Ya Zhou

MicroRNA-7-regulated TLR9 signaling-enhanced growth and metastatic potential of human lung cancer cells by altering the phosphoinositide-3-kinase, regulatory subunit 3/Akt pathway.

This article reports that TLR9 signaling can reduce intrinsic microRNA-7 (miR-7) expression in human lung cancer cells and that overexpression of miR-7 can significantly inhibit TLR9 signaling–enhanced growth and metastatic potential of lung cancer cells in vitro and in vivo.

TLR9 Signaling Promotes Tumor Progression of Human Lung Cancer Cell In Vivo

Toll like receptor 9 (TLR9) was identified mainly in cells of the immune system, and CpG oligonucleotides (CpG ODN), which induces signaling through TLR9, are currently under investigation as adjuvants in clinical therapies against cancer. However, accumulating data suggested that functional TLR9 was also expressed in tumor cells and the effects of TLR9 signaling on the progression of tumor cells remain undefined. Our previous study demonstrated that the TLR9 signaling could significantly enhance the metastatic potential of human lung cancer cells in vitro. Here we carefully evaluated the direct effect of TLR9 signaling on tumor progression of human lung cancer cells in vitro and in vivo. We observed that TLR9 agonist CpG ODN could robustly enhance the tumor progression of 95D cells which expressed high level of TLR9 in nude mice. Furthermore, the CpG ODN could effectively induce the proliferation and IL-10 secretion of 95D cells in vitro. Finally, we demonstrated that CpG ODN could significantly elevate the tumor progression of TLR9 modifying 95C cells in vitro and in vivo, which could be dramatically abrogated by the inhibitory CpG ODN. Our findings indicated that the TLR9 signaling could promote the tumor progression of human tumor cells, which might provide novel insight into the implications for CpG based anti-tumor therapies.

MicroRNA-126 regulates the induction and function of CD4+ Foxp3+ regulatory T cells through PI3K/AKT pathway

Recent evidence showed that limited activation of PI3K/Akt pathway was critical for induction and function sustainment of CD4+Foxp3+ regulatory T cells (Tregs). However, the underlying mechanism remains largely unknown. In this study, we reported that miR‐126 was expressed in mouse and human Tregs. Further study showed that silencing of miR‐126 using miR‐126 antisense oligonucleotides (ASO) could significantly reduce the induction of Tregs in vitro. Furthermore, miR‐126 silencing could obviously reduce the expression of Foxp3 on Tregs, which was accompanied by decreased expression of CTLA‐4 and GITR, as well as IL‐10 and TGF‐β, and impair its suppressive function. Mechanistic evidence showed that silencing of miR‐126 enhanced the expression of its target p85β and subsequently altered the activation of PI3K/Akt pathway, which was ultimately responsible for reduced induction and suppressive function of Tregs. Finally, we further revealed that miR‐126 silencing could impair the suppressive function of Tregs in vivo and endow effectively antitumour effect of CD8+T cells in adoptive cell transfer assay using a murine breast cancer model. Therefore, our study showed that miR‐126 could act as fine‐tuner in regulation of PI3K‐Akt pathway transduction in the induction and sustained suppressive function of Tregs and provided a novel insight into the development of therapeutic strategies for promoting T‐cell immunity by regulating Tregs through targeting specific miRNAs.

Antisense Oligonucleotide Treatment Enhances the Recovery of Acute Lung Injury through IL-10–Secreting M2-like Macrophage-Induced Expansion of CD4+ Regulatory T Cells

MicroRNAs (miRNAs) have been shown as an important regulator in the pathologies of acute lung injury (ALI). However, the potential effect of miRNA-based therapeutic studies in ALI remains poorly understood. We assessed the effect of antisense oligonucleotides (ASOs) against miR-155 on the development of ALI using a murine ALI model. We found that miR-155 ASO treatment could enhance the recovery of ALI as evidenced by accelerated body weight back, reduced level of bronchoalveolar lavage (BAL) protein and proinflammatory cytokines, and reduced number of BAL cells. Adoptive cell transfer assay in RAG1−/− mice showed that CD4+CD25+ regulatory T cells (Tregs) mediated the enhanced recovery of ALI. Mechanistic evidence showed that enhanced expansion of Tregs in vivo, dominantly induced by IL-10–secreting M2-like macrophages, was critical for their elevated proportion in miR-155 ASO-treated ALI mice. Finally, we report that C/EBPβ, a target molecule of miR-155, was upregulated and associated with IL-10 secretion and M2-like phenotype of macrophages. These data provided a previously unknown mechanism for miRNA-based therapy against ALI, which could ultimately aid the understanding of recovery of ALI and the development of new therapeutic strategies against clinical inflammatory lung disease.

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.

