The nm23-H1 gene is an important tumor metastatic suppressor gene. Our previous study showed that the downregulation of nm23-H1 gene expression using small interfering RNA (siRNA) in NL9980 lung cancer cells greatly enhanced their invasiveness. To further explore the molecular mechanisms after nm23-H1 gene knockdown, we established transgene NL9980 and A549 lung cancer cell lines with stable nm23-H1 gene silencing through the lentivirus-mediated short hairpin RNA (shRNA) method.
The human large cell lung cancer NL9980 and human lung adenocarcinoma A549 cells were transfected with shRNA lentiviral particles specific for the nm23-H1 gene, and were then selected through puromycin. Puromycin-resistant clones were generated and screened using reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time polymerase chain reaction (qPCR), and Western blot analysis. shRNA rescue experiments were performed to restore the nm23-H1 gene expression in the shRNA-expressing cells. Invasiveness was determined through a Boyden chamber assay.
The puromycin-resistant clones (NL9980-99 and A549-99) showed very low levels of nm23-H1 mRNA and protein expression under RT-PCR, qPCR, and Western blot analysis. Meanwhile, the shRNA rescue experiment restored the nm23-H1 expression in the NL9980-99 and A549-99 cells detected by Western blot. Downregulation of nm23-H1 gene expression enhanced the invasiveness of the NL9980-99 and A549-99 cells compared with the controls.
The lung cancer cell lines NL9980-99 and A549-99 with stable nm23-H1 gene silencing were successfully established and their invasiveness was greatly increased after nm23-H1 gene knockdown.
[Show abstract][Hide abstract] ABSTRACT: Persistent STAT3 activation is a critical event in tumorigenesis and metastatic progression. Recent studies have found higher levels of STAT3 in metastatic tissues than in primary tumor tissues. We speculated that such increased STAT3 activity might be attributed to a loss of function or reduction in expression of metastasis inhibitory protein during cancer progression, and we therefore examined the role of tumor metastasis-suppressor nm23-H1 in the activation of STAT3 in the A549 lung cancer cell line. We found that IL-6-dependent induction of tyrosine phosphorylation and activation of STAT3 were influenced by nm23-H1 inhibition. IL-6-induced STAT3(Tyr705) phosphorylation was significantly enhanced in A549 cells transfected with siRNA specific for nm23-H1, and the effect of nm23-H1 depletion on IL-6-induced STAT3(Tyr705) phosphorylation was reversed by ectopic expression of shRNA-resistant nm23-H1 protein. Moreover, STAT3 directly bound to the STAT3 binding site on the nm23-H1 promoter and activated its expression. Thus, we have identified a new feedback mechanism that might provide insight into an in-built metastasis-suppression function in tumor cells and which could be a logical new target for treatment of early metastatic disease.
Biochemical and Biophysical Research Communications 04/2013; 434(3). DOI:10.1016/j.bbrc.2013.03.110 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Uveal melanoma (UM) is the most common primary intraocular malignancy and the leading potentially fatal primary intraocular disease in adults. Melanoma antigen recognized by T-cells (MART-1) has been studied extensively as a clinically important diagnostic marker for melanoma, however, its biological function remains unclear. In the present study, the UM cell line SP6.5, which showed a high level of MART-1 expression, was subjected to small interfering RNA-mediated silencing of MART-1. Silencing of MART-1 expression increased the migration ability of SP6.5 cells and down-regulated the expression of the metastasis suppressor NM23. Our results suggest that MART-1 is a candidate target for the development of therapeutic strategies for UM and in particular for the suppression of metastasis associated with this malignancy.
International Journal of Molecular Sciences 07/2013; 14(7):15092-104. DOI:10.3390/ijms140715092 · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: C-X-C chemokine receptor types 1 (CXCR1), a cell-surface G-protein-coupled receptor has been found to be associated with tumorigenesis, development, and progression of some tumors. Previously, we have found that CXCR1 overexpression is associated with late-stage gastric adenocarcinoma. We also have demonstrated that knockdown of CXCR1 could inhibit cell proliferation in vitro and in vivo. In this study, we compared the changes of protein expression profile between gastric carcinoma MKN45 cell line and CXCR1-knockdown MKN45 cell line by 2D electrophoresis. Among the 101 quantified proteins, 29 spots were significantly different, among which 13 were down-regulated and 16 were up-regulated after CXCR1 knockdown. These proteins were further identified by mass spectrometry analysis. Among them, several up-regulated proteins such as hCG2020155, Keratin8, heterogeneous nuclear ribonucleoprotein C (C1/C2), and several down-regulated proteins such as Sorcin, heat shock protein 27, serpin B6 isoform b, and heterogeneous nuclear ribonucleoprotein K were confirmed. These proteins are related to cell cycle, the transcription regulation, cell adherence, cellular metabolism, drug resistance, and so on. These results provide an additional support to the hypothesis that CXCR1 might play an important role in proliferation, invasion, metastasis, and prognosis, and drug resistance of gastric carcinoma.
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