Reduced frequencies of Foxp3+GARP+ regulatory T cells in COPD patients are associated with multi-organ loss of tissue phenotype.

BACKGROUND: Expression of glycoprotein A dominant repeat (GARP) has been reported to occur only in activated human naturally occurring regulatory T cells (Tregs) and their clones, and not in activated effector T cells, indicating that GARP is a marker for bona fide Tregs. A different phenotype of chronic obstructive pulmonary disease (COPD) may have a different immunologic mechanism. OBJECTIVE: To investigate whether the distribution of Tregs defined by GARP is related to the multi-organ loss of tissue phenotype in COPD. METHODS: GARP expression on T cells from peripheral blood and bronchoalveolar lavage (BAL) collected from patients with COPD was examined by flow cytometry. The correlation of GARP expression to clinical outcomes and clinical phenotype, including the body mass index, lung function and quantitative computed tomography (CT) scoring of emphysema, was analyzed. RESULTS: Patients with more baseline emphysema had lower forced expiratory volume, body mass index (BMI), worse functional capacity, and more osteoporosis, thus, resembling the multiple organ loss of tissue (MOLT) phenotype. Peripheral Foxp3+GARP+ Tregs are reduced in COPD patients, and this reduction reversely correlates with quartiles of CT emphysema severity in COPD. Meanwhile, the frequencies of Foxp3+GARP- Tregs, which are characteristic of pro-inflammatory cytokine production, are significantly increased in COPD patients, and correlated with increasing quartiles of CT emphysema severity in COPD. Tregs in BAL show a similar pattern of variation in peripheral blood. CONCLUSION: Decreased GARP expression reflects more advanced disease in MOLT phenotype of COPD. Our results have potential implications for better understanding of the immunological nature of COPD and the pathogenic events leading to lung damage.

Dexamethasone reduces autoantibody levels in MRL/lpr mice by inhibiting Tfh cell responses.

Previous studies have shown that dexamethasone (Dex) reduces the levels of anti-nuclear (ANA) and anti-dsDNA antibodies in MRL/lpr mice (a mouse model of SLE). However, the effect of Dex on T follicular helper (Tfh) cells is less documented. Here, using the MRL/lpr mouse model, we investigated the influence of Dex on Tfh cells and potential underlying mechanisms. The data showed that the proportion of Tfh cells, identified as CD4+ CXCR5+ ICOS+ , CD4+ CXCR5+ PD-1+ or CD4+ BCL-6+ cells, markedly decreased after treatment with the Dex, in both Balb/c mice and MRL/lpr mice. Dex significantly inhibited IL-21 expression at both the mRNA and the protein levels. Dex also significantly reduced the proportion of germinal centre B cells and decreased serum IgG, IgG2a/b and IgA levels. Moreover, a positive correlation between the proportion of Tfh cells (CD4+ CXCR5+ ICOS+ , CD4+ CXCR5+ PD-1+ or CD4+ BCL-6+ ) and autoantibodies was observed. Dex significantly increased the Prdm1 and Stat5b mRNA expression and decreased the Bcl-6 and c-Maf mRNA expression of CD4+ T cells. In brief, Dex inhibited the Tfh development, which relies on many other transcription factors in addition to Bcl-6. Our data indicate that Dex can be used as a Tfh cell inhibitor in SLE.

Use of aspirin in the prevention of colorectal cancer through TIGIT-CD155 pathway.

Colorectal cancer (CRC) is one of the most widespread malignant cancers, with a high incidence and mortality all over the world. Aspirin (ASA) otherwise known as acetylsalicylic acid, is a non-steroidal anti-inflammatory drug that has shown promising results in the prevention of chronic diseases, including several cancers. In previous studies, aspirin has been shown to reduce the incidence of CRC. Immune checkpoint blockade of T cell Ig and ITIM domain receptor (TIGIT) alone or combined with other immune checkpoint blockades moleculars has gained impressive results in the treatment of the melanoma and glioblastoma. Here, we found that TIGIT and Poliovirus receptor (PVR, CD155) are expressed in tumour cells; the TIGIT and CD155 protein expression in cancer tissue has been found to be significantly higher than that in the precancerous tissue. T cell Ig and ITIM domain receptor and CD226 were expressed in the lymphocytes near the tumour tissue and the adjacent tissues. Aspirin has been found to inhibit cancer cell viability and promote CRC cell apoptosis.Similarly, aspirin has also been found to increase pro-apoptotic protein Bax's expression. We found that the expression of TIGIT decreased with an increase in the concentration of aspirin and that the suppression of TIGIT can affect the effect of aspirin on cell proliferation. In this paper, we found that aspirin attenuates cancer cell proliferation and induces CRC cells apoptosis by down-regulating the expression of TIGIT, which provides new evidence for the application of aspirin in cancer treatment.

