Clinical and gonadal transcriptome analysis of 38,XX disorder of sex development pigs†.

Pigs serve as a robust animal model for the study of human diseases, notably in the context of disorders of sex development (DSD). This study aims to investigate the phenotypic characteristics and molecular mechanisms underlying the reproductive and developmental abnormalities of 38,XX ovotestis-DSD (OT-DSD) and 38,XX testis-DSD (T-DSD) in pigs. Clinical and transcriptome sequencing analyses were performed on DSD and normal female pigs. Cytogenetic and SRY analyses confirmed that OT/T-DSD pigs exhibited a 38,XX karyotype and lacked the SRY gene. The DSD pigs had higher levels of follicle-stimulating hormone, luteinizing hormone, and progesterone, but lower testosterone levels when compared with normal male pigs. The reproductive organs of OT/T-DSD pigs exhibit abnormal development, displaying both male and female characteristics, with an absence of germ cells in the seminiferous tubules. Sex determination and development-related differentially expressed genes shared between DSD pigs were identified in the gonads, including WT1, DKK1, CTNNB1, WTN9B, SHOC, PTPN11, NRG1, and NXK3-1. DKK1 is proposed as a candidate gene for investigating the regulatory mechanisms underlying gonadal phenotypic differences between OT-DSD and T-DSD pigs. Consequently, our findings provide insights into the molecular pathogenesis of DSD pigs and present an animal model for studying into DSD in humans.

Characterization of a membrane Fcγ receptor in largemouth bass (Micropterus saloumoides) and its response to bacterial challenge.

Fc receptors (FcRs), specific to the Fc portion of immunoglobulin (Ig), are required to regulate immune responses against pathogenic infections. However, FcγR is a member of FcRs family, whose structure and function remains to be elucidated in teleost fish. In this study, the FcγRII, from largemouth bass (Micropterus saloumoides), named membrane MsFcγRII (mMsFcγRII), was cloned and identified. The opening reading frame (ORF) of mMsFcγRII was 750 bp, encoding 249 amino acids with a predicted molecular mass of 27 kDa. The mMsFcγRII contained a signal peptide, two Ig domains, a transmembrane domain, and an intracellular region, which was highly homology with FcγR from other teleost fish. The mRNA expression analysis showed that mMsFcγRII was widely distributed in all tested tissues and with the highest expression level in spleen. After bacterial challenge, the expression of mMsFcγRII was significantly upregulated in vivo (spleen and head kidney), as well as in vitro (leukocytes from head kidney). The subcellular localization assay revealed that mMsFcγRII was mostly observed on the membrane of HEK293T cells which were transfected with mMsFcγRII overexpression plasmid. Flow cytometric analysis showed that natural mMsFcγRII protein was highly expressed in head kidney lymphocytes. Moreover, indirect immunofluorescence assay and pull-down assay indicated that mMsFcγRII could bind to IgM purified from largemouth bass serum. These results suggested that mMsFcγRII was likely to play an influential role in the immune response against pathogens and provided valuable insights for studying the function of FcRs in teleost.

Transcriptome analysis reveals a potential regulatory mechanism of the lnc-5423.6/IGFBP5 axis in the early stages of mouse thymic involution.

Age-related thymic involution is one of the significant reasons for induced immunity decline. Recent evidence has indicated that lncRNAs are widely involved in regulating organ development. However, the lncRNA expression profiles in mouse thymic involution have not been reported. In this study, we collect mouse thymus at the ages of 1 month, 3 months, and 6 months for sequencing to observe the lncRNA and gene expression profiles in the early stages of thymic involution. Through bioinformatics analysis, a triple regulatory network of lncRNA-miRNA-mRNA that contains 29 lncRNAs, 145 miRNAs and 12 mRNAs that may be related to thymic involution is identified. Among them, IGFBP5 can reduce the viability, inhibit proliferation and promote apoptosis of mouse medullary thymic epithelial cell line 1 (MTEC1) cells through the p53 signaling pathway. In addition, miR-193b-3p can alleviate MTEC1 cell apoptosis by targeting IGFBP5. Notably, lnc-5423.6 can act as a molecular sponge of miR-193b-3p to regulate the expression of IGFBP5. In summary, lnc-5423.6 enhances the expression of IGFBP5 by adsorption of miR-193b-3p, thereby promoting MTEC1 cell apoptosis.

