Runx1-deficient afferents impair visceral nociception, exacerbating dextran sodium sulfate-induced colitis.

Colitis is a group of inflammatory and auto-immune disorders that affect the tissue lining of the gastrointestinal (GI) system. Studies of chemically-induced animal models of colitis have indicated that nociceptive afferents or neuropeptides have differing effects on GI inflammation. However, the molecular mechanisms involved in visceral pain and the role of visceral sensory afferents involved in the modulation of colitis remains unclear. A previous study demonstrated that Runx1, a Runt domain transcription factor, is restricted to nociceptors. In these neurons, Runx1 regulates the expression of numerous ion channels and receptors, controlling the lamina-specific innervation patterns of nociceptive afferents in the spinal cord. Moreover, mice that lack Runx1 exhibit specific defects in thermal and neuropathic pain. To examine the function of Runx1 in visceral nociception, we employed double-transgenic mice (WntCre: Runx1(F/F)), in which the expression of Runx1 was specifically disrupted in the sensory neurons. To determine the role of Runx1 in visceral pain sensation, the WntCre: Runx1(F/F) mice and their control littermates (Runx1(F/F)) were treated using dextran sodium sulfate (DSS) to induce colitis. The results indicated that disrupted Runx1 in the sensory afferents resulted in: (1) impairment of the visceral pain sensation in murine DSS-induced colitis; (2) exacerbating the phenotypes in murine DSS-induced colitis; (3) a differential effect on the production of pro- and anti-inflammatory cytokines in the colon tissues isolated from mice treated using DSS and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis; and (4) alteration of the distribution of lymphocytes and mast cells in mucosa. These results show that the function of Runx1 in sensory afferents is vital for modulating visceral pain and the neuro-immune axis.

Disrupting the CXCL12/CXCR4 axis disturbs the characteristics of glioblastoma stem-like cells of rat RG2 glioblastoma.

BACKGROUND: Glioblastoma stem-like cells (GSC) have been shown to promote tumor growth, tumor-associated neovascularization, therapeutic resistance, and metastasis. CXCR4 receptors have been found involved in the proliferation, metastasis, angiogenesis, and drug-resistant characteristics of glioblastoma. However, the role of CXCR4 in modulating the stem-like cell properties of rat glioblastoma remains ambiguous. METHODS: To explore the role of the CXCL12/CXCR4 axis in maintaining rat GSC properties, we disrupted the CXCR4 signaling by using small hairpin interfering RNA (shRNA). To investigate the role of the CXCL12/CXCR4 axis in maintaining rat GSC properties, we used a spheroid formation assay to assess the stem cell self-renewal properties. A western blot analysis and PCR arrays were used to examine the genes involved in proliferation, self-renewal, and cancer drug resistance. Finally, DNA content and flow cytometry, an immunohistochemical analysis, and methylcellulose colony formation, in vitro invasive and intracranial injection xenograft assays were employed to examine the disruptive effect of CXCR4 on the characteristics of GSCs of the RG2 cell line. RESULTS: Disrupting CXCR4 inhibited the proliferation of RG2 cells both in vitro and in vivo. The spheroid formation assay indicated that CXCR4 was vital for the self-renewal of RG2 GSCs. Disrupting the CXCL12/CXCR4 pathway also reduced the expression of GSC cell markers, including Nestin, ABCG2, and musashi (Msi), and the expression of genes involved in regulating stem cell properties, including Oct4, Nanog, maternal embryonic leucine zipper kinase (MELK), MGMT, VEGF, MMP2, and MMP9. CONCLUSION: The chemokine receptor CXCR4 is crucial for maintaining the self-renewal, proliferation, therapeutic resistance, and angiogenesis of GSCs of rat RG2 glioblastoma.

Lymphoid Enhancer Factor 1 Contributes to Hepatocellular Carcinoma Progression Through Transcriptional Regulation of Epithelial-Mesenchymal Transition Regulators and Stemness Genes.

Lymphoid enhancer factor 1 (LEF1) activity is associated with progression of several types of cancers. The role of LEF1 in progression of hepatocellular carcinoma (HCC) remains poorly known. We investigated LEF1 expression in HCC and its interactions with epithelial-mesenchymal transition (EMT) regulators (e.g., Snail, Slug, Twist) and stemness genes (e.g., octamer-binding transcription factor 4 [Oct4], sex determining region Y-box 2 [Sox2], Nanog homeobox [Nanog]). Microarray analysis was performed on resected tumor samples from patients with HCC with or without postoperative recurrence. LEF1 expression was associated with postoperative recurrence as validated by immunohistochemical staining in another HCC cohort. Among 74 patients, 44 displayed a relatively high percentage of LEF1 staining (>30% of HCC cells), which was associated with a reduced recurrence-free interval (P < 0.001) and overall survival (P = 0.009). In multivariate analysis, a high percentage of LEF1 staining was significantly associated with low albumin level (P = 0.035), Twist overexpression (P = 0.018), Snail overexpression (P = 0.064), co-expression of Twist and Snail (P = 0.054), and multinodular tumors (P = 0.025). Down-regulation of LEF1 by short hairpin RNA decreased tumor sphere formation, soft agar colony formation, and transwell invasiveness of HCC cell lines Mahlavu and PLC. Xenotransplant and tail vein injection experiments revealed that LEF1 down-regulation in Mahlavu reduced tumor size and metastasis. LEF1 up-regulation in Huh7 increased sphere formation, soft agar colony formation, and transwell invasiveness. LEF1 was shown to physically interact with and transcriptionally activate promoter regions of Oct4, Snail, Slug, and Twist. Furthermore, Oct4, Snail, and Twist transactivated LEF1 to form a regulatory positive-feedback loop. Conclusion: LEF1 plays a pivotal role in HCC progression through transcriptional regulation of Oct4 and EMT regulators.

Screening large numbers of expression patterns of transcription factors in late stages of the mouse thymus.

Transcription factor families are well known to be involved in the intrinsic pathways that regulate the organogenesis, early development, and microenvironment of the thymus. However, identification of the transcription factors (TFs) involved in the late development of the thymus, particularly later than embryonic day 15.5 (E15.5), is progressing slowly. In this study, we used in situ hybridization to screen numerous expression patterns of the TFs involved in the development of the mouse thymus. More than 400 members, including unique TFs and some transcription co-factors, were tested. Among the screened TFs, 160 were found to be expressed in the thymus after E15.5, and 74 of these were expressed in restricted areas.