Immunological features and efficacy of a multi-epitope vaccine CTB-UE against H. pylori in BALB/c mice model.

Epitope vaccine is a promising option for prophylactic and therapeutic vaccination against Helicobacter pylori infection. Urease is an essential virulence factor and colonization factor for H. pylori. In this study, we constructed a multi-epitope vaccine named CTB-UE with mucosal adjuvant cholera toxin B subunit (CTB) and tandem copies of Th and B cell epitopes from H. pylori urease A and B subunits. The immunogenicity, specificity, ability to induce neutralizing antibodies against H. pylori urease, and prophylactic and therapeutic efficacy of the CTB-UE vaccine were evaluated in BALB/c mice model after purification. The experimental results indicated that CTB-UE could induce comparatively high levels of specific antibodies against native H. pylori urease, UreA, UreB, or the selected B cell epitopes UreA183₋203 and UreB327₋334 involved with the active site of urease and showed an effectively inhibitory effect on the enzymatic activity of urease. Besides, oral prophylactic or therapeutic immunization with CTB-UE significantly decreased H. pylori colonization compared with oral immunization with rUreB or PBS, and the protection was correlated with antigen-specific CD4⁺ T cells and IgG, IgA, and mucosal sIgA antibody responses. This CTB-UE vaccine may be a promising vaccine candidate for the control of H. pylori infection.

Aspirin prevents colorectal cancer by regulating the abundance of Enterococcus cecorum and TIGIT+Treg cells.

Although aspirin can reduce the incidence of colorectal cancer (CRC), there is still uncertainty about its significance as a treatment for CRC, and the mechanism of aspirin in CRC is not well understood. In this study, we used aspirin to prevent AOM/DSS-induced CRC in mice, and the anti-CRC efficacy of aspirin was assessed using haematoxylin and eosin (H&E) staining and by determining the mouse survival rate and tumour size. 16S rDNA sequencing, flow cytometry (FCM), and Western blotting were also conducted to investigate the changes in the gut microbiota, tumour immune microenvironment, and apoptotic proteins, respectively. The results demonstrated that aspirin significantly exerted anti-CRC effects in mice. According to 16S rDNA sequencing, aspirin regulated the composition of the gut microbiota and dramatically reduced the abundance of Enterococcus cecorum. FCM demonstrated that there were more CD155 tumour cells and CD4 + CD25 + Treg cells showed increased TIGIT levels. Moreover, increased TIGIT expression on Treg cells is associated with reduced Treg cell functionality. Importantly, the inhibition of Treg cells is accompanied by the promotion of CD19 + GL-7 + B cells, CD8 + T cells, CD4 + CCR4 + Th2 cells, and CD4 + CCR6 + Th17 cells. Overall, aspirin prevents colorectal cancer by regulating the abundance of Enterococcus cecorum and TIGIT + Treg cells.

The changes of intestinal flora and its relevance with memory Tfh and B cells in rheumatoid arthritis patients treated with csDMARDs and csDMARDs + bDMARDs.