Fcγ receptor-mediated phagocytosis pathway was involved in phagocytosis of mIgM+ B lymphocytes from largemouth bass (Micropterus salmoides).

The potential for phagocytosis has been proven in teleost B cells, but the research on the regulatory mechanism of phagocytosis remains lacking. In this study, three largemouth bass (Micropterus salmoides) (15 ± 5 g) were injected intraperitoneally with Nocardia seriolae (105 CFU/100 µl/fish) in vivo, and their spleen was collected at 72 h post-infection for mRNA-seq. After the de novo assembly of the paired-end reads, 73,622 unigenes were obtained. Gene expression profiling revealed that 2043 unigenes were differentially expressed after N. seriolae infection, comprising 1285 upregulated and 758 downregulated unigenes (q-value <0.05, log2FC > |2|) of which 181 genes were involved in phagocytosis. The Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis demonstrated that 12 differentially expressed genes (DEG) associated with phagocytosis were enriched in the Fcγ receptor-mediated phagocytosis signalling pathway. In vitro, the phagocytic ability of mIgM+ B lymphocytes was validated using indirect immunofluorescence assay (IIFA) and fluorescence activating cell sorter (FACS), and the phagocytosis rates of the mIgM+ B lymphocytes incubated with a Lyn inhibitor had decreased from 18.533 ± 6.00% to 11.610 ± 4.236% compared with the unblocked group. These results suggested that the Fcγ receptor-mediated phagocytosis signalling pathway had participated in the phagocytosis of B cells and provide further insight into the role of B cells in innate immunology.

Effects of PAMK on lncRNA, miRNA, and mRNA expression profiles of thymic epithelial cells.

Polysaccharides from Atractylodes macrocephala Koidz (PAMK) can promote the proliferation of thymocytes and improve the body's immunity. However, the effect of PAMK on thymic epithelial cells has not been reported. Studies have shown that miRNAs and lncRNAs are key factors in regulating cell proliferation. In this study, we found that PAMK could promote the proliferation of mouse medullary thymic epithelial cell line 1 (MTEC1) cells through CCK-8 and EdU experiments. To further explore its mechanism, we detected the effect of PAMK on the expression profiles of lncRNAs, miRNAs, and mRNAs in MTEC1 cells. The results showed that PAMK significantly affected the expression of 225 lncRNAs, 29 miRNAs, and 800 mRNAs. Functional analysis showed that these differentially expressed genes were significantly enriched in cell cycle, cell division, NF-kappaB signaling, apoptotic process, and MAPK signaling pathway. Finally, we used Cytoscape to visualize lncRNA-miRNA-mRNA(14 lncRNAs, 17 miRNAs, 171 mRNAs) networks based on ceRNA theory. These results suggest that lncRNAs and miRNAs may be involved in the effect of PAMK on the proliferation of MTEC1 cells, providing a new research direction for exploring the molecular mechanism of PAMK promoting the proliferation of thymic epithelial cells.

iTRAQ-based proteomic analysis of 17ß-Estradiol-induced anti-proliferation and apoptosis in mouse thymic epithelial cells by disturbing ribosomal biogenesis.

Many evidences have suggested that estrogen was associated with thymic atrophy and suppressed thymocyte functions. Thymic epithelial cells (TECs), as a crucial constituent of thymic stroma support a unique microenvironment for thymocyte maturation, but the effects of estrogen on TECs were poorly understood. In our study, we found that 17ß-Estradiol (17ß-E2), one of the primary estrogens, could significantly inhibit cell proliferation, and cause cell cycle arrest in G2/M phase and apoptosis in mouse thymic epithelial cell line 1 (MTEC1 cells) with time- and dose- dependent. Above all, we provided the systemic and sufficient proteomic profiling of 17ß-E2 (50 nmol/L) acting on MTEC1 cells through isobaric tags for relative and absolute quantitation and LC-MS/MS (Liquid Chromatography Mass Spectrometry/Mass Spectrometry). A total of 71 differentially expressed proteins were identified, of which 61 were up-regulated and 10 were down-regulated. Particularly, the differential expression of abundant ribosomal proteins (RPs) was drawing our attention, including RPL3, RPL4, RPS11, RPL17, RPL5, RPS9, RPL13, RPL23A, RPLP2, RPS15A, and RPL29. Most of these proteins have been widely reported exerting extra-ribosomal function associated with the proliferation and apoptosis of distinct cell types, but not yet observed in TECs. Moreover, bioinformatics analysis revealed that disturbance of ribosomal biogenesis was closely related to the anti-proliferation and apoptosis in MTEC1 cells upon 17ß-E2. These data highlighted the possible mechanisms of 17ß-E2 on MTEC1 cells through showing adequate differential protein expression profiles. We inferred that 17ß-E2 induced anti-proliferation and apoptosis in MTEC1 cells in response to alterations of ribosome biogenesis and RPs expression, which will contribute to gaining insight into the internal mechanism of thymic degeneration and exploiting to treat autoimmune diseases in the future.

