siRNA targeting HIF-1alpha induces apoptosis of pancreatic cancer cells through NF-kappaB-independent and -dependent pathways under hypoxic conditions.
OBJECTIVE
The primary objective was to explore the molecular mechanism of small interference RNA (siRNA) targeting hypoxic inducible factor-1alpha (HIF-1alpha) for inducing apoptosis of pancreatic cancer cells through a nuclear factor-kappaB (NF-kappaB)-independent or -dependent pathway under hypoxic conditions.


METHODS
A cassette encoding siRNA targeting HIF-1alpha mediated by recombinant adeno-associated virus (rAAV) was constructed, giving rAAV-siHIF. rAAV-siHIF or rAAV-hrGFP were transfected into exponentially growing MiaPaCa2 cells under hypoxic conditions. The expression of HIF-1alpha mRNA and protein and the activity of NF-kappaB were observed by real-time PCR, Western blot and electromobility shift assay (EMSA). The proliferation and apoptosis of MiaPaCa2 cells with or without PDTC, as an inhibitor of NF-kappaB, were investigated by MTT and TUNEL.


RESULTS
rAAV-siHIF inhibited the expression of HIF-1alpha mRNA and protein and the activity of NF-kappaB in MiaPaCa2 cells under hypoxic conditions. At the same time, rAAV-siHIF decreased MiaPaCa2 cell proliferation and induced apoptosis, but these effects were not abrogated by PDTC. Moreover, PDTC also inhibited MiaPaCa2 cell proliferation and induced apoptosis while rAAV-hrGFP did not have these effects.


CONCLUSIONS
Under hypoxic conditions, HIF-1alpha plays a key role in the proliferation of MiaPaCa2 cells and inhibition of HIF-1alpha expression may lead to MiaPaCa2 cell apoptosis through NF-kappaB-independent and -dependent pathways.
Department of Esophageal Cancer, TianJin Medical University Cancer Institute and Hospital, TianJin 300060, China.
Chuangui
C
Chen
Zhentao
Z
Yu
D017209:Apoptosis; D015153:Blotting, Western; D015687:Cell Hypoxia; D049109:Cell Proliferation; D000229:Dependovirus / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020287:In Situ Nick-End Labeling; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016328:NF-kappa B; D012333:RNA, Messenger; D034741:RNA, Small Interfering