BACKGROUND: A growing body of experimental and clinical evidence has implicated gut microbiota in the onset and course of rheumatoid arthritis (RA). The imbalance of intestinal flora in RA patients may lead to abnormal expression of immune cells and related cytokines. PURPOSE: Conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) and conventional synthetic disease-modifying antirheumatic drugs combined with biological disease-modifying antirheumatic drugs (csDMARDs + bDMARDs) are widely used to treat RA, but the characteristics of gut microbiota before and after treatment and their relationship with memory Tfh/B cells and cytokines remain unclear. METHODS: Stool samples were collected from 50 RA patients and 25 healthy controls (HCs) for 16SrRNA gene sequencing. We examined the proportion of lymphocyte subsets in healthy controls and RA patients. Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of related cytokines in serum. The α and ß diversity of intestinal flora, and the correlation between intestinal flora and clinical indicators, lymphocyte subsets, cytokines were analyzed. RESULT: At the genus level, Ruminococcaceae_Ruminococcus was decreased in the csDMARDs and csDMARDs + bDMARDs treatment group, whereas Faecalibacterium was reduced in the csDMARDs treatment group, compared to untreated group. CD4+CD45RO+CCR7+CXCR5+central memory Tfh cells and CD4+CD45RO+CCR7-CXCR5+effector memory Tfh cells were significantly lower in the csDMARDs + bDMARDs treatment group than in untreated group. CD19+CD27+IgD+pre-switched memory B cells were higher in the csDMARDs and csDMARDs + bDMARDs treatment groups, whereas CD19+CD27+IgD-switched memory B cells were significantly lower than in untreated group. Ruminococcaceae_Ruminococcus was negatively correlated with CD19+CD27+IgD+ pre-switched memory B cells but positively correlated with CD4+CD45RO+CCR7-CXCR5+effector memory Tfh and CD19+CD27+IgD-switched memory B cells in patients with RA treated with DMARDs. CONCLUSION: The gut microbiota, memory Tfh cells, memory B cells, and cytokines of patients with RA changed significantly under different treatment regimens and had certain correlations with the clinical indicators of RA.

Bioluminescence imaging of caspase-3 activity in mouse liver.

Apoptosis is an essential process for the maintenance of liver physiology. The ability to noninvasively image apoptosis in livers would provide unique insights into its role in liver disease processes. In the present work, we established a stable mouse model by hydrodynamics methods to study the activity of caspase-3 and evaluate the effect of the apoptosis inhibitors in mouse livers under true physiological conditions by bioluminescence imaging. The reporter plasmid attB-ANLuc(DEVD)BCLuc that contains fragment of attB and ANLuc(DEVD)BCLuc was codelivered with the mouse-codon optimized φC31 (φC31o) integrase plasmids specifically to mouse liver by hydrodynamic injection procedure. Then, φC31o integrase mediated intramolecular recombination between wild-type attB and attP site in mice, and thus the reporter expression cassette attB-ANLuc(DEVD)BCLuc was integrated permanently into mouse liver chromosome. We used these mice to characterize in vivo activation of caspase-3 upon treatment with LPS/D-GalN. Our data show that liver apoptosis could be reflected by the activity of luciferase. The shRNA targeting caspase-3 protein or apoptosis inhibitors could effectively downregulate luciferase activity in vivo. Also, this model could be used to measure caspase-3 activation during inflammatory and infectious events in vivo as verified by infected with MHV-3. This model could be used for screening anti-apoptosis compounds target mouse livers.

Probiotic powder ameliorates colorectal cancer by regulating Bifidobacterium animalis, Clostridium cocleatum, and immune cell composition.

Based on the relationship between the gut microbiota and colorectal cancer, we developed a new probiotic powder for treatment of colorectal cancer. Initially, we evaluated the effect of the probiotic powder on CRC using hematoxylin and eosin staining, and evaluated mouse survival rate and tumor size. We then investigated the effects of the probiotic powder on the gut microbiota, immune cells, and apoptotic proteins using 16S rDNA sequencing, flow cytometry, and western blot, respectively. The results showed that the probiotic powder improved the intestinal barrier integrity, survival rate, and reduced tumor size in CRC mice. This effect was associated with changes in the gut microbiota. Specifically, the probiotic powder increased the abundance of Bifidobacterium animalis and reduced the abundance of Clostridium cocleatum. In addition, the probiotic powder resulted in decreased numbers of CD4+ Foxp3+ Treg cells, increased numbers of IFN-γ+ CD8+ T cells and CD4+ IL-4+ Th2 cells, decreased expression of the TIGIT in CD4+ IL-4+ Th2 cells, and increased numbers of CD19+ GL-7+ B cells. Furthermore, the expression of the pro-apoptotic protein BAX was significantly increased in tumor tissues in response to the probiotic powder. In summary, the probiotic powder ameliorated CRC by regulating the gut microbiota, reducing Treg cell abundance, promoting the number of IFN-γ+ CD8+ T cells, increasing Th2 cell abundance, inhibiting the expression of TIGIT in Th2 cells, and increasing B cell abundance in the immune microenvironment of CRC, thereby increasing the expression of BAX in CRC.

