP1 activity. To verify that the role of E6 on hTERT was mediated by SP1, siRNA knockdown of SP1 was performed on both H1299 and A549 cell lines. Inhibition of SP1 downregulated hTERT expression. Our results indicate that HPV16 E6 indirectly upregulated the expression of hTERT by inhibition of LBK1 expression and upregulation of Sp1 expression, thus suggesting a HPV-LKB1-SP1-hTERT axis for the tumorigenesis of lung cancer. Our study also provides new evidence to support the critical role of SP1 and LKB1 in the pathogenesis of HPV-related lung cancer, and suggests novel therapeutic targets.
人乳头瘤病毒16型E6蛋白上调肺癌细胞中的端粒酶逆转录酶（hTERT）表达。然而，其潜在的分子机制尚不清楚。在本文中，通过定量逆转录聚合酶链反应（qRT-PCR）检测了106例肺癌患者和68例良性肺病患者的刷检细胞中E6、肝脏激酶B1（LKB1）、特异性蛋白1（SP1）和hTERT的mRNA表达水平。与良性组相比，恶性组中E6、SP1和hTERT的mRNA表达水平显著升高（P<0.01）。相反，恶性组中LKB1的mRNA表达水平显著降低（P<0.01）。此外，对E6、Sp1、hTERT和LKB1之间的相关性进行分析，结果表明E6、Sp1和hTERT呈正相关，而LKB1呈负相关（P<0.01）。为了研究这些基因之间的潜在关系，我们采用双向基因操作，结果显示在H1299细胞中过表达E6会下调LKB1的mRNA和蛋白表达，但上调SP1和hTERT以及Sp1的转录活性。相反，通过小干扰RNA（siRNA）敲低A549细胞中的E6会上调LKB1的表达，但下调SP1和hTERT的表达以及Sp1的活性。LKB1缺失在蛋白和mRNA水平以及Sp1活性上均上调了SP1和hTERT。为了验证E6对hTERT的作用是由SP1介导的，我们在H1299和A549细胞系中对SP1进行了siRNA敲低。抑制SP1会下调hTERT的表达。我们的结果表明，人乳头瘤病毒16型E6蛋白通过抑制LBK1表达和上调Sp1表达间接上调hTERT的表达，从而提示了一条HPV-LKB1-SP1-hTERT轴参与肺癌的肿瘤发生。我们的研究还为支持SP1和LKB1在人乳头瘤病毒相关肺癌发病机制中的关键作用提供了新证据，并提示了新的治疗靶点。
