MicroRNAs 15A and 16-1 Activate Signaling Pathways That Mediate Chemotaxis of Immune Regulatory B cells to Colorectal Tumors

Abstract

Background & aims: B cells infiltrate tumors, but little is known about how they affect tumor growth and progression. microRNA15A (MIR15A or miRNA15A) and microRNA16-1 (MIR16-1 or miRNA16-1) regulate cell proliferation, apoptosis, and drug resistance. We investigated their involvement in B-cell-mediated immune suppression by colorectal tumors.

Methods: Mice with disruptions of the gene cluster that encodes MIR15A and MIR16-1 (knockout mice), and control (C57BL/B6) mice were given azoxymethane with dextran sodium sulfate (AD) to induce formation of colorectal tumors. Mice were given anti-CD20 to delete B cells, or injections of agomir to increase MIR15A and MIR16-1. Proliferation of CD8⁺T cells was measured by carboxyfluorescein-succinimidyl-ester analysis. Colon tissues were collected from mice and analyzed by flow cytometry, microRNA (miRNA) sequencing, and for cytokine production. Intestinal epithelial cells (IECs) were isolated and transfected with miRNA mimics, to identify their targets. We analyzed miRNA expression patterns and quantified B cells in colorectal cancer tissue microarrays derived from 90 patients who underwent surgical resection, from July 2006 through April 2008, in Shanghai, China; expression data were compared with clinical outcomes.

Results: Tumors that developed in knockout mice following administration of AD were larger and contained greater numbers of B cells than tumors that grew in control mice. Most of the B cells in the tumors were positive for immunoglobulin A (IgA⁺). IgA⁺ B cells expressed high levels of immune regulatory molecules (programmed death ligand 1, interleukin 10, and transforming growth factor beta), and repressed the proliferation and activation of CD8⁺ T cells. Levels of MIR15A and MIR16-1 were reduced in colon tumors from mice, compared with nontumor colon tissue. Incubation of IECs with IL17A reduced expression of MIR15A and MIR16-1. Transgenic expression of MIR15A and MIR16-1 in IECs decreased activation of NF-κB and STAT1 by reducing expression of I-kappaB kinases; this resulted in reduced production of chemokine (C-X-C motif) ligands 9 and 10 and decreased chemotaxis of IgA⁺ B cells. Tumors in mice injected with AD and agomir grew more slowly than tumors in mice not given in agomir and contained fewer IgA⁺ B cells. We found a negative correlation between levels of MIR15A and MIR16-1 and numbers of IgA⁺B cells in human colorectal tumor tissues; high levels of MIR15A and MIR16-1 and low numbers of IgA⁺B cells were associated with longer survival times of patients.

Conclusions: We found increased levels of MIR15A and MIR16-1 to reduce numbers of IgA⁺ B cells in colorectal tumor tissues and correlate with increased survival time of patients. In mice that lack MIR15A and MIR16-1, colon tumors grow more rapidly and contain increased numbers of IgA⁺ B cells. MIR15A and MIR16-1 appear to activate signaling pathways required for B-cell-mediated immune suppression.

Keywords: Colon Cancer Model; Immune Regulation; Tumor Immunosuppression; microRNA15A/16–1.