Posttranscriptional inhibition of γ-adducin promotes the proliferation and migration of osteosarcoma cells.

OBJECTIVE: The expression of cytoskeleton-related protein γ-adducin (ADD3) was abnormally reduced in some tumors. Functional experiments demonstrated that it could inhibit the malignant progression of lung cancer and glioma, whereas the involvement of ADD3 in osteosarcoma was not clear. This study aimed to investigate the role of ADD3 in osteosarcoma and its upstream regulatory mechanisms. METHODS: ADD3 was knocked down by siRNA transfection and the expression level of ADD3 was determined using quantitative real-time PCR assay and Western blot. CCK-8 assay and colony formation were performed to detect the capacity of cell proliferation. Transwell assay and PI and Annexin V-FITC staining were used to determine cell migration and apoptosis, respectively. Luciferase reporter experiment was performed to investigate the interaction between ADD3 and miR-23b-3p. RESULTS: Based on gene silencing assays, we showed that knockdown of ADD3 suppressed apoptosis and promoted the proliferation and migration of osteosarcoma cells, revealing inhibitory effects of ADD3 in osteosarcoma. Luciferase reporter gene assays confirmed that miR-23b-3p could bind to the 3'-UTR of ADD3. Upregulation of miR-23b-3p not only inhibited the expression of ADD3, but also released the tumor suppressive role of ADD3 on the proliferation and migration of osteosarcoma cells. CONCLUSIONS: Our study found that ADD3 functioned as a tumor suppressor gene during osteosarcoma development. The abnormal upregulation of miR-23b-3p targeted the expression of ADD3 and resulted in accelerated osteosarcoma cell proliferation and migration. Thus, the miR-23b-3p/ADD3 axis contributes to the development of osteosarcoma and ADD3 is a key driver of malignancy.

Long-term aspirin intervention can inhibit TIGIT, regulating T cells to reverse damage to intestines.

Aspirin is one of the most commonly prescribed medications. Evidence shows that it can even treat and prevent intestinal tumors. However, it has also caused a great deal of controversy due to its intestinal side effects. We therefore explored whether aspirin was beneficial or harmful to the intestines. We used aspirin continuously interfered with C57BL/6 J mice for 48 weeks, examining their intestinal tissues at 13, 26 and 48 weeks to determine the drug's effect on the intestines. In addition, we used flow cytometry (FCM) used to detect T cells and expression of T-cell immunoreceptor with immunoglobulin (Ig)- and tyrosine-based inhibitory motif (ITIM) domain (TIGIT) on their surfaces to determine aspirin's immunomodulatory effects. The results showed that long-term aspirin intervention could reverse damage to the intestines, an effect related to the drug's significant inhibitory effect on TIGIT. The change in TIGIT level could regulate T-cell subsets, so that counts of Cluster of Differentiation 4 (CD4)+/chemokine (C-X3-C motif) receptor 3 (CXCR3)+ T-helper 1 (Th1) cells and CD4+/interleukin-4 (IL-4)+ Th2 cells increased, while those of CD4+/C-C chemokine receptor type 6 (CCR6)+ Th17 cells and CD4+/CD25+ regulatory T cells (Tregs) decreased. In summary, we demonstrated that long-term aspirin intervention could inhibit TIGIT, regulating T cells to reverse damage to the intestines. Furthermore, aspirin is a potential therapy for diseases related to an increase in TIGIT.

Lycium barbarum Polysaccharides Promote Maturity of Murine Dendritic Cells through Toll-Like Receptor 4-Erk1/2-Blimp1 Signaling Pathway.

