COX-2 silencing inhibits cell proliferation in A549 cell

The Chinese-German Journal of Clinical Oncology 07/2011; 10(7):423-427. DOI: 10.1007/s10330-011-0829-0


ObjectiveThe aim of this study was to explore the effects on malignant proliferation of A549 cell by silencing cyclooxygenase (COX)-2.

MethodsIn the present study, we constructed three siRNA vectors producing small interference RNA. The siRNA vectors and the vacant
vectors were transfected into A549 cell with lipofectamine respectively and the transfected cell strains were constructed.
The change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of lung cancer
cells were studied by cell growth curve, clonogenic assay and xenograft assays.

ResultsThe siRNA expression vectors produced marked effects in A549 cell but the inhibited effects were different. The effect of
psi-10 was best and the mRNA and protein levels of COX-2 reduced 61.2% and 56.2% respectively in A549-si10 cell in contrast
to the control. The growth of A549 cell slowed and the colony formation rate reduced after silencing COX-2. In xenograft assays,
the growth speeds of tumor became slow and the numbers of tumor reduced after silencing COX-2.

ConclusionThe si10 target of COX-2 has the best silencing effect in A549 cell and the best inhibition effect on malignant proliferation
of A549 cell in vivo and in vitro.

Key wordscyclooxygenase (COX)-2–A549 cell–malignant proliferation

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    ABSTRACT: Overexpression of cyclooxygenase 2 (COX-2) is thought to promote survival of transformed cells. Transforming growth factor β (TGF-β) exerts anti-proliferative effects on a broad range of epithelial cells. In the current study, we investigated whether TGF-β can regulate COX-2 expression in A549 human lung adenocarcinoma cells, which are TGF-β-responsive and overexpress COX-2. Western blotting, Northern blotting, and mRNA stability assays were performed to demonstrate that COX-2 protein and mRNA expression were suppressed by TGF-β. We also evaluated the effects of tristetraprolin (TTP) on COX-2 mRNA using RNA interference. We demonstrated that COX-2 mRNA and protein expression were both significantly suppressed by TGF-β. An actinomycin D chase experiment demonstrated that COX-2 mRNA was more rapidly degraded in the presence of TGF-β, suggesting that TGF-β-induced inhibition of COX-2 expression is achieved via decreased mRNA stability. We also found that TGF-β rapidly and transiently induced the expression of TTP, a well-known mRNA destabilizing factor, before suppression of COX-2 mRNA expression was observed. Using RNA interference, we confirmed that increased TTP levels play a pivotal role in the destabilization of COX-2 mRNA by TGF-β. Furthermore, we showed that Smad3 is essential to TTP-dependent down-regulation of COX-2 expression in response to TGF-β. The results of this study show that TGF-β down-regulated COX-2 expression via mRNA destabilization mediated by Smad3/TTP in A549 cells.
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