Analysis of phagocytosis by mIgM+ lymphocytes depending on monoclonal antibodies against IgM of largemouth bass (Micropterus salmoides).

The phagocytic actives of B cells in fish have been proven in recent years. In this study, five positive hybridomas secreting monoclonal antibodies (MAbs) against largemouth bass IgM were produced. Indirect immunofluorescence assay (IFA) demonstrated that five MAbs could specifically recognize membrane-bound IgM (mIgM) molecule of largemouth bass. Indirect ELISA and Western blotting analysis showed that all the five MAbs had no cross-reactions with the other two teleost IgMs. Flow cytometry analysis (FCM) revealed that the percentages of largemouth bass mIgM+ lymphocytes in head kidney, peripheral blood and spleen were 51.66 ± 0.608%, 16.5 ± 1.235% and 42.92 ± 1.091%, respectively. In addition, the phagocytosis rates of mIgM + lymphocytes ingesting Nocardia seriolae from head kidney, peripheral blood and spleen were calculated to be 5.413 ± 0.274%, 16.6 ± 0.289% and 26.3 ± 0.296%, respectively. The qPCR results of sorted cells indicated that most inflammatory cytokines (IFNγ, IL-1ß, IL-2, IL-12ß, IL-34, IL-10), chemokine (CXCL12), chemokines receptors (CXCR2, CXCR4) and genes (FcγRⅠa, NCF1, CFL, ARP2/3, CD45, Syk, MARCKS) related to FcγR-mediated phagocytic signaling pathway in phagocytic mIgM+ lymphocytes were up-regulated significantly (P < 0.05). Taken together, the results suggested that the MAb (MM06H) produced in this paper could be used as a tool to study mIgM+ lymphocytes of largemouth bass, and FcγR may participate in the phagocytosis of mIgM+ lymphocytes, which is helpful to further study the role of mIgM+ lymphocytes in innate immunity.

Proteomic analysis of zearalenone toxicity on mouse thymic epithelial cells.

Zearalenone (ZEA) is one of the most major food contaminants in cereal crops worldwide, risking health of both livestock and humans. This study aimed to assess the cytotoxicity and the underlying mechanism of ZEA on thymic epithelial cells. By using proteomics analysis, we identified 596 differentially expressed proteins in MTEC1 cells upon zearalenone exposure, of which 245 were upregulated and 351 were downregulated. Gene ontology (GO) analysis suggested that differentially expressed proteins were participated in protein synthesis, oxidative phosphorylation, and ATP binding. KEGG pathway enrichment analysis showed that differentially expressed proteins were mainly related to mitochndrial metabolism, such as citrate cycle (TCA cycle) and oxidative phosphorylation. We demonstrated that ZEA treatment was able to increase the intracellular reactive oxygen species (ROS) level, to decrease ΔΨm, ATP level, and the copy number of mtDNA, leading to necrotic cell death. Moreover, we showed that ZEA treatment inhibited cell proliferation and induced G2/M phase arrest by downregulation of proliferation-associated proteins ERK, p-ERK, CDK1, and p-CHK1. Taken together, we found that the toxicity of ZEA on thymic epithelial cells is mainly caused by the inhibition of mitochondrial dysfunction and cell proliferation. Our study might open new avenues for treatment strategies.

Comprehensive analysis of lncRNAs, miRNAs and mRNAs related to thymic development and involution in goose.

