Yijie Zheng

Glucocorticoids inhibit lipopolysaccharide-mediated inflammatory response by downregulating microRNA-155: a novel anti-inflammation mechanism.

Glucocorticoids (GCs) are among the most widely used and effective therapies for many chronic inflammatory diseases. Although attempts have been made to identify important protein-coding genes and pathways involved in the anti-inflammatory effect of GCs, knowledge of genomic aberrations associated with noncoding genes, such as micro-RNAs (miRNAs), and their contributions is relatively limited. In this study, a systematic screening of the miRNA expression profile by microarray showed that GCs inhibited the expression of miR-155 in lipopolysaccharide (LPS)-induced macrophage inflammatory responses. Overexpression of miR-155 markedly reversed the suppressive action of GCs, whereas inhibition of miR-155 exhibited an effect similar to that of GCs on LPS-treated RAW264.7 cells, indicating miR-155 to be a functional regulator in the anti-inflammatory effect of GCs. Furthermore, GCs inhibited miR-155 expression in a GC receptor- and NF-κB-dependent manner. Bioinformatics analysis and luciferase assay revealed that the NF-κB binding site located in the promoter region of the B-cell integration cluster was important in mediating the GC-driven suppression of miR-155 in response to LPS stimulation. In addition, the combination of treatment with GCs and inhibition of miR-155 enhanced the anti-inflammatory effect of GCs on LPS-stimulated RAW264.7 cells. Therefore, we identify miR-155 to be a novel target through which GCs exert their anti-inflammatory effect on the LPS-induced macrophage inflammatory response. These findings may provide a basic rationale for new approaches in the effort to develop anti-inflammatory therapeutics.

DNMT1-microRNA126 epigenetic circuit contributes to esophageal squamous cell carcinoma growth via ADAM9-EGFR-AKT signaling

Purpose: MicroRNAs (miRNA) are involved in and are controlled by epigenetic regulation, and thereby form a reciprocal regulatory circuit. Using next-generation sequencing (NGS)–based miRNA profiling, this study aimed to discover esophageal squamous cell carcinoma (ESCC)–specific miRNAs and miRNA-related epigenetic modulations. Experimental Design: NGS-based miRNA profiles were generated for four pairs of ESCC tissues and adjacent normal tissues. In situ hybridization was used to assess miRNA expression and its correlation with prognosis. miRNA-related DNA methylations were identified using bisulfite genomic sequencing, and the role of DNA methyltransferase 1 (DNMT1) was investigated using RNA interference. miRNA targets were screened by mRNA sequencing, and functional validation was performed in vitro and in vivo. Results: NGS-based miRNA profiling identified 78 differentially expressed miRNAs in ESCC. Among them, microRNA126-3p (miR-126) was significantly downregulated, and its downregulation correlated with poor ESCC prognosis. Downregulation of miR-126 was due to promoter hypermethylation of its host gene, Egfl7. DNMT1 was aberrantly upregulated in ESCC and responsible for the hypermethylation of Egfl7. Intriguingly, DNMT1 was suppressed by overexpression of miR-126, indicating the existence of a regulatory feedback circuit. ADAM9 was identified as a key target of miR-126. Ectopic expression of miR-126 or silencing of ADAM9 reduced ESCC cell proliferation and migration by inhibiting epidermal growth factor receptor–AKT signaling. Conclusions: Our results indicate that miR-126 is a potential prognostic indicator for ESCC and suggest that a novel “DNMT1–miR-126 epigenetic circuit” is involved in ESCC progression. Consequently, miR-126–based epigenetic modulations may provide a basic rationale for new approaches to antitumor therapeutics. Clin Cancer Res; 21(4); 854–63. ©2014 AACR.

CD4+Foxp3+ regulatory T-cell impairment by paclitaxel is independent of toll-like receptor 4.

Paclitaxel (PTX) is one of the most widely used clinical antitumour drugs in chemotherapy nowadays. Its effect on immune system has become a hot spot of research in recent years. Here, we demonstrated that PTX not only decreased the percentage of CD4+Foxp3+ regulatory T (Treg) cells both in vitro and in vivo but also impaired cell viability and cytokine production of Treg cells rather than CD4+Foxp3− effector T (Teff) cells. As PTX has been reported to mimic the activity of LPS to trigger the toll‐like receptor 4 (TLR4) signalling pathway in macrophages, we investigated the possible role of TLR4 in the effect of PTX. However, although TLR4 expression on Treg cells was higher than that on Teff cells, the expression level remained unaltered in both Treg and Teff cells after PTX treatment. Surface molecules and activation markers in Treg and Teff cells did not change, either. Further study showed that the effect of PTX on TLR4−/− mice deficient in TLR4 signalling was similar to that on C57BL/6 mice both in vivo and in vitro. These data indicate that the selective impairment of Treg cells by PTX is independent of TLR4.

MicroRNA-7 sensitizes non-small cell lung cancer cells to paclitaxel

Paclitaxel (PTX) is the front-line chemotherapeutic agent against human non-small cell lung cancer (NSCLC). However, its therapeutic efficacy is restricted by the increasing frequency of chemotherapeutic resistance in NSCLC. Accumulating evidence has shown the potential role of microRNAs (miRNAs) in the chemotherapeutic sensitivity of cancer cells. Previously it was reported that microRNA-7 (miR-7) acts as an important tumor suppressor in NSCLC. Therefore, the present study was conducted to determine the regulatory role of miR-7 in PTX chemotherapy for NSCLC. Four NSCLC cell lines were used to analyze the correlation of the PTX-sensitivity and endogenoaus miR-7 expression. miR-7 expression was up- and downregulated using miR-7 mimics and inhibitors respectively, and the role of miR-7 in sensitizing NSCLC cells to PTX was assessed by cell viability and apoptosis assays. The molecular mechanism of PTX sensitivity was determined by quantitative polymerase chain reaction and western blotting. It was found that the sensitivity of NSCLC cells to PTX was dependent on endogenous miR-7. Upregulation of miR-7 enhanced the PTX-sensitivity of NSCLC cells by suppressing cell proliferation and promoting cell apoptosis, while the inhibition of miR-7 abrogated the antiproliferative proapoptotic effects of PTX. Pretreatment of miR-7 mimics enhanced the PTX-mediated downregulation of epidermal growth factor receptor (EGFR) in NSCLC cells. These results have identified miR-7 as a potential EGFR-targeting sensitizer in PTX therapy. These data may facilitate the development of novel chemotherapeutic approaches for NSCLC.

Trichosanthin enhances anti-tumor immune response in a murine Lewis lung cancer model by boosting the interaction between TSLC1 and CRTAM.

Trichosanthin (TCS), extracted from the Chinese medicinal herb Trichosanthes kirilowi, has shown promise for the inhibition of tumor growth. However, its immunomodulatory effect on tumor–host interaction remains unknown. In this study, we focused on the effect of TCS on murine anti-tumor immune response in the 3LL Lewis lung carcinoma tumor model and explored the possible molecular pathways involved. In addition to inhibiting cell proliferation and inducing apoptosis in the 3LL tumor, TCS retarded tumor growth and prolonged mouse survival more significantly in C57BL/6 immunocompetent mice than in nude mice. This reflected the fact that the host immune system was involved in tumor eradication. Using FACS analysis, we found that TCS increased the percentage of effector T cells, particularly Interferon-gamma (IFN-γ) producing CD4+ and CD8+ T cells from tumor-bearing mice. TCS also promoted the vigorous proliferation of antigen-specific effector T cells, markedly increased Th1 cytokine secretion and elicited more memory T cells in tumor-bearing mice, consequently enhancing the anti-tumor response and inducing immune protection. Furthermore, we found that TCS upregulated the expression of tumor suppressor in lung cancer 1 (TSLC1) in 3LL tumor cells and the expression of its ligand, class I-restricted T cell-associated molecule (CRTAM), in effector T cells. Blocking TSLC1 expression with small interfering RNA (siRNA) significantly eliminated the effects of TCS on the proliferation and cytokine secretion of effector T cells, suggesting that TCS enhances anti-tumor immune response at least partially by boosting the interaction between TSLC1 and CRTAM. Collectively, our data demonstrate that TCS not only affects tumor cells directly, but also enhances anti-tumor immunity via the interaction between TSLC1 and CRTAM. These findings may lead to the development of a novel approach for tumor regression.

B cells expressing CD11b effectively inhibit CD4+ T-cell responses and ameliorate experimental autoimmune hepatitis in mice.

Increasing evidence in recent years has suggested that B cells act as a crucial regulator in autoimmune diseases. However, little is known about their role in autoimmune hepatitis (AIH) and the underlying regulatory mechanisms. In this study, we show that B cells ameliorated experimental AIH (EAH) by suppressing CD4+ T‐cell responses and that CD11b expression on B cells was required for the regulatory function of B cells. In vitro studies reveal that the suppressive function of CD11b was mediated by the impairment of T‐cell antigen receptor (TCR) signaling transduction and the promotion of TCR down‐regulation. Moreover, we show that the increased CD11b expression on B cells was interleukin (IL)−10 dependent and that additional IL‐10 stimulation promoted CD11b expression on B cells, thereby enhancing B‐cell regulatory effects. Conclusion: These findings reveal a previously unrecognized role for CD11b in B‐cell regulatory function and its protective effect on EAH. (Hepatology 2015;62:1563–1575)

Selective Impairment of CD4 + CD25 + Foxp3 + Regulatory T cells by paclitaxel is explained by Bcl-2/Bax mediated apoptosis

Paclitaxel has become one of the most effective and widely used chemotherapeutic agents over the past decades. Although it has shown promise to selectively deplete regulatory T (Treg) cells in our previous study, the underlying molecular mechanism remains to be further elucidated. The present study focused on the effect of paclitaxel on Treg cells in 3LL Lewis tumor model and explored the possible molecular pathways involved in this process. We found that paclitaxel significantly decreased the percentage of Treg cells in CD4(+) cells and impaired their suppressive functions, but effector T (Teff) cells remained unaffected. Compared with Teff cells, Treg cells exhibited a high sensitivity to paclitaxel-mediated apoptosis in vitro. Interestingly, though paclitaxel has been characterized as a mitotic inhibitor, tubulin was not involved in the selective function of paclitaxel. Treg cells exposed to paclitaxel displayed downregulation of Bcl-2 and upregulation of Bax. Blocking the Bcl-2 pathway eliminated the difference between Treg and Teff cells responding to paclitaxel. These results suggest that Bcl-2 rather than tubulin contributes to the distinctive effect of paclitaxel on Treg cells. Therefore, we here identify a molecular pathway through which paclitaxel selectively ablates Treg cells.

Neddylation pathway regulates the proliferation and survival of macrophages

Neddylation is a new type of protein post-translational modification which adds the ubiquitin-like molecule Nedd8 to target proteins. The well-identified targets of neddylation are cullins, which serve as essential components of Cullin-RING E3 ligases (CRL). It is reported that inhibition of neddylation repressed NF-κB-mediated proinflammatory cytokine production in macrophages. However, the role of neddylation in the proliferation and survival of macrophages has not been well defined. Here we report that partial inactivation of the neddylation pathway by a specific Nedd8-activating enzyme E1 (NAE) inhibitor MLN4924 reduced LPS-induced production of the proinflammatory cytokines TNF-α and IL-6 without obvious impairment of cell viability. However, persistent and severe inactivation of neddylation by MLN4924 significantly inhibited cell proliferation by inducing G2 phase cell-cycle arrest and further triggered cell death by inducing apoptosis in RAW264.7 macrophages. Mechanistic analysis revealed that inactivation of neddylation blocked cullin neddylation, inhibited CRL E3 ligase activity, and thus led to the accumulation of CRL substrates, resulting in cell-cycle arrest, DNA damage response and apoptosis. The findings revealed that neddylation serves as an important signaling pathway regulating the proliferation and survival of macrophages.

Abnormal DNA methylation of ITGAL (CD11a) in CD4+ T cells from infants with biliary atresia

Recent evidence indicates that alterations to epigenetic DNA methylation patterns contribute to many autoimmune diseases. Biliary atresia (BA) is a virus-induced autoimmune disease characterized by impaired T cells, which may be due to aberrant DNA methylation. CD11a, a subunit of the β2-integrin LFA-1 (CD11a/CD18) with costimulatory functions, is overexpressed due to hypomethylation of its promoter regulatory elements in CD4+ T cells from patients with many autoimmune diseases. However, it is unknown whether aberrant expression and methylation of CD11a occur in T cells from infants with BA. We aimed to compare the CD11a expression level and the methylation status of the CD11a promoter region in CD4+ T cells from BA infants and healthy controls (HC). We used real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) to examine CD11a mRNA levels in CD4+ T cells from BA and HC infants. Bisulfite sequencing was used to determine the methylation status of the CD11a promoter and flanking regions in CD4+ T cells from BA and HC infants, and in CD4+ T cells with DNA methylation inhibitors. We found that CD11a expression is significantly decreased in BA CD4+ T cells (P=0.007). This was associated with hypermethylation of the CD11a promoter region in CD4+ T cells from infants with BA. Treatment with a DNA methylation inhibitor decreased CD11a promoter methylation and increased CD11a mRNA. Therefore, DNA hypermethylation at the CD11a locus contributes to the lowered expression of CD11a in BA CD4+ T cells.

miR-222 overexpression may contribute to liver fibrosis in biliary atresia by targeting PPP2R2A.

Objective: Biliary atresia (BA) is a devastating liver disease in infants. Progressive hepatic fibrosis is often observed in postoperative patients with BA even after a successful Kasai portoenterostomy procedure. MicroRNA-222 (miRNA) has been linked to the activation of stellate cells and the progression of liver fibrosis. Methods: In this study, the miR-222 expression profile in BA and infants with anicteric choledochal cyst (CC) was determined. The functional effect of miR-222 inhibition on the growth of the human hepatic stellate cell line LX-2 was also evaluated. The downstream signaling pathways and target of miR-222 were determined by coupling gene expression profiling and pathway analysis and by in silico prediction, respectively. In addition, we demonstrated miR-222 overexpression in patients with BA compared with choledochal cyst controls. Results: Inhibition of miR-222 in the LX-2 cell line significantly decreased cell proliferation. We also identified protein phosphatase 2A subunit B as a target of miR-222. The downstream signaling pathway, Akt, was also influenced by miR-222. A consistent reduction of Akt phosphorylation and Ki67 in the LX-2 line was shown following miR-222 suppression. Conclusions: Our results show that miR-222 overexpression is common in BA and contributes to LX-2 cell proliferation by targeting protein phosphatase 2A subunit B and Akt signaling.