Spontaneous CD8 T cell responses against the melanocyte differentiation antigen RAB38/NY-MEL-1 in melanoma patients

The melanocyte differentiation Ag RAB38/NY-MEL-1 was identified by serological expression cloning (SEREX) and is expressed in the vast majority of melanoma lesions. The immunogenicity of RAB38/NY-MEL-1 has been corroborated previously by the frequent occurrence of specific Ab responses in melanoma patients. To elucidate potential CD8 T cell responses, we applied in vitro sensitization with overlapping peptides spanning the RAB38/NY-MEL-1 protein sequence and the reverse immunology approach. The identified peptide RAB38/NY-MEL-1(50-58) exhibited a marked response in ELISPOT assays after in vitro sensitization of CD8 T cells from HLA-A *0201(+) melanoma patients. In vitro digestion assays using purified proteasomes provided evidence of natural processing of RAB38/NY-MEL-1(50-58) peptide. Accordingly, monoclonal RAB38/NY-MEL-1(50-58)-specific T cell populations were capable of specifically recognizing HLA-A2(+) melanoma cell lines expressing RAB38/NY-MEL-1. Applying fluorescent HLA-A2/RAB38/NY-MEL-1(50-58) multimeric constructs, we were able to document a spontaneously developed memory/effector CD8 T cell response against this peptide in a melanoma patient. To elucidate the Ag-processing pathway, we demonstrate that RAB38/NY-MEL-1(50-58) is produced efficiently by the standard proteasome and the immunoproteasome. In addition to the identification of a RAB38/NY-MEL-1-derived immunogenic CD8 T cell epitope, this study is instrumental for both the onset and monitoring of future RAB38/NY-MEL-1-based vaccination trials.     

Hypoxia induces osteogenic/angiogenic responses of bone marrow‐derived mesenchymal stromal cells seeded on bone‐derived scaffolds via ERK1/2 and p38 pathways

Osteogenesis and angiogenesis are tightly coupled processes during bone development and formation. It is thus well known that the enhancement of vascularization is of great importance in bone tissue engineering. As a potential approach for repairing bone defects, bone tissue constructs should therefore replicate the essential components in vivo microenvironments to promote cell osteogenic differentiation while at same time induce angiogenic response. In light of standpoint above, a combination of human bone‐derived scaffolds and BMSCs that subjected to hypoxia was used to mimic in vivo conditions. Also the underlying cellular/molecular regulation was fully investigated. The results showed that hypoxia (5–10% O₂) greatly enhanced the proliferation of BMSCs seeded in scaffolds, although the hypoxia (5% O₂)‐induced proliferative effect on BMSC cellular scaffolds was not apparent to those cultured in plates. However, such a kind of model was able to significantly induce the osteogenic/angiogenic responses of BMSCs as reflected by osteogenesis or angiogenesis‐related highly expressed genes or proteins, such as alkaline phosphatase, osteocalcin, hypoxia‐inducible factor‐1α and vascular endothelial growth factor. Moreover, ERK1/2 and/or p38 pathways were demonstrated to play essential roles in hypoxia‐induced osteogenic/angiogenic responses. Our results indicated that the combination of bone‐derived scaffolds, a material that has a three dimensional network structure, and hypoxia, an environment that replicates in vivo BMSCs hypoxic living conditions, may be a potential approach for creating functional tissue‐engineered bone. Biotechnol. Bioeng. 2013; 110: 1794–1804. © 2013 Wiley Periodicals, Inc.     

Dihydroartemisinin induces apoptosis in human gastric cancer cell line BGC-823 through activation of JNK1/2 and p38 MAPK signaling pathways

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, is associated with a broad range of biological properties including antitumor activity. However, the effect of DHA on gastric cancer has not been clearly clarified. The aim of this study was to investigate the role and mechanism of DHA in human gastric cancer cell line BGC-823. Cell viability was assessed by MTT assay. Cell apoptosis was analyzed with flow cytometry. The expressions of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and their phosphorylated forms as well as apoptosis related proteins were examined by western blot analysis. The results demonstrated that DHA inhibited cell viability of BGC-823 cells in a dose- and time-dependent manner. DHA treatment upregulated the expression of Bax, cleaved caspase-3 and -9, and degraded form of PARP, and downregulated the Bcl-2 expression and Bcl-2/Bax ratio. Meanwhile, DHA increased the phosphorylation of ERK1/2, JNK1/2 and p38 MAPK. Synthetic inhibitors of JNK1/2 or p38 MAPK kinase activity, but not inhibitor of ERK1/2, significantly abolished the DHA-induced activation of caspase-3 and -9. In vivo tumor-suppression assay further indicated that DHA displayed significant inhibitory effect on BGC-823 xenografts in tumor growth. These results indicate that DHA induces apoptosis of BGC-823 cells through JNK1/2 and p38 MAPK signaling pathways and DHA could serve as a potential additional chemotherapeutic agent for treatment of gastric cancer.     

