Department of Internal Medicine, The Ohio State University, Columbus, Ohio.
J Surg Res. 2013 Oct;184(2):855-60. doi: 10.1016/j.jss.2013.04.061. Epub 2013 May 18.
Pancreatic cancer cells exist in a hypoxic microenvironment containing numerous factors that impact tumor survival, proliferation, and metastasis. MicroRNAs (miRs) are differentially expressed in cancer but also altered by hypoxia. We hypothesized that hypoxia could induce expression of miR-21, an oncomir in pancreatic cancer cells.
We examined how hypoxia regulates miR-21 expression in pancreatic cancer cell lines (BxPC-3, AsPC-1) by stem-loop RT-PCR. Chromatin immunoprecipitation assays were used to study how hypoxia alters hypoxia-inducible factor (HIF)-1α binding to the hypoxia response element of miR-21. BxPC-3 and AsPC-1 cells were transfected with a constitutively stable HIF-1α subunit or vector control (pcDNA3.1) to determine the influence of miR-21 in normoxia. The effect of mature miR-21 sense and antisense oligonucleotides on proliferation and apoptosis in hypoxic and normoxic conditions was assessed via WST-1 assay and flow cytometry.
MiR-21 levels increased in all cell lines grown in hypoxic conditions versus normoxia, whereas siRNA targeting HIF-1α reduced miR-21 expression. Hypoxic conditions resulted in direct binding of HIF-1α to the predicted binding site in miR-21. Transfection with a constitutively stable HIF-1α expression plasmid in normoxia resulted in upregulated miR-21, similar to that seen in hypoxia. Cells transfected with antisense constructs targeting miR-21 had reduced proliferation and increased apoptosis in normoxia, whereas miR-21 overexpression abrogated hypoxia-associated reductions in proliferation.
MiR-21 is induced by hypoxia in pancreatic cancer cells via HIF-1α upregulation. MiR-21 overexpression allows cells to avoid apoptosis in a hypoxic microenvironment. Inhibition of miR-21 expression may increase cellular susceptibility to hypoxia in pancreatic cancer.
胰腺癌细胞存在于富含多种影响肿瘤存活、增殖和转移的因素的缺氧微环境中。微小 RNA（miRs）在癌症中表达不同，但也受缺氧影响而改变。我们假设缺氧可诱导胰腺癌细胞中致癌 miRNA-21 的表达。
我们通过茎环 RT-PCR 检测了缺氧如何调节胰腺癌细胞系（BxPC-3、AsPC-1）中 miR-21 的表达。染色质免疫沉淀实验用于研究缺氧如何改变缺氧诱导因子（HIF）-1α 与 miR-21 缺氧反应元件的结合。BxPC-3 和 AsPC-1 细胞转染组成型稳定的 HIF-1α 亚单位或空载体（pcDNA3.1），以确定 miR-21 在常氧条件下的影响。通过 WST-1 检测和流式细胞术评估成熟 miR-21 正义和反义寡核苷酸在缺氧和常氧条件下对增殖和凋亡的影响。
与常氧相比，所有在缺氧条件下培养的细胞系中 miR-21 水平均增加，而靶向 HIF-1α 的 siRNA 则降低了 miR-21 的表达。缺氧条件下导致 HIF-1α 直接与 miR-21 的预测结合位点结合。在常氧条件下转染组成型稳定的 HIF-1α 表达质粒导致 miR-21 上调，与缺氧时相似。转染靶向 miR-21 的反义构建体的细胞在常氧条件下增殖减少，凋亡增加，而 miR-21 过表达则消除了缺氧引起的增殖减少。
miR-21 是通过 HIF-1α 上调在胰腺癌细胞中由缺氧诱导的。miR-21 过表达使细胞能够在缺氧微环境中避免凋亡。抑制 miR-21 表达可能会增加胰腺癌细胞对缺氧的敏感性。
