A region within the 5'-untranslated region of hypoxia-inducible factor-1alpha mRNA mediates its turnover in lung adenocarcinoma cells.
Previously, we showed that CL1-5 cells express more hypoxia-inducible factor-1alpha (HIF-1alpha) than the parental CL1 cells, which bestows CL1-5 cells a stronger invasive activity. Here, we investigated the mechanisms underlying the differential expression of HIF-1alpha mRNA in CL1 and CL1-5 cells. Data showed that the transcription rate of HIF-1alpha gene in CL1 cells was slightly higher than that of CL1-5 cells, suggesting that the expression of HIF-1alpha mRNA in CL1 cells was repressed by post-transcriptional mechanisms. RNA electrophoretic mobility shift assays revealed a 61-base segment (designated as D5) within the 5'-untranslated repeat of HIF-1alpha mRNA, with which the CL1 cell lysates formed more prominent complexes (including complex I) than did CL1-5 cell lysates. Insertion of D5 into a reporter construct reduced the half-life of the chimeric transcripts in transfected CL1 but not CL1-5 cells; conversely, overexpression of D5-containing reporter construct in CL1 cells increased HIF-1alpha mRNA. We also identified the polypyrimidine tract-binding protein (PTB) as a required component of complex I. Deletion of the RNA recognition motif 1 (RRM1) or RRM3 of PTB abolished the formation of complex I. Our data showed that CL1 cells expressed more PTB than CL1-5 cells. Inhibition of PTB expression in CL1 cells decreased the formation of complex I, whereas overexpression of PTB in CL1-5 cells increased the levels of complex I, decreased the stability of HIF-1alpha and D5-containing chimeric mRNAs, and decreased cell invasiveness. In sum, we have identified in lung adenocarcinoma cells a mechanism that regulates HIF-1alpha expression by modulating HIF-1alpha mRNA stability.
Wang
Ming Jen
MJ
Institute of Cellular and System Medicine, National Health Research Institutes, Number 35, Keyan Road, Zhunan Town, Miaoli County 35053, Taiwan.
Lin
Shankung
S
Institute of Cellular and System Medicine, National Health Research Institutes, Number 35, Keyan Road, Zhunan Town, Miaoli County 35053, Taiwan. Electronic address: shankung@nhri.org.tw.
D020121:5' Untranslated Regions; D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis* / Q000235:genetics; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics; D038941:Polypyrimidine Tract-Binding Protein / Q000235:genetics / Q000378:metabolism; D020871:RNA Stability; D012262:Ribonucleoside Diphosphate Reductase; D014158:Transcription, Genetic / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
D020121:5' Untranslated Regions; C497442:HIF1A protein, human; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D046912:Multiprotein Complexes; D009363:Neoplasm Proteins; C450006:PTBP1 protein, human; D025521:Tumor Suppressor Proteins; D038941:Polypyrimidine Tract-Binding Protein; C475286:RRM1 protein, human; D012262:Ribonucleoside Diphosphate Reductase