Thymic involution is a sign of immunosenescence, but little is known about it in goose. miRNAs and lncRNAs are critical factors regulating organ growth and development. In this study, we comprehensively analyzed the profiles of lncRNAs, miRNAs and mRNAs during the development and involution of the thymus in Magang goose. The results showed that 2436 genes, 16 miRNAs and 417 lncRNAs were differentially co-expressed between the developmental (20-embryo age, 3-day post-hatch and 3-month age) and degenerative (6-month age) stages. The functional analysis showed that these differentially expressed genes were significantly enriched in cell proliferation, cell adhesion, apoptotic signaling pathway, and Notch signaling pathway. In addition, we established a gene-gene network through the STRING database and identified 50 key genes. Finally, we constructed a miRNA-mRNA network followed by a lncRNA-miRNA-mRNA network. These results suggest that lncRNAs and miRNAs may be involved in the regulation of thymic development and involution in goose.

Functional Characterization of Largemouth Bass (Micropterus salmoides) Soluble FcγR Homolog in Response to Bacterial Infection.

Fc receptors (FcRs) are key players in antibody-dependent cellular phagocytosis (ADCP) with their specific recognition of the Fc portion of an immunoglobulin. Despite reports of FcγR-mediated phagocytosis in mammals, little is known about the effects of soluble FcγRs on the immune response. In this study, FcγRIα was cloned from the largemouth bass (Micropterus salmoides) (MsFcγRIα). Without a transmembrane segment or a cytoplasmic tail, MsFcγRIα was identified as a soluble form protein and widely distributed in the spleen, head kidney, and intestine. The native MsFcγRIα was detected in the serum of Nocardia seriolae-infected largemouth bass and the supernatants of transfected HEK293 cells. Additionally, it was verified that the transfected cells' surface secreted MsFcRIα could bind to largemouth bass IgM. Moreover, the expression changes of MsFcγRIα, Syk, and Lyn indicated that MsFcγRIα was engaged in the acute phase response to bacteria, and the FcγR-mediated phagocytosis pathway was activated by Nocardia seriolae stimulation. Furthermore, recombinant MsFcγRIα could enhance both reactive oxygen species (ROS) and phagocytosis to Nocardia seriolae of leukocytes, presumably through the interaction of MsFcγRIα with a complement receptor. In conclusion, these findings provided a better understanding of the function of soluble FcγRs in the immune response and further shed light on the mechanism of phagocytosis in teleosts.

miR-199b-5p enhances the proliferation of medullary thymic epithelial cells via regulating Wnt signaling by targeting Fzd6.

Thymic epithelial cells (TECs) are essential regulators of T-cell development and selection. miRNAs play critical roles in regulating TEC proliferation during the process of thymic aging. Our previous studies revealed that miR-199b-5p was upregulated in TECs from 1- to 3-month-old mice. But its function and potential mechanism are not clear. We hypothesized that miR-199b-5p may play an important role in age-related thymus involution via targeting some genes. To confirm it, the murine thymic epithelial cell line 1 (MTEC1) cells were used. Our results showed that overexpression of miR-199b-5p can enhance MTEC1 cell proliferation. On the contrary, repression of miR-199b-5p can inhibit MTEC1 cell proliferation. Meanwhile, it was confirmed that frizzled receptor 6 (Fzd6) is the direct target gene of miR-199b-5p. Furthermore, overexpression of miR-199b-5p can upregulate the expressions of ß-catenin, Tcf7, Wnt4, and C-myc to activate Wnt signaling and cell cycle signaling. Silence of Fzd6 and co-transfection with siFzd6 and miR-199b-5p mimic/inhibitor confirmed that the biological function of miR-199b-5p is indeed by targeting Fzd6 in medullary TECs. Overall, miR-199b-5p is an important regulator in medullary TEC proliferation through targeting Fzd6 to activate Wnt signaling and cell cycle signaling. Our data indicate that miR-199b-5p may block the process of thymic aging and be a potential therapeutic target for thymus involution.

Characterization of a novel brain cell line from Jian carp (Cyprinus carpio var. Jian).

