Knockdown of HMGB1 Suppresses Hypoxia-Induced Mitochondrial Biogenesis in Pancreatic Cancer Cells
Knockdown of HMGB1 Suppresses Hypoxia-Induced Mitochondrial Biogenesis in Pancreatic Cancer Cells
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DOI：
10.2147/ott.s234530
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发表时间：
2020-02
影响因子：
4
通讯作者：
Liangchun Yang;Fanghua Ye;L. Zeng;Yanling Li;W. Chai
中科院分区：
文献类型：
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作者：
Liangchun Yang;Fanghua Ye;L. Zeng;Yanling Li;W. Chai
目的探讨高迁移率族蛋白1（HMGB 1）对缺氧诱导的胰腺癌PANC 1/CFPAC 1细胞线粒体生物合成的调控作用。方法利用慢病毒介导的RNAi技术下调HMGB 1的表达，观察HMGB 1基因敲减对缺氧诱导的线粒体生物合成的影响。采用定量逆转录聚合酶链反应（qRT-PCR）、Western blot、线粒体DNA和ATP检测试剂盒和电镜分别检测缺氧处理后胰腺癌细胞NRF-1/TFAM表达、线粒体DNA拷贝数、ATP含量和线粒体数量/形态。通过MTS测定来测量细胞增殖。采用Western blot、免疫共沉淀（IP）和SIRT 1活性试剂盒检测PGC-1α蛋白和乙酰化水平及SIRT 1活性。结果缺氧诱导胰腺癌细胞NRF-1/TFAM表达增强，线粒体DNA拷贝数和ATP含量增加，线粒体数量增加，而HMGB 1的敲低则抑制了NRF-1/TFAM的表达。敲低HMGB 1表达可降低缺氧诱导的PGC-1α/SIRT 1的表达和活性以及AMPK的磷酸化。通过质粒转染过表达PGC-1α未能增加HMGB 1敲低细胞中mtDNA拷贝数或ATP含量。敲低HMGB 1可减弱缺氧诱导的NRF-1、TFAM、PGC-1α、SIRT 1和复合物Ⅰ、Ⅲ蛋白的表达，并降低PGC-1α/SIRT 1活性的乙酰化水平。此外，SRT 1720（一种SIRT 1激活剂）诱导的SIRT 1活性升高促进了缺氧诱导的PGC-1α脱乙酰化，但HMGB 1敲减细胞除外。结论缺氧条件下HMGB 1通过AMPK/SIRT 1途径调控线粒体生物合成，可能成为胰腺癌治疗的潜在药物靶点。
Purpose To explore the regulatory effect of HMGB1 upon hypoxia-induced mitochondrial biogenesis in pancreatic cancer PANC1/CFPAC1 cells. Methods After a down-regulation of HMGB1 expression by lentivirus-mediated RNAi, the effect of knocking down HMGB1 on hypoxia-induced mitochondrial biogenesis was examined. NRF-1/TFAM expression, mtDNA copy number, ATP content and mitochondrial number/morphology in hypoxia-treated pancreatic cancer cells were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot, mtDNA and ATP assay kits and electron microscopy, respectively. Cell proliferation was measured by MTS assay. And protein and acetylation levels of PGC-1α and SIRT1 activity were detected by Western blot, immunoprecipitation (IP) and SIRT1 activity kit. Results Hypoxia enhanced the expressions of NRF-1/TFAM, boosted mtDNA copy number and ATP content and increased the number of mitochondria in pancreatic cancer cells while induction was suppressed by a knockdown of HMGB1. Knocking down HMGB1 expression lowered hypoxia-induced PGC-1α/SIRT1 expression and activity, phosphorylation of AMPK. PGC-1α over-expression by a plasmid transfection failed to boost mtDNA copy number or ATP content in HMGB1-knockdown cells. A knockdown of HMGB1 attenuated hypoxia with AICAR (an AMPK activator)-induced expression of NRF-1, TFAM, PGC-1α, SIRT1 and the proteins of complexes Ⅰ& Ⅲ and reduced the acetylation level of PGC-1α/SIRT1 activity. Additionally, SRT1720 (a SIRT1 activator)-induced elevation in SIRT1 activity boosted hypoxia-induced PGC-1α deacetylation, except in HMGB1-knockdown cells. Conclusion As a novel regulator of mitochondrial biogenesis via AMPK/SIRT1 pathway under hypoxia, HMGB1 may become a potential drug target for therapeutic interventions in pancreatic cancer.