In previous studies, Lycium barbarum polysaccharides (LBP), a traditional Chinese medicine, can promote immature dendritic cells (DCs) to mature. However, the molecular mechanisms by which LBP works are not yet elucidated. Here, we found that LBP can induce DCs maturation, which is mainly characterized by the upregulation of MHCII and costimulatory molecules (CD80, CD86), and increase the production of IL-6 and IL-4. Furthermore, we found that LBP could increase the mRNA and protein expression of TLR4, p38, Erk1/2, JNK, and Blimp1 signal molecules. More interestingly, after blocking by Toll-like receptor 4 inhibitor, Resatorvid (TAK 242), the mRNA and protein expression of TLR4, Erk1/2, and Blimp1 was significantly decreased while the expression of p38 and JNK has not changed. Then, we found that after blocking by p38 inhibitor (SB203580), Erk inhibitor (PD98059), and JNK inhibitor (SP603580) separately, Blimp1 protein expression was significantly reduced; after downregulating Blimp1 by Blimp1-siRNA, the production of IL-6 was reduced. In conclusion, our results indicate that LBP can induce maturation of DCs through the TLR4-Erk1/2-Blimp1 signal pathway instead of the JNK/p38-Blimp1 pathway. Our findings may provide a novel evidence for understanding the molecular mechanisms of LBP on activating murine DCs.

1IFN-α Modulates Memory Tfh Cells and Memory B Cells in Mice, Following Recombinant FMDV Adenoviral Challenge.

Follicular helper T (Tfh) cells regulate high-affinity antibody production. Some findings have indicated that Tfh cells could be differentiated into memory cells. Here we have investigated the effects of IFN-α, as an adjuvant, on the generation of memory Tfh cell and memory B cell responses. The data showed that adenoviral vectors expressing: (i) foot-and-mouth disease virus (FMDV) VP1 proteins and porcine IFN-α, or (ii) porcine IFN-α alone, potently enhanced the generation of memory Tfh cells, especially the CCR7 lo memory Tfh subset. Upon rechallenge with FMD recombinant adenoviral vaccines, IFN-α enhances Tfh cells activity, rapidly upregulating their signature Bcl-6, CXCR5, and IL-21 markers. The results suggest that IFN-α enhances the levels of the transcription factor Bcl-6 within Tfh cells, potentially by regulating STAT1. Additionally, IFN-α substantially increased the number of IgG1+ and CD86+ memory B cells, which are responsible for inducing the rapid effector functions of memory Tfh cells after vaccine reactivation, establishing the close relationship between memory B cell and memory Tfh cell subsets. In brief, IFN-α enhances the potency of FMD recombinant adenoviral vaccines to induce memory Tfh and memory B cell responses, thus elevating serum antibody titers. IFN-α administration therefore represents an attractive strategy for enhancing responses to vaccination.

Toll-like receptor 7 agonist imiquimod prevents the progression of SLE in MRL/lpr mice via inhibiting the differentiation of T follicular helper cells.

Previous research has recently indicated that TLR7 is able to induce CD4+T cell anergy, which is the opposite of the role it plays in innate immune cells. Therefore, TLR7 ligands may be used as a manner in which to induce CD4+T cells "tolerance" in autoimmune diseases. T follicular helper (Tfh) cells were demonstrated to be a subset of CD4+T cells that help B cells produce antibodies. The abnormal activity of Tfh cells, though, is their function as a primary pathogenic factor in systemic lupus erythematosus (SLE). However, the role of TLR7 in Tfh cells is not clear. Our study was aimed at determining the influence of TLR7 on Tfh cells in a murine model of SLE (MRL/lpr mice). We were surprised to find that the frequency of Tfh cells and germinal center (GC) B cells was significantly reduced after treatment with the TLR7 agonist imiquimod. Imiquimod also significantly reduced the expression of inducible costimulatory molecule (ICOS) and programmed death 1(PD-1) in Tfh cells and decreased IL-21 secretion. Moreover, imiquimod significantly reduced the mRNA expression of several transcription factors, including Bcl-6, c-Maf, Batf3, Nfatc2 and Stat3, and enhanced the expression of Prdm1 and Stat5b in CD4+T cells. Imiquimod also ameliorated the progression of SLE in MRL/lpr mice by inhibiting anti-dsDNA antibodies and antinuclear antibody (ANA) secretion in the serum. Our findings indicated that TLR7 inhibited the development of Tfh cells both in vivo and ex vivo, which depended on many transcription factors aside from Bcl-6. Our results demonstrated that a TLR7 agonist has the potential to be used to inhibit Tfh cell responses during SLE.