Jian carp (Cyprinus carpio var. Jian) is an economically important cultured fish in China. Currently, it is facing threats from infectious diseases including koi herpesvirus (KHV). Here, we established a new cell line, designated CCB-J, derived from the brain tissue of the Jian carp. CCB-J cells grew well in Leibovitz's L-15 medium containing 20% fetal bovine serum at 25 °C and have been subcultured for more than 60 passages. At the 30th passage, analysis showed that the number of chromosomes was 100, which is identical to that of other carp variants. Sequencing of the 18S ribosomal DNA confirmed that CCB-J originated from Jian carp. After transfection with the pEGFP-N1 plasmid, green fluorescence was observed in CCB-J. The replication of KHV in CCB-J cells was confirmed by RT-PCR and transmission electron microscopy. The viral titers of KHV in CCB-J cells and CCB cells, which have been widely used in the study of KHV, reached 103.9 and 101.8 median tissue culture infectious dose (TCID50/mL), respectively, within 14 days. The result of TaqMan PCR revealed that CCB-J cells were more sensitive to KHV than CCB cells. Meanwhile, a cytopathic effect (CPE) was also observed in the CCB-J cells in a shorter time post-infection compared with CCB cells. In summary, the CCB-J cell line will be a useful tool in the study of viral pathogenesis and vaccine research.

Protective immunity against CyHV-3 infection via different prime-boost vaccination regimens using CyHV-3 ORF131-based DNA/protein subunit vaccines in carp Cyprinus carpio var. Jian.

Cyprinid Herpesvirus 3 (CyHV-3), also known as Koi Herpesvirus (KHV), causes Koi Herpesvirus Disease (KHVD) which leads to serious economic losses worldwide. To exploit DNA/subunit vaccine candidates, CyHV-3 ORF131 gene and cDNA was cloned and analyzed in the present study. Major B cell epitopes of deduced CyHV-3 pORF131 was also predicted. Then the complete CDS of CyHV-3 ORF131 was inserted into pEGFP-N1 vector and a modified pYD1/EBY100 system to construct the DNA and subunit vaccine, respectively. Subsequently, carp were immunized with homologous and heterologous prime-boost regimens relying on the constructed DNA and oral subunit vaccines. Then the protective immunity generated from different vaccines and regimens as well as the capacity of yeast (Saccharomyces cerevisiae) as an oral vaccine vehicle was evaluated. Our study confirmed that CyHV-3 ORF131 gene consisted of 2 introns and 3 exons encoding a 428 amino acids peptide. Further analysis indicated that four fragments of CyHV-3 pORF131 contained the major B cell epitopes (Cys20~Val140, Ser169~Tyr245, Thr258~Pro390, Phe414~Gln428), which could be linked and expressed in E. coli (BL21) as a truncated pORF131. The expression of full-length CyHV-3 pORF131 by pEGFP-N1 and yeast surface display was verified by In vitro assays before vaccination. Immunization of carp with CyHV-3 ORF131 DNA and subunit vaccines could evoke the activation of immune-related genes such as CXCa, CXCR1, IL-1ß, TNF-α, INF-a1, Mx-1, IgM, IgT1 and production of specific serum IgM measured by ELISA. RPS (relative percent of survival) ranging from 53.33% to 66.67% was acquired post challenge test. Moreover, flow cytometry analysis illustrated the delivery of surface-displayed CyHV-3 pORF131 to midgut after oral gavage. Thus, our findings suggest that CyHV-3 ORF131 can serve as DNA/subunit vaccines candidate and the yeast as an ideal oral vaccine vehicle.

Profiling analysis of 17ß-estradiol-regulated lncRNAs in mouse thymic epithelial cells.

Thymus is the primary organ for T cell differentiation and maturation. Many studies have demonstrated that estrogen plays a crucial role in thymic epithelial cell (TEC) proliferation during thymic involution. LncRNAs are involved in various biological processes; however, estrogen-mediated lncRNA expression in TECs has not been yet reported. To address this question, the mouse medullary thymic epithelial cell line 1 (MTEC1) was treated with 17ß-estradiol (E2). By using CCK8 assay and flow cytometry, we found that E2 was able to inhibit viability and proliferation of MTEC1 cells. The expression profiles of lncRNAs in MTEC1 cells with or without E2 treatment were then measured by RNA-Seq, and a total of 962 lncRNAs and 2,469 mRNAs were shown to be differentially expressed. The reliability of RNA-Seq was confirmed by quantitative RT-PCR. Correlation analysis was conducted to investigate the potential function of lncRNAs. According to gene ontology (GO) analysis, differentially expressed lncRNAs were mainly related to cell proliferation, cell cycle and cell apoptosis. KEGG pathway analysis indicated that these lncRNAs were associated with several pathways, namely immunological activity, metabolism and cytokine-cytokine receptor interaction. In conclusion, our study provided a novel direction for studying the relationship between lncRNAs and E2 in the thymus.

