Enyu Huang

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.

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.

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)

NKT cells mediate the recruitment of neutrophils by stimulating epithelial chemokine secretion during colitis.

Ulcerative colitis (UC) is a kind of inflammatory bowel diseases characterized by chronic inflammation and ulcer in colon, and UC patients have increased risk of getting colorectal cancer. NKT cells are cells that express both NK cell markers and semi-invariant CD1d-restricted TCRs, can regulate immune responses via secreting a variety of cytokines upon activation. In our research, we found that the NKT cell-deficient CD1d(-/-) mice had relieved colitis in the DSS-induced colitis model. Further investigations revealed that the colon of CD1d(-/-) mice expressed less neutrophil-attracting chemokine CXCL 1, 2 and 3, and had decreased neutrophil infiltration. Infiltrated neutrophils also produced less reactive oxygen species (ROS) and TNF-α, indicating they may cause less epithelial damage. In addition, colitis-associated colorectal cancer was also relieved in CD1d(-/-) mice. During colitis, NKT cells strongly expressed TNF-α, which could stimulate CXCL 1, 2, 3 expressions by the epithelium. In conclusion, NKT cells can regulate colitis via the NKT cell-epithelium-neutrophil axis. Targeting this mechanism may help to improve the therapy of UC and prevent colitis-associated colorectal cancer.

MicroRNA 15a/16‐1 suppresses aryl hydrocarbon receptor–dependent interleukin‐22 secretion in CD4+ T cells and contributes to immune‐mediated organ injury

Interleukin‐22 (IL‐22), as a link between leukocytic and nonleukocytic cells, has gained increasing attention for its pronounced tissue‐protective properties. MicroRNAs, emerging as crucial immune modulators, have been reported to be involved in the production and action of various cytokines. However, the precise control of IL‐22 by microRNAs and its subsequent actions remained to be elucidated. In this study, we found a negative correlation between the expression of microRNA 15a/16‐1 (miR‐15a/16‐1) and IL‐22 in the model of concanavalin A–induced, immune‐mediated liver injury. Knockout of miR‐15a/16‐1 ameliorated liver injury in an IL‐22‐dependent manner. Further results revealed that cluster of differentiation 4–positive (CD4+) T cells were the major source of IL‐22 during liver injury and that the aryl hydrocarbon receptor was the direct target of miR‐15a/16‐1 in CD4+ T cells. In vivo and in vitro data showed that miR‐15a/16‐1 knockout CD4+ T cells produced more IL‐22, while overexpression of miR‐15a/16‐1 down‐regulated the IL‐22 production by inhibiting the aryl hydrocarbon receptor. Moreover, transfer of miR‐15a/16‐1 knockout CD4+ T cells promoted tissue repair compared to wild‐type CD4+ T cells by up‐regulating IL‐22. In addition, as a synergistic effect, IL‐22 could down‐regulate miR‐15a/16‐1 expression by activating phosphorylated signal transducer and activator of transcription 3‐c‐myc signaling, and the decrease of miR‐15a/16‐1 in damaged hepatocytes contributed to IL‐22‐mediated tissue repair by reducing cell apoptosis and promoting cell proliferation. As further proof, we demonstrated the role of miR‐15a/16‐1 in controlling IL‐22 production and IL‐22‐mediated reconstruction of the intestinal epithelial barrier in a dextran sodium sulfate–induced colitis model. Conclusion: Our results suggest that miR‐15a/16‐1 acts as a essential regulator of IL‐22 and that the miR‐15a/16‐1–aryl hydrocarbon receptor–IL‐22 regulatory axis plays a central role in tissue repair; modulation of miR‐15a/16‐1 might hold promise in developing new strategies to enhance IL‐22‐mediated tissue repair. (Hepatology 2018;67:1027–1040)

Eukaryotic translation initiation factor 3B accelerates the progression of esophageal squamous cell carcinoma by activating β-catenin signaling pathway