Novel recombinant BCG coexpressing Ag85B,ESAT‐6 and mouse TNF‐α induces significantly enhanced cellular immune and antibody responses in C57BL/6 mice

The recent emergence of multidrug‐resistant and extensively drug‐resistant strains of Mtb and the epidemic of TB in populations co‐infected with human immunodeficiency virus demonstrate that TB remains a leading infectious disease. Moreover, the failure of BCG to protect against this disease indicates that new vaccines against TB are urgently needed. Experimental evidence has revealed that TNF plays a major role in host defense against Mtb in both active and latent phases of infection. Release of TNF, which would induce mycobacteria‐mediated macrophage apoptosis and thus reduce the spread of mycobacteria, is one of the most important and early responses of macrophages challenged with Mtb. In order to identify the usefulness of TNF in improving the effectiveness of TB vaccine, in the current study a novel rBCG strain expressing the fusion gene of Ag85B‐Esat6‐TNF‐α in BCG Danish strain was constructed, and its ability to induce an immune response in C57BL/6 mice evaluated. It was found that immunization with strains of rBCG‐Ag85B‐Esat6‐TNF‐α can induce a stronger immune response than does immunization with rBCG‐Ag85B‐Esat6 or parental BCG. The results indicate that rBCG‐Ag85B‐Esat6‐TNF‐α is a promising candidate for further study.     

Resveratrol enhances proliferation and osteoblastic differentiation in human mesenchymal stem cells via ER-dependent ERK1/2 activation

In the present study, we investigated the in vitro effect of resveratrol (RSVL), a polyphenolic phytoestrogen, on cell proliferation and osteoblastic maturation in human bone marrow-derived mesenchymal stem cell (HBMSC) cultures. RSVL (10⁻⁸–10⁻⁵ M) increased cell growth dose-dependently, as measured by [³H]-thymidine incorporation, and stimulated osteoblastic maturation as assessed by alkaline phosphatase (ALP) activity, calcium deposition into the extracellular matrix, and the expression of osteoblastic markers such as RUNX2/CBFA1, Osterix and Osteocalcin in HBMSCs cell cultures. Further studies found that RSVL (10⁻⁶ M) resulted in a rapid activation of both extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) signaling in HBMSCs cultures. The effects of RSVL were mimicked by 17β-estrodial (10⁻⁸ M) and were abolished by estrogen receptor (ER) antagonist ICI182780. An ERK1/2 pathway inhibitor, PD98059, significantly attenuated RSVL-induced ERK1/2 phosphorylation, consistent with the reduction of cell proliferation and osteoblastic differentiation as well as expression of osteoblastic markers. In contrast, SB203580, a p38 MAPK pathway blocker, blocked RSVL-induced p38 phosphorylation, but resulted in an increase of cell proliferation and a more osteoblastic maturation. These data suggest that RSVL stimulates HBMSCs proliferation and osteoblastic differentiation through an ER-dependent mechanism and coupling to ERK1/2 activation.     

SIRT1/FoxO3 axis alteration leads to aberrant immune responses in bronchial epithelial cells