Integrated microRNA and mRNA sequencing analysis of age-related changes to mouse thymic epithelial cells.

MicroRNAs (miRNAs) play key roles in the regulation of gene expression during multiple physiological processes, including early development, differentiation, and ageing. However, their involvement in age-related thymic involution is not clear. In this study, we profiled the global transcriptome and miRNAome of thymic epithelial cells in 1- and 3-month-old male and female mice, and predicted the possible transcription factors and target genes of the four most significantly differentially expressed miRNAs (DEMs) (miR-183-5p, miR-199b-5p, miR-205-5p, and miR-200b-3p) by performing bioinformatics analyses. We also evaluated the relationships between the significantly DEMs and mRNAs. We performed quantitative polymerase chain reaction to confirm the changes in the expression of the miRNAs and their predicted target genes. We found that miR-183-5p, miR-199b-5p, miR-205-5p, and miR-200b-3p can be used as a biomarker group for mouse thymus development and involution. In addition, the predicted target genes (Ptpn4, Slc2a9, Pkib, Pecam1, and Prkdc), which were identified by mRNA sequencing analysis, were mainly involved in growth, development, and accelerated senescence. In conclusion, miRNAs and their predicted target genes likely play important roles in thymus development and involution. To the best of our knowledge, this is the first study to systematically analyze the relevance of miRNAs and their targets by mRNA sequencing in mouse thymic epithelial cells. © 2018 IUBMB Life, 70(7):678-690, 2018.

Transcriptional profiling analysis of Zearalenone-induced inhibition proliferation on mouse thymic epithelial cell line 1.

Zearalenone (ZEA) was a mycotoxin biosynthesized by a variety of Fusarium fungi via a polypeptide pathway. ZEA has significant toxic reaction on immune cells. Thymic epithelial cells (TECs) as a crucial constituent of thymic stroma can provide unique microenvironment for thymocyte maturation, but the mechanism of ZEA affecting the TECs is poorly understood. The basic data about gene expression differences for the ZEA on thymic epithelial cell line 1 (MTEC1) will help us to elucidate this mechanism. Here, cell viability and proliferation assay and transcriptome sequencing on MTEC1 treated with ZEA were performed. 4188 differentially expressed genes (DEGs) between ZEA treated and control groups were identified, confirmed and analyzed. Our results showed that 10-50µg/ml ZEA significantly inhibited MTEC1 proliferation and arrested cell cycle at G2/M phase. Gene ontology and KEGG pathway analysis revealed that Chemokine, JAK-STAT and Toll-like receptor signaling pathway, were involved in the cell cycle pathway. 16 key genes involved in the cell cycle processes were validated and the results suggested that Mitotic catastrophe (MC) may take part in ZEA inhibition of METC1 cell proliferation. These data highlighted the importance of cell cycle pathway in MTEC1 treated with ZEA, and will contribute to get the molecular mechanisms of ZEA inhibition of MTEC1 cell proliferation.

Age and sex differences in microRNAs expression during the process of thymus aging.

The gender-biased thymus involution and the importance of microRNAs (miRNAs, miRs) expression in modulating the thymus development have been reported in many studies. However, how males and females differ in so many ways in thymus involution remains unclear. To address this question, we investigated the miRNA expression profiles in both untreated 3- and 12-month-old female and male mice thymuses. The results showed that 7 and 18 miRNAs were defined as the sex- and age-specific miRNAs, respectively. The expression of miR-181c-5p, miR-20b-5p, miR-98b-5p, miR-329-3p, miR-341-5p, and miR-2137 showed significant age-difference in mice thymus by quantitative polymerase chain reaction. High expression levels of miR-2137 were detected in mice thymic epithelial cells and gradually increased during the process of thymus aging. MiR-27b-3p and miR-378a-3p of the female-biased miRNAs were confirmed as the sex- and estrogen-responsive miRNAs in mice thymus in vivo. Their potential target genes and the pathway were identified by the online software. Possible regulation roles of sex- and age-specific miRNA expression during the process of thymus aging were discussed. Our results suggested that these miRNAs may be potential biomarkers for the study of sex- and age-specific thymus aging and involution.

