This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Background: A standardized extract of cultured Lentinula edodes mycelia (ECLM), an extract from cultured Lentinula edodes, has been reported to suppress breast cancer stem cell proliferation by regulating microRNA (miR) expression. Natural antisense RNAs (ASs), a type of protein non-coding RNA, can regulate the expression of protein-coding genes by acting as a competing endogenous RNA (ceRNA) that adsorbs miRNAs, resulting in the prevention of mRNA degradation, and can also form a transient RNA duplex with mRNA. EphA2, a receptor tyrosine kinase, is typically expressed at low levels in normal epithelial cells, whereas its overexpression has been widely observed in numerous solid tumors and is associated with cell transformation, primary tumor initiation, and tumor progression. Objective: This study aimed to investigate the effect of ECLM on the expression of both EphA2 mRNA and endogenous AS to this mRNA, which could negatively affect human breast carcinoma cell proliferation. Methods: We used MCF7 and MDA-MB-231 human breast carcinoma cells, which were sub-cultured three times in the presence of optimized concentrations of ECLM. The effect of ECLM on the expression of EphA2 AS and mRNA was analyzed by RT-qPCR. miRNAs targeting both EphA2 AS and EphA2 mRNA and their RNA miR response elements (MREs) were predicted and analyzed by RT-qPCR and luciferase reporter assays. Results: ECLM suppressed the proliferation of MCF7 and MDA-MB-231 cells in a dose-dependent manner. In cells for which proliferation was negatively affected by ECLM, EphA2 AS and mRNA expression was also significantly inhibited by ECLM. Although neutralization of miR-335 led to the de-repression of both EphA2 AS and mRNA, results did not fully support the possibility that EphA2 AS might function as a ceRNA to regulate EphA2 mRNA levels. Conclusion: ECLM suppressed the proliferation of breast carcinoma cells in a specific dose-dependent manner. This suppressive effect was associated with a concordant reduction in both EphA2 AS and mRNA expression. These effects were not thought to occur via the reported ceRNA effect. These results thus suggest that ECLM could regulate EphA2 AS and mRNA expression by forming a transient RNA duplex formation, thereby stabilizing EphA2 mRNA. Keywords: ECLM, regulatory RNA, antisense RNA, microRNA, EphA2

Animal models are essential for basic and clinical research on virus diseases. Humanized mice (mice reconstituted with human hematopoietic cells) have been effectively used for various virus studies as small animal models. Studies on human-tropic HIV-1 have also been performed using different humanized mouse models. Various humanized mice have been generated using distinct mouse strains and engraftment methods. These different techniques affect the reconstitution of human hematopoietic cells in individual mice, and in turn the HIV-1 replication in vivo. In this report, we describe the details of the generation method of humanized mice, i.e., severely immunodeficient mice (NSG mice) transplanted with human CD133-positive cells via intra-bone marrow injection (IBMI). It has been shown that the CD133-positive cells are highly capable to generate CD34-positive cells in vivo and IBMI is an excellent methodology for lymphoid and myeloid cell repopulation. In humanized mice transplanted with CD133-positive cells into the bone marrow, human lymphocytes were increased 3 months after the transplantation and a steady increase in CD4-positive cells was observed until 6–8 months after the transplantation. In order to test the utility of our system, CXCR4-tropic and CCR5-tropic HIV-1 clones were intraperitoneally inoculated into the resultant humanized mice 6–8 months after the transplantation. Upon inoculation at the same dose of viruses, the plasma viral load in CCR5-tropic HIV-1-inoculated mice peaked earlier than that in CXCR4-tropic HIV-1-inoculated mice (2–3 weeks vs 5–10 weeks post-inoculation). While a rapid decrease in CD4-positive cells was observed at the peak or prior to the peak of viremia for CXCR4-tropic HIV-1-inoculated mice, CD4-positive cells were gradually decreased in CCR5-tropic HIV-1-inoculated mice. Upon inoculation at the same dose of viruses, a Nef-deleted R5-tropic HIV-1 exhibited retarded growth kinetics in the inoculated mice compared to the parental virus (around 8 weeks vs 2–3 weeks post-inoculation), which appears to reflect the decrease in replication potential in primary cells. Taken all together, in addition to the humanized mice reported so far, our humanized mice generated by transplanting CD133-positive cells with the IBMI method would be an appropriate prototype model for understanding HIV-1 biology in vivo.

Immune responses in humanized mice are generally inefficient without co-transplantation of human thymus or HLA transgenes. Previously, we generated humanized mice via the intra-bone marrow injection of CD133+ cord blood cells into irradiated adult immunodeficient mice (IBMI-huNSG mice), which could mount functional immune responses against HTLV-1, although the underlying mechanisms were still unknown. Here, we investigated thymocyte development in IBMI-huNSG mice, focusing on the roles of human and mouse MHC restriction. IBMI-huNSG mice had normal developmental profiles but aberrant thymic structures. Surprisingly, the thymic medulla-like regions expanded after immunization due to enhanced thymocyte expansion in association with the increase in HLA-DR+ cells, including CD205+ dendritic cells (DCs). The organ culture of thymus from immunized IBMI-huNSG mice with a neutralizing antibody to HLA-DR showed the HLA-DR-dependent expansion of CD4 single positive thymocytes. Mature peripheral T-cells exhibited alloreactive proliferation when co-cultured with human peripheral blood mononuclear cells. Live imaging of the thymus from immunized IBMI-huNSG mice revealed dynamic adhesive contacts of human-derived thymocytes and DCs accompanied by Rap1 activation. These findings demonstrate that an increase in HLA-DR+ cells by immunization promotes HLA-restricted thymocyte expansion in humanized mice, offering a unique opportunity to generate humanized mice with ease.