Antisense oligonucleotides against microRNA-21 reduced the proliferation and migration of human colon carcinoma cells

BackgroundColon carcinoma is one of the commonly tumors that threaten human beings as its highly morbidity and mortality. Recent evidences suggested that microRNA-21 (miR-21) played an important role in the development of colon carcinoma and might be a potential biological marker for the diagnosis and prognosis of colon carcinoma. However, the potential effect of miR-21 based therapeutic studies in colon carcinoma remains to be fully elucidated.MethodsIn present study, we constructed an eukaryotic expression vector encoding antisense oligonucleotides against miR-21 (termed as p-miR-21-ASO) and the expression of miRNA-21 in human colon cancer was detected by Real-time PCR. To assess its possible effect on the proliferation and migration capacity of human colon carcinoma cells in vitro, CCK-8 assay, colony formation assay and cell invasion, as well as migration assay, were performed respectively. Moreover, PTEN, one of target molecules of miRNA-21, was analyzed by Western blot and Fluorescence activated cell sorter assay. Finally, the transduction of AKT and ERK pathways in human colon carcinoma cells was determined by Western blot.ResultsWe found that transiently transfection of p-miR-21-ASO could efficiently decrease the relative expression of miR-21 in human colon carcinoma HCT116 cells, accompanied by impaired proliferation and clone formation. Furthermore, we found that down-regulation of miR-21 also could significantly abrogate the invasion and migration capacity in vitro, as well as the expression of vascular endothelial growth factor which is critical for the metastatic capacity of colon carcinoma cells. Mechanistic evidence showed that down-regulation of miR-21 increased the expression of its target molecule PTEN in HCT116 cells. Finally, we revealed that the expression level of both phosphor-ERK1/2 and phosphor-AKT also were altered.ConclusionsTherefore, our data suggested miR-21 ASO against miR-21 might be a useful strategy to alter the expression of miR-21 in colon carcinoma cells, which was helpful for the development of miR-21-based therapeutic strategies against clinical colon carcinoma.

MicroRNA-7: a promising new target in cancer therapy.

The incidence of tumors with life-threatening effects has increased gradually over time; however, the mechanisms involved in tumor development have not been fully elucidated. Recent studies have shown that microRNA-7 (miR-7), which is endogenous non-coding RNA molecules of approximately 23 nucleotides, plays an important role in the occurrence and development of tumors as a key tumor suppressor. Mechanistic evidence showed that miR-7 is closely related to the growth, metastasis, and prognosis of various malignant tumors through regulating different target molecules, which suggest that miR-7 may be a new target for the clinical diagnosis and treatment of various tumors. In this review, we summarize current knowledge of the relationship between miR-7 and tumor development, diagnosis, and treatment.

CpG oligonucleotides induce the differentiation of CD4+Th17 cells by triggering plasmacytoid dendritic cells in adoptively cell transfer immunotherapy

Our previous data showed that CpG-ODNs could significantly enhance the anti-tumor efficacy of adoptively cell transfer (ACT), which was closely correlated to accumulation of Th17 cells in tumor mass. Here we further investigated that CpG-ODNs had no significant effect on the migration and proliferation capacity of Th17 cells in tumor mass. Instead, we showed that CpG-ODNs could induce the differentiation of Th17 cells via dendritic cells (DCs) in tumor infiltrating lymphocytes (TILs). Notably, we found that plasmacytoid dendritic cells (pDCs), but not myeloid dendritic cells (mDCs), were responsible for the Th17 differentiation induced by CpG-ODNs via IL-6, TGF-β and IFN-α in vitro. Finally, we revealed that CpG-ODNs could stimulate pDCs to induce the differentiation of Th17 cells in vivo, which subsequently reduced the tumor size and prolonged the survival of tumor bearing nude mice. These data provided a novel insight into the mechanism of anti-tumor efficacy of CpG-ODNs based therapeutic strategy.

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

BackgroundOur 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.MethodsThe 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.ResultsOur 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.ConclusionOur 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.

MiR-21 controls in situ expansion of CCR6⁺ regulatory T cells through PTEN/AKT pathway in breast cancer.

Our recent evidence showed that prior expansion of CCR6+ Foxp3+ regulatory T cells (Tregs) was important for their dominant enrichment in tumor tissue, which was closely related to poor prognosis of breast cancer patients. However, the underlying regulation mechanism of expansion of CCR6+ Tregs in situ remains largely unknown. In this study, we reported that miR‐21 was highly expressed in CCR6+ Tregs in tumor tissues from a murine breast cancer model. And silencing of miR‐21 could significantly reduce the proliferation of CCR6+ Tregs in vitro. Adoptive cell‐transfer assay further showed that silencing of miR‐21 could alter the enrichment of CCR6+ Tregs in the tumor mass and endow effectively antitumor effect of CD8+ T cells using a murine breast cancer model. Mechanistic evidence showed that silencing of miR‐21 enhanced the expression of its target phosphatase and tensin homolog deleted on chromosome ten (PTEN) and subsequently altered the activation of Akt pathway, which was ultimately responsible for reduced proliferation activity of CCR6+ Tregs. Finally, we further revealed that miR‐21 was also highly expressed on CCR6+ Tregs in clinical breast cancer patients. Therefore, miR‐21 can act as a fine tuner in the regulation of PTEN/Akt pathway transduction in the expansion of CCR6+ Tregs in tumor sites and provided a novel insight into the development of therapeutic strategies for promoting T‐cell immunity by regulating distinct subset of Tregs through targeting specific miRNAs.