MicroRNA-181a-5p enhances cell proliferation in medullary thymic epithelial cells via regulating TGF-ß signaling.

The expression profiles of miRNAs in thymus tissues from mice of different age have been demonstrated in our previous study. After an integrated analysis of the miRNA expression profiles, we demonstrated that the expression of miR-181a-5p was significantly decreased in thymic epithelial cells (TECs) from 10- to 19-month-old mice when compared with that in TECs from 1-month-old mice by quantitative reverse transcriptase polymerase chain reaction. We hypothesized that miR-181a-5p in TECs might be associated with the age-related thymus involution through regulating some genes or signaling pathway. To test this hypothesis, the mouse medullary thymic epithelial cells (MTEC1) were used. Transfection with miR-181a-5p mimic promoted the proliferation of MTEC1 cells, but did not affect apoptosis. The effect was reversed when the expression of miR-181a-5p was suppressed in MTEC1 cells. Furthermore, the transforming growth factor beta receptor I (Tgfbr1) was confirmed as a direct target of miR-181a-5p by luciferase assay. Moreover, it was found that overexpression of miR-181a-5p down-regulated the phosphorylation of Smad3 and blocked the activation of the transforming growth factor beta signaling. Nevertheless, an inversely correlation was observed between the expression of Tgfbr1 and miR-181a-5p in TECs derived from mice of different age. Collectively, we provide evidence that miR-181a-5p may be an important endogenous regulator in the proliferation of TECs, and the expression levels of miR-181a-5p in TECs may be associated with the age-related thymus involution.

MiR-181a-5p regulates 3T3-L1 cell adipogenesis by targeting Smad7 and Tcf7l2.

MicroRNAs are highly conserved non-coding small RNAs participating in almost all kinds of biological activities. MiR-181a has been reported to be involved in the differentiation of porcine primary preadipocytes, but the profound effect of miR-181a-5p on 3T3-L1 adipocyte differentiation and proliferation is still unclear. In this study, we found that supplementation of miR-181a-5p in 3T3-L1 cells significantly promoted the adipogenesis and inhibited cell proliferation with increased expression of adipogenic marker genes including peroxisome proliferator-activated receptor gamma (Pparγ), CCAAT/enhancer-binding protein alpha (C/ebpα), fatty acid-binding protein 4 (Fabp4), and Adiponectin, accompanied by an accumulation of lipid droplet, an increase of triglyceride content, and a decrease of cell proliferation. Furthermore, by using the luciferase assay, Smad7 and Tcf7l2, two important members of transforming growth factor-ß (TGFß) and Wnt signaling pathway, were proven to be the direct target genes of miR-181a-5p. Moreover, supplementation of miR-181a-5p in 3T3-L1 cells altered the expressions of proteins involved in the TGFß signaling pathway, such as TGFBR1, p-SMAD3, SMAD4, c-MYC, and p15. Taken together, these results indicate that miR-181a-5p promotes 3T3-L1 preadipocyte differentiation and adipogenesis through regulating TGFß/Smad and Wnt signaling pathway by directly targeting Smad7 and Tcf7l2.

MicroRNA-195a-5p inhibits mouse medullary thymic epithelial cells proliferation by directly targeting Smad7.

MiR-195 has been implicated in inhibiting cell proliferation in different types of tumors. Whether it contributes to the process of thymic epithelial cells (TECs) proliferation remains unclear. In this study, we found that miR-195a-5p was highly up-regulated in the TECs isolated from the aging mice. Further experiments showed that miR-195a-5p mimic transfection inhibited the proliferation of mouse medullary thymic epithelial cell line 1 (MTEC1), whereas the transfection of miR-195a-5p inhibitor in MTEC1 had the opposite effect. In addition, miR-195a-5p had no obvious effect on MTEC1 apoptosis. Furthermore, Smad7, a negative regulator of transforming growth factor ß pathway, was confirmed as a direct target of miR-195a-5p by luciferase assays. Taken together, our results indicate that miR-195a-5p inhibits MTEC1 proliferation, at least in part, via down-regulation of Smad7.