Inflammation and ageing are intertwined in chronic obstructive pulmonary disease (COPD). The histone deacetylase SIRT1 and the related activation of FoxO3 protect from ageing and regulate inflammation. The role of SIRT1/FoxO3 in COPD is largely unknown. This study evaluated whether cigarette smoke, by modulating the SIRT1/FoxO3 axis, affects airway epithelial pro‐inflammatory responses. Human bronchial epithelial cells (16HBE) and primary bronchial epithelial cells (PBECs) from COPD patients and controls were treated with/without cigarette smoke extract (CSE), Sirtinol or FoxO3 siRNA. SIRT1, FoxO3 and NF‐κB nuclear accumulation, SIRT1 deacetylase activity, IL‐8 and CCL20 expression/release and the release of 12 cytokines, neutrophil and lymphocyte chemotaxis were assessed. In PBECs, the constitutive FoxO3 expression was lower in patients with COPD than in controls. Furthermore, CSE reduced FoxO3 expression only in PBECs from controls. In 16HBE, CSE decreased SIRT1 activity and nuclear expression, enhanced NF‐κB binding to the IL‐8 gene promoter thus increasing IL‐8 expression, decreased CCL20 expression, increased the neutrophil chemotaxis and decreased lymphocyte chemotaxis. Similarly, SIRT1 inhibition reduced FoxO3 expression and increased nuclear NF‐κB. FoxO3 siRNA treatment increased IL‐8 and decreased CCL20 expression in 16HBE. In conclusion, CSE impairs the function of SIRT1/FoxO3 axis in bronchial epithelium, dysregulating NF‐κB activity and inducing pro‐inflammatory responses.     

Establishment of a monoclonal antibody directed against Gb3Cer/CD77: a useful immunochemical reagent for a differentiation marker in Burkitt's Iymphoma and germinal centre B cells

A new monoclonal antibody (TU-1) directed against the Galα1-4Galβ1-4Glc residue of the Gb3Cer/CD77 antigen was prepared by the hybridoma technique following immunization of mice with an emulsion composed of monophosphoryl lipid A, trehalose dimycolate, and Gb3Cer isolated from porcine erythrocytes. TU-1 showed reactivity towards Gb3Cer and lyso-Gb3Cer (Galα1-4Galβ1-4Glcβ1-1′Sph), although the reactivity towards lyso-Gb3Cer was about 10-fold lower than that to Gb3Cer. But it did not react with other structurally-related glycolipids, such as LacCer (Galβ1-4Glcβ1-1′Cer), Gg3Cer, Gg4Cer, Gb4Cer (GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1′Cer), galactosylparagloboside (Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer), sulfatide (HSO3-3Galβ1-1′Cer), other gangliosides (GM3, GM2, GM1a, GD1a and GT1b), or P1 antigen (Galα1-4Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer) among neutral glycolipids prepared from P1 phenotype red blood cells. Furthermore, TU-1 reacted with viable lymphoma cells, such as human Burkitt lymphoma cell line, Daudi, and Epstein-Barr virus (EBV)-transformed B cells by the immunofluorescence method, and also with germinal centre B cells in human tonsil and vessel endothelial cells in human thymus histochemically. These results indicate that TU-1 is a monoclonal antibody directed against Gb3Cer/CD77 antigen and can be utilized as a diagnostic reagent for Burkitt's lymphoma and also for detection of the blood group Pk antigen in glycolipid extracts of erythrocytes. Abbreviations: ATL, adult T-cell leukaemia; BSA, bovine serum albumin; Cer, ceramide; DPPC, L-α-dipalmitoylphosphatidylcholine; EBV, Epstein-Barr virus; FCS, fetal calf serum; GalCer, Galβ1-1′Cer; GlcCer, Glcβ1-1′Cer; LacCer, Galβ1-4Glcβ1-1′Cer; Gb3Cer, Galα1-4Galβ1-4Glcβ1-1′Cer; Iyso-Gb3Cer, Galα1-4Galβ1-4Glc1-1′Sph; Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glc1-1′Cer; galactosylparagloboside, Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer; Gg3Cer, GalNAcβ1-4Galβ1-4Glcβ1-1′Cer; Gg4Cer, Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1′Cer; GM3, Neu5Acα2-3Galβ1-4Glcβ1-1′Cer; GM2, GalNAcβ1-4(Neu5Acα2-3) Galβ1-4Glcβ1-1′Cer; GM1a, Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GD1a, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GD1b, Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GT1b, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3) Galβ1-4Glcβ1-1′Cer; HRP, horseradish peroxidase; LDH, lactate dehydrogenase; MAb, monoclonal antibody; MPL, monophosphoryl lipid A; P1 antigen, Galα1-4Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer; PVP, polyvinylpyrolidone; Sph, sphingosine; sulfatide, HSO3-Galβ1-1′Cer; TDM, trehalose dimycolate; TLC, thin-layer chromatography This revised version was published online in November 2006 with corrections to the Cover Date.     

