Li Sha-sha, Sheng Jun, Yuan Zhan-na, Wang Xiu-chao, Zhao Tian-suo, Ren He, Hao Ji-hui
Department of Pancreatic Oncology, Tianjin Medical University, Tianjin, China.
Zhonghua Yi Xue Za Zhi. 2012 Sep 11;92(34):2386-8.
To discussion the in vitro molecular mechanism of leptin promoting the expression of hTERT in breast cancer cells.
The hTERT mRNA expression of STAT3 knockdown on leptin-induced hTERT was measured by reverse transcription-polymerase chain reaction (RT-PCR). Determine the expression of hTERT protein after different treatments in MCF7 by Western blot. Chromatin immunoprecipitation assay (ChIP) was performed to detect the binding of STAT3 to hTERT promoter in MCF7. Luciferase assay was used to confirm the effects of leptin and STAT3 phosphorylation inhibitor on the transcriptional activity of hTERT promoter.
The RT-PCR analysis showed that knockdown of STAT3 significantly reduced the leptin-induced transcription of hTERT. Western blot showed that the expression of hTERT were 3.109 ± 0.051 and 1.025 ± 0.031 after leptin or both of leptin and AG490 treatments. The results of CHIP showed that the mRNA of control and leptin (160 ng/ml) treatment were 1 and 3.311 ± 0.017. Leptin increased the combination of STAT3 and hTERT promoter. Luciferase assay showed that when the concentration of leptin was 160 ng/ml, the hTERT promoter activity was 80.98 ± 0.18 while the control was 20.76 ± 0.31. After AG490 treatment, the hTERT promoter activity was 18.65 ± 0.32,significantly reduced the leptin-induced activity of hTERT promoter.
Leptin/STAT3 signaling is a novel pathway for the up-regulation of hTERT expression in breast cancer cells.
探讨瘦素促进乳腺癌细胞中hTERT表达的体外分子机制。
采用逆转录-聚合酶链反应（RT-PCR）检测STAT3基因敲低对瘦素诱导的hTERT mRNA表达的影响。通过蛋白质免疫印迹法检测不同处理后MCF7细胞中hTERT蛋白的表达。进行染色质免疫沉淀实验（ChIP）以检测MCF7细胞中STAT3与hTERT启动子的结合情况。利用荧光素酶报告基因检测法确认瘦素和STAT3磷酸化抑制剂对hTERT启动子转录活性的影响。
RT-PCR分析显示，敲低STAT3可显著降低瘦素诱导的hTERT转录。蛋白质免疫印迹法显示，瘦素处理或瘦素与AG490联合处理后，hTERT的表达分别为3.109±0.051和1.025±0.031。ChIP结果显示，对照组和瘦素（160 ng/ml）处理组的mRNA分别为1和3.311±0.017。瘦素增加了STAT3与hTERT启动子的结合。荧光素酶报告基因检测法显示，当瘦素浓度为160 ng/ml时，hTERT启动子活性为80.98±0.18，而对照组为20.76±0.31。AG490处理后，hTERT启动子活性为18.65±0.32，显著降低了瘦素诱导的hTERT启动子活性。
瘦素/STAT3信号通路是乳腺癌细胞中上调hTERT表达的新途径。