Expression of TIGIT/CD155 and correlations with clinical pathological features in human hepatocellular carcinoma.

T cell immunoglobulin and ITIM domain (TIGIT) is a recently identified T cell coinhibitory receptor. Studies have shown that TIGIT is expressed in colon adenocarcinoma, uterine corpus endometrioid carcinoma, breast carcinoma and kidney renal clear cell carcinoma. However, the role of the TIGIT/human poliovirus receptor (CD155) pathway in the pathogenesis of hepatocellular carcinoma (HCC) remains to be elucidated. In the present study, the expression of TIGIT and CD155 in HCC tissues and peripheral blood were determined, and correlations among TIGIT, CD155, TIGIT+ CD4+ T cells, TIGIT+ regulatory T (Treg) cells and α­fetoprotein (AFP) were investigated in order to identify a potential target for diagnosing and treating HCC. Immunohistochemistry, reverse transcription­quantitative PCR analysis and western blotting were used to examine the expression of TIGIT and CD155 in cancerous tissues and peripheral blood collected from patients with HCC. The frequency of TIGIT+ CD4+ T cells and TIGIT+ Treg cells and the concentration of inflammatory cytokines secreted by T cell subsets were analyzed by flow cytometry and a Merck Milliplex assay. Correlations between the frequency of TIGIT+ CD4+ T and TIGIT+ Treg cells and AFP were analyzed using Spearman's rank correlation test. With the degree of cancerous differentiation from high to low, the expression levels of TIGIT and CD155 were upregulated in the cancerous tissues from patients with HCC. TIGIT+ CD4+ T cell and TIGIT+ Treg cell frequencies were decreased in peripheral blood from postoperative patients with HCC. The increased expression of TIGIT was positively correlated with the level of AFP. These results indicate that co­inhibitory receptor TIGIT may be involved in the pathogenesis of HCC and represent a novel target for the diagnosis and treatment of HCC.

Bursin as an adjuvant is a potent enhancer of immune response in mice immunized with the JEV subunit vaccine.

Adjuvants play an important role in the formulation of effective and appropriate vaccines. A series of synthetic bursin and bursin-like peptides was heterologously expressed in Escherichia coli. The use of bursin as an adjuvant enhanced the specific immune responses of mice immunized with a recombinant Japanese encephalitis virus E-binding domain (JEV E-BD) fusion protein. The effect was determined in the form of protective anti-JEV E titers, antibodies (IgG1 and IgG2a), spleen cell lymphocyte proliferation, the levels of interferon-gamma and interleukin-4 cytokines, and the T-lymphocyte sub-type composition. The IgG2a titer and interferon-gamma level suggested that the E-BD protein potentiates the Th1 immune response. These responses were changed when the immunogen was combined with one of the synthetic peptides as adjuvant. JEV-neutralization assay results show that the presence of bursin significantly enhance the JEV-neutralizing titer. We conclude that bursin as an adjuvant is a potent enhancer of immune response in mice immunized with the JEV subunit vaccine, and represents a promising adjuvant for vaccination.

Heterologous expression of FMDV immunodominant epitopes and HSP70 in P. pastoris and the subsequent immune response in mice.

Mycobacterium tuberculosis heat shock protein70 (HSP70) is a major antigen with both chaperone and cytokine functions. It has been used as an adjuvant to induce or potentiate humoral and cellular immunity, both in the form of a mixture with peptide antigens, and as a fusion protein. We have evaluated the effects of HSP70 on foot and mouth virus (FMDV) subunit vaccines. FMDV VP1, and a synthetic multi-epitope FMDV (EG), and VP1-HSP70 and EG-HSP70 fusion proteins were all heterologously expressed in the yeast Pichia pastoris, and used as antigen in mice. The recombinant VP1 and EG alone was able to induce both humoral and marginal cell-mediated immune responses, while the HSP70 fusions markedly enhanced both the humoral and cell-mediated immune responses. The most prominent immune responses arose from vaccination with the EG-HSP70 fusion product. Both fusion protein-induced Th1-like cytokine (IFN-gamma) and Th2-like cytokine (IL-4) were identified.