Laminin-1 induces neurite outgrowth in human mesenchymal stem cells in serum/differentiation factors-free conditions through activation of FAK-MEK/ERK signaling pathways

Mesenchymal stem cells (MSCs) can be differentiated into cell types derived from all three germ layers by manipulating culture conditions in vitro. A multitude of growth and differentiation factors have been employed for driving MSCs towards a neuronal phenotype. In the present study, we investigated the potential of extracellular matrix (ECM) proteins—fibronectin, collagen-1, collagen-IV, laminin-1, and laminin-10/11, to induce a neuronal phenotype in bone marrow derived human MSCs in the absence of growth factors/differentiating agents. All of the ECM proteins tested were found to support adhesion of MSCs to different extents. However, direct interaction only with laminin-1 triggered sprouting of neurite-like processes. Cells plated on laminin-1 exhibited neurite out growth as early as 3 h, and by 24 h, the cells developed elaborate neurites with contracted cell bodies and neuronal-like morphology. Function-blocking antibodies directed against α6 and β1 integrin subunits inhibited neurite formation on laminin-1 which confirmed the involvement of integrin α6β1 in neurite outgrowth. Mechanistic studies revealed that cell adhesion to laminin-1 activated focal adhesion kinase (FAK), and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways. Abrogation of FAK phosphorylation by herbimycin-A inhibited neurite formation and also decreased activities of MEK and ERK. Pharmacological inhibitors of MEK (U0126) and ERK (PD98059) also blocked neurite outgrowth in cells plated on laminin-1. Our study demonstrates the involvement of integrin α6β1 and FAK-MEK/ERK signaling pathways in laminin-1-induced neurite outgrowth in MSCs in the absence of serum and differentiation factors.     

Association of the ERK1/2 and p38 kinase pathways with nitric oxide-induced apoptosis and cell cycle arrest in colon cancer cells

To investigate the mechanism by which nitric oxide (NO) induces cell death in colon cancer cells, we compared two types of colon cancer cells with different p53 status: HCT116 (p53 wild-type) cells and SW620 (p53-deficient) cells. We found that S-nitrosoglutathione (GSNO), the NO donor, induced apoptosis in both types of colon cancer cells. However, SW620 cells were much more susceptible than HCT116 cells to apoptotic death by NO. We investigated the role of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 kinase on NO-induced apoptosis in both types of colon cancer cells. GSNO treatment effectively stimulated activation of the ERK1/2 and p38 kinase in both types of cells. In HCT116 cells, pretreatment with PD98059, an inhibitor of ERK1/2, or SB203580, an inhibitor of p38 kinase, had no marked effect on GSNO-induced apoptosis. However, in SW620 cells, SB203580 significantly reduced the NO-induced apoptosis, whereas PD098059 increases NO-induced apoptosis. Furthermore, we found evidence of cell cycle arrest of the G₀/G₁ phase in SW620 cells but not in HCT116 cells. Inhibition of ERK1/2 with PD098059, or of p38 kinase with SB203580, reduced the GSNO-induced cell cycle arrest of the G₀/G₁ phase in SW620 cells. We therefore conclude that NO-induced apoptosis in colon cancer cells is mediated by a p53-independent mechanism and that the pathways of ERK1/2 and p38 kinase are important in NO-induced apoptosis and in the cell cycle arrest of the G₀/G₁ phase.     

人参多糖注射液联合TP方案对晚期非小细胞肺癌患者Th1/Th2免疫平衡和血清肿瘤标志物的影响

摘要 目的：探讨人参多糖注射液联合多西他赛和顺铂（TP方案）治疗晚期非小细胞肺癌（NSCLC）的疗效及对患者辅助性T细胞（Th）1/Th2免疫平衡和血清肿瘤标志物的影响。方法：选取我院2019年3月~2021年2月收治的80例晚期NSCLC患者。按随机数字表法分为对照组（TP方案治疗,40例）和观察组（人参多糖注射液联合TP方案治疗,40例）。对比两组疗效、血清肿瘤标志物、Th1/Th2免疫平衡指标、生存质量和不良反应发生率。结果：观察组的客观缓解率、疾病控制率分别为55.00%、85.00%,分别高于对照组的32.50%、57.50%,差异有统计学意义（P<0.05）。两组治疗4个疗程后肺癌患者生存质量量表（FACT-L）各领域评分均较治疗前升高,且观察组高于对照组（P<0.05）。两组治疗4个疗程后血清肿瘤标志物糖类抗原19-9（CA19-9）、细胞角蛋-19片段（CYFRA21-1）、癌胚抗原（CEA）水平下降,且观察组低于对照组（P<0.05）。两组治疗4个疗程后血清γ-干扰素（INF-γ）、白介素-2（IL-2）水平升高,且观察组高于对照组（P<0.05）。两组治疗4个疗程后血清白介素-4（IL-4）、白介素-5（IL-5）水平降低,且观察组低于对照组（P<0.05）。两组不良反应发生率对比无统计学差异（P>0.05）。结论：晚期NSCLC在TP化疗基础上结合人参多糖注射液治疗,可更好的降低血清肿瘤标志物水平,控制疾病进展,同时还可调节Th1/Th2免疫平衡,促进患者生存质量改善。     

