TLR9 signaling repressed tumor suppressor miR-7 expression through up-regulation of HuR in human lung cancer cells.

BACKGROUND: Our recent evidence showed that Toll like receptor 9 (TLR9) signaling could enhance the growth and metastatic potential of human lung cancer cells through repressing microRNA-7 (miR-7) expression. Human antigen R (HuR) has been involved in stabilizing multiple mRNAs in cellular biology. However, whether HuR also contributed to the altered expression of miR-7 in TLR9 signaling stimulated human lung cancer cells remains to be elucidated. METHODS: The expression of HuR in human lung cancer 95D cells treated with TLR9 agonist CpG Oligonucleotides (ODNs) was detected by Real-time PCR and Western blot assay. To explore the possible role of HuR on miR-7 expression, eukaryotic expression vector encoding HuR was transiently transfected into 95D cells and then the expression of miR-7 was detected by Real-time PCR assay. Moreover, RNA interference, western blot, Real-time PCR, MTT assay, BrdU labeling, invasion assay and scratch assay were employed to examine the disrupt effect of HuR on miR-7 expression in human lung cancer cells treated with CpG ODNs. Finally, inhibitors for PI3K, Akt or Erk respectively, and western blot were performed to explore the possible signaling pathway related to HuR expression in CpG ODNs treated human lung cancer cells. RESULTS: Our data showed that TLR9 agonist CpG ODNs could induce the expression of HuR in human lung cancer cells. Moreover, overexpression of HuR could reduce the expression of miR-7 in lung cancer cells. Notably, down-regulation of HuR using RNA interference restored miR-7 expression in CpG ODNs treated lung cancer cells, accompanied by enhanced growth and metastatic potential. Finally, CpG ODNs could induce HuR expression through Akt pathway. CONCLUSION: Our findings indicated that HuR could act as regulator in regulating TLR9 signaling associated biological effect in human lung cancer cells, which might be helpful for the understanding of the potential role of HuR in tumor biology.

Trehalose inhibited the phagocytosis of refrigerated platelets in vitro via preventing apoptosis.

BACKGROUND: The sole reason that platelets (PLTs) are currently stored at room temperature is that refrigeration of PLTs results in their rapid clearance after transfusion. Recent data have suggested that trehalose was an effective cryoprotective reagent for human PLTs. This study evaluated the effect of trehalose on the phagocytosis of refrigerated PLTs. STUDY DESIGN AND METHODS: Phagocytosis of PLTs was evaluated with THP-1 cells. Phosphatidylserine exposure and caspase-3 activity in PLTs were measured with flow cytometry. PLT mitochondrial transmembrane potentials were studied by staining PLTs with JC-1. Expression of Bcl-XL and Bax was determined by Western blot. PLT aggregation was studied in vitro with a PLT aggregation profiler. RESULTS: It was demonstrated that trehalose significantly inhibited the phagocytosis of refrigerated PLTs by showing that the phagocytotic ratio of PLTs refrigerated with trehalose for 9 days was generally comparable with that of PLTs stored at room temperature for 5 days, which was significantly lower than that of PLTs refrigerated without trehalose. Further studies revealed that trehalose could prevent the apoptosis of refrigerated PLTs, which was determined by the phosphatidylserine exposure, caspase-3 activities, mitochondrial transmembrane potentials, and expression of Bcl-XL and Bax. Finally, it was shown that PLTs refrigerated with trehalose for 9 days also maintained their activity to aggregate when exposed to agonists in a standard aggregometry assay. CONCLUSION: Our results suggest that trehalose could inhibit the phagocytosis of refrigerated PLTs in vitro through preventing apoptosis, implying that trehalose is a promising candidate to optimize the refrigeration of human PLTs.

CXCR4/SDF-1 pathway is crucial for TLR9 agonist enhanced metastasis of human lung cancer cell.

Accumulating data suggested that CXCR4/SDF-1 pathway may play an important role in the metastasis of tumor. We previously demonstrated that CpG ODN could enhance the metastasis of human lung cancer cell via TLR9. Here we further evaluated the possible role of CXCR4/SDF-1 pathway in the enhanced metastasis of human lung cancer 95D cells induced by CpG ODN. Our data showed down-regulation of CXCR4 expression using siRNA against CXCR4 could significantly reduce the enhanced metastasis of 95D cells induced by CpG ODN both in vitro and in vivo. These results suggested that TLR9 agonist might promote the metastasis of human lung cancer cells via CXCR4/SDF-1 pathway.

Targeting toll-like receptor 9 with CpG oligodeoxynucleotides enhances anti-tumor responses of peripheral blood mononuclear cells from human lung cancer patients.

CpG-oligonucleotides (CpG-ODN), which induce signaling through Toll-like receptor 9 (TLR9), are currently under investigation as adjuvants in therapy against infections and cancer. However, whether the CpG-ODN alone could enhance the anti-tumor immunity and the underlying mechanisms remains unclear. Here, we investigated that stimulation of peripheral blood mononuclear cells (PBMCs) from human lung cancer patients with CpG-ODN induced proliferation responses of the PBMCs, accompanied by the elevated cytokine secretion, including IFN-alpha, IL-12 and TNF-alpha. In addition, after treatment with CpG-ODN, the cytotoxic activity of the PBMCs and the production of IFN-gamma in CD8(+) T cells were dramatically enhanced. Furthermore, we found that adoptive transfer of CpG-ODN treated PBMCs significantly inhibited the tumor progression in nude mice, which were challenged with the autologuous tumor cells from human lung cancer patients. Finally, we demonstrated that the inhibitory CpG ODN or chloroquine could dramatically abrogate the enhanced anti-tumor responses of the CpG ODN treated PBMCs. Our findings suggest that the CpG-ODN is promising as a preventive and therapeutic anti-tumor measure against pulmonary carcinoma.

Functional expression of TLR9 is associated to the metastatic potential of human lung cancer cell: functional active role of TLR9 on tumor metastasis.

CpG-oligonucleotides (CpG-ODN), which induce signaling through Toll-like receptor 9 (TLR9), are widely used as adjuvants in therapy against cancer. However, tumor cells express functional TLR9 were recently reported and the immune effect of CpG ODN on tumor cells remains unclear. Here we investigated the direct effects of CpG ODN on human tumor cell line 95D cells using flow cytometric analysis and Western blotting. We found strongly high expression of TLR9 in 95D cells. Stimulation of 95D cells with CpG ODN induced significantly elevated secretion of IL-1alpha and IL-8, as well as the expression of CXCR4, ICAM-1 and MMP-2. Furthermore, the invasion of 95D cells and TLR9 modifying 95C cells were significantly enhanced by stimulation of CpG ODN, which could be abrogated by inhibitory CpG ODN and chloroquine. These results suggest that functionally active TLR9 is expressed on human tumor cell lines, and may represent a novel insight on the role of TIL9 agonist used in tumor immunotherapy.