MicroRNA-508-5p suppresses metastasis in human gastric cancer by targeting S-phase kinase­associated protein 2.

S-phase kinase-associated protein 2 (SKP2), a potent oncogene was revealed to be upregulated in gastric cancer (GC) tissue samples, in which SKP2 was inversely correlated with microRNA (miR)­508­5p transcripts. In present study, the functional effect of miR­508­5p on SKP2 and its metastatic potential were investigated in SGC­7901 GC cells. Significant downregulation of the miR­508­5p transcript was associated with the progression of GC. Furthermore, the overexpression of miR­508­5p was demonstrated to inhibit the proliferation, migration and invasion of SGC­7901 cells, as well as induced cell apoptosis and cell cycle arrest at the G0/G1 phase in vitro. The overexpression of miR­508­5p was able to downregulate the expression of the SKP2 oncogene, through a mechanism by which miR­508­5p directly targeted the SKP2 gene. Thus, regulating transcriptional and post­transcriptional SKP2 expression, as demonstrated using luciferase reporter assays, reverse transcription­quantitative polymerase chain reaction analysis and immunoblotting assays. The results of the present study identified that miR­508­5p functionally affects the SKP2 gene and reduces metastatic potential in GC, suggesting a novel role of miR­508­5p in the regulation of SKP2 and cell cycle.

miR-1247-5p functions as a tumor suppressor in human hepatocellular carcinoma by targeting Wnt3.

Increasing evidence suggests that aberrant expression of certain microRNAs (miRNAs) may participate in the genesis and progression of tumors. Several studies have indicated that miR-1247-5p plays different roles in various types of cancer cells. The effects of miR-1247-5p on human hepatocellular carcinoma (HCC) cells are elusive. In the present study, we investigated the effects of miR-1247-5p on the progression of HCC. The transcript of miR-1247-5p was markedly downregulated in clinical samples of patients with HCC and HCC cell lines, and ectopic overexpression of miR­1247-5p markedly inhibited the proliferation and invasion of HepG2 cells, induced cell apoptosis in vitro, and suppressed the growth of transplanted tumors in vivo. Wnt3 was found to be a potential target of miR-1247-5p and overexpression of miR-1247-5p was able to significantly downregulate the expression of Wnt3 by directly targeting the 3'UTR of this gene, which was verified by luciferase reporter assay and western blotting. Furthermore, we found that the miR-1247-5p gene was hypermethylated in HepG2 cells, and the transcript of miR-1247-5p was increased significantly after treatment with the demethylation drug 5-azacytidine. These findings demonstrated that miR-1247-5p functions as a tumor suppressor in human HCC by targeting Wnt3 and that the expression of miR-1247-5p can be regulated by DNA methylation, which indicates that miR-1247-5p has the potential to be a therapeutic target as well as a diagnostic marker of HCC.

Hepatic NK cell-mediated hypersensitivity to ConA-induced liver injury in mouse liver expressing hepatitis C virus polyprotein.

The role of hepatic NK cells in the pathogenesis of HCV-associated hepatic failure is incompletely understood. In this study, we investigated the effect of HCV on ConA-induced immunological hepatic injury and the influence of HCV on hepatic NK cell activation in the liver after ConA administration. An immunocompetent HCV mouse model that encodes the entire viral polyprotein in a liver-specific manner based on hydrodynamic injection and φC31o integrase was used to study the role of hepatic NK cells. Interestingly, the frequency of hepatic NK cells was reduced in HCV mice, whereas the levels of other intrahepatic lymphocytes remained unaltered. Next, we investigated whether the reduction in NK cells within HCV mouse livers might elicit an effect on immune-mediated liver injury. HCV mice were subjected to acute liver injury models upon ConA administration. We observed that HCV mice developed more severe ConA-induced immune-mediated hepatitis, which was dependent on the accumulated intrahepatic NK cells. Our results indicated that after the administration of ConA, NK cells not only mediated liver injury through the production of immunoregulatory cytokines (IFN-γ, TNF-α and perforin) with direct antiviral activity, but they also killed target cells directly through the TRAIL/DR5 and NKG2D/NKG2D ligand signaling pathway in HCV mice. Our findings suggest a critical role for NK cells in oversensitive liver injury during chronic HCV infection.