槐耳颗粒联合GP方案治疗晚期非小细胞肺癌的疗效及对Th1/Th2免疫平衡和血清肿瘤标志物的影响

摘要 目的：观察晚期非小细胞肺癌（NSCLC）采用吉西他滨+顺铂（GP方案）联合槐耳颗粒治疗的疗效及对Th1/Th2免疫平衡和血清肿瘤标志物的影响。方法：选取2020年01月~2022年02月期间来成都市第六人民医院接受治疗的晚期NSCLC患者80例。采用双色球法将患者分为对照组（40例,GP方案治疗）和研究组（40例,槐耳颗粒联合GP方案治疗）。对比两组临床疗效、血清肿瘤标志物[糖类抗原125（CA125）、癌胚抗原（CEA）、细胞角蛋白19片段21-1（CYFRA21-1）]、Th1/Th2免疫平衡和不良反应。结果：研究组客观缓解率、疾病控制率高于对照组（P<0.05）。两组不良反应发生率组间对比无差异（P>0.05）。研究组治疗后卡式评分（KPS）、Th1、Th1/Th2高于对照组（P<0.05）,Th2低于对照组（P<0.05）。治疗后研究组血清CA125、CYFRA21-1、CEA水平较对照组低（P<0.05）。结论：槐耳颗粒联合GP方案治疗晚期NSCLC,可有效降低血清CA125、CEA、CYFRA21-1水平,改善Th1/Th2免疫平衡,安全可靠。     

Cathepsin D non-proteolytically induces proliferation and migration in human omental microvascular endothelial cells via activation of the ERK1/2 and PI3K/AKT pathways

Epithelial ovarian cancer (EOC) frequently metastasises to the omentum, a process that requires pro-angiogenic activation of human omental microvascular endothelial cells (HOMECs) by tumour-secreted factors. We have previously shown that ovarian cancer cells secrete a range of factors that induce pro-angiogenic responses e.g. migration, in HOMECs including the lysosomal protease cathepsin D (CathD). However, the cellular mechanism by which CathD induces these cellular responses is not understood. The aim of this study was to further examine the pro-angiogenic effects of CathD in HOMECs i.e. proliferation and migration, to investigate whether these effects are dependent on CathD catalytic activity and to delineate the intracellular signalling kinases activated by CathD. We report, for the first time, that CathD significantly increases HOMEC proliferation and migration via a non-proteolytic mechanism resulting in activation of ERK1/2 and AKT. These data suggest that EOC cancer secreted CathD acts as an extracellular ligand and may play an important pro-angiogenic, and thus pro-metastatic, role by activating the omental microvasculature during EOC metastasis to the omentum.     

Leishmania major: immune response in BALB/c mice immunized with stress-inducible protein 1 encapsulated in liposomes

Protection against leishmaniasis is depending upon generation of a Th1 type of immune response. Field trials of first generation Leishmania vaccine showed a limited efficacy even with multiple doses mainly due to lack of an appropriate adjuvant. In this study, susceptible BALB/c mice were immunized with rLmSTI1 encapsulated in liposomes to explore the extent of protection induced by Leishmania antigen encapsulated in the liposomes against challenge with Leishmania major. The results showed that s.c. immunization of BALB/c mice with liposomal rLmSTI1 induced a significant protection against challenge and a significant lower parasite burden in spleen up to 14 weeks after challenge. The protected animals showed a significantly smaller footpad thickness after challenge, and a higher level of anti-SLA IgG antibodies before and after challenge with a predominant IgG2a titer. The data supports the possibility of using liposomal Leishmania antigens as a vaccine.     

A new mutation in the gene ROR2 causes brachydactyly type B1

Brachydactyly type B, an autosomal dominant disorder that is characterized by hypoplasia of the distal phalanges and nails, can be divided into brachydactyly type B1 (BDB1) and brachydactyly type B2