Introduction Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive malignant tumors. Eukaryotic translation initiation factors 3B (EIF3B) is considered to influence tumor proliferation, invasion, apoptosis and cell cycle, which act together to promote the progression of tumors. However, the role of EIF3B in ESCC is unknown. This study aims to explore the clinical and biological role of EIF3B in ESCC. Results EIF3B expressions were up-regulated in both ESCC tissues and cell lines. Overexpression of EIF3B was associated with tumor depth, lymph node metastasis and advanced TNM stage. Importantly, patients with high EIF3B expression suffered shorter overall and disease-free survival. Knockdown of EIF3B could inhibit cell proliferation and invasion, promote cell apoptosis, and interfere the cell cycle in vitro. EIF3B-knockdown cells could form smaller subcutaneous tumors in vivo. Finally, we demonstrated EIF3B could activate β-catenin signaling pathway. Methods Immunohistochemical staining and Western blot were performed to detect the EIF3B expression in ESCC patient tissues and cell lines. The association between EIF3B expression and patients’ prognosis was analyzed by Kaplan-Meier and Cox regression. Then, CCK-8, colony-formation, Transwell and wound-healing assay were performed to compare the bio-functional change after knockdown of EIF3B. Flow cytometry was applied to analyze the change of cell apoptosis and cycle induced by EIF3B knockdown. Tumor xenograft assay was done to verify the in-vitro results. Conclusions EIF3B might serve as a novel marker for predicting prognosis of ESCC patients and as a potential therapeutic target, individually or together with other subunits of EIF3 complex.

Biological Response Modifier in Cancer Immunotherapy

Biological response modifiers (BRMs) emerge as a lay of new compounds or approaches used in improving cancer immunotherapy. Evidences highlight that cytokines, Toll-like receptor (TLR) signaling, and noncoding RNAs are of crucial roles in modulating antitumor immune response and cancer-related chronic inflammation, and BRMs based on them have been explored. In particular, besides some cytokines like IFN-α and IL-2, several Toll-like receptor (TLR) agonists like BCG, MPL, and imiquimod are also licensed to be used in patients with several malignancies nowadays, and the first artificial small noncoding RNA (microRNA) mimic, MXR34, has entered phase I clinical study against liver cancer, implying their potential application in cancer therapy. According to amounts of original data, this chapter will review the regulatory roles of TLR signaling, some noncoding RNAs, and several key cytokines in cancer and cancer-related immune response, as well as the clinical cases in cancer therapy based on them.

MicroRNAs: New regulators of IL-22

Interleukin-22 (IL-22) is a cytokine that belongs to the IL-10 family of interleukins. It can be produced by T helper 22 (Th22) cells, T helper 1 (Th1) cells, T helper 17 (Th17) cells, natural killer 22 (NK22) cells, natural killer T (NKT) cells, innate lymphoid cells (ILCs), and γδ T cells. IL-22 acts via binding to a heterodimeric transmembrane receptor complex that consists of IL-22R1 and IL-10R2 and mainly contributes to the tissue repair and host defense. Transcription factors such as retinoid orphan receptor γt (RORγt) and signal transducer and activator of transcription 3 (STAT3), have been reported to play important roles in regulation of IL-22 expression. Recently, it has been demonstrated in several studies that microRNAs (miRNAs) potently regulate expression of interleukins, including production of IL-22. Here, we review current knowledge about regulators of IL-22 expression with a particular emphasis on the role of miRNAs.

Deceleration of glycometabolism impedes IgG-producing B-cell-mediated tumor elimination by targeting SATB1

B lymphocytes, known as antibody producers, mediate tumor cell destruction in the manner of antibody‐dependent cell‐mediated cytotoxicity; however, their anti‐tumor function seems to be weakened during tumorigenesis, while the underlying mechanisms remain unclear. In this study, we found that IgG mediated anti‐tumor effects, but IgG‐producing B cells decreased in various tumors. Considering the underlying mechanism, glycometabolism was noteworthy. We found that tumor‐infiltrating B cells were glucose‐starved and accompanied by a deceleration of glycometabolism. Both inhibition of glycometabolism and deprivation of glucose through tumor cells, or glucose‐free treatment, reduced the differentiation of B cells into IgG‐producing cells. In this process, special AT‐rich sequence‐binding protein‐1 (SATB1) was significantly silenced in B cells. Down‐regulating SATB1 by inhibiting glycometabolism or RNA interference reduced the binding of signal transducer and activator of transcription 6 (STAT6) to the promoter of germline Cγ gene, subsequently resulting in fewer B cells producing IgG. Our findings provide the first evidence that glycometabolic inhibition by tumorigenesis suppresses differentiation of B cells into IgG‐producing cells, and altering glycometabolism may be promising in improving the anti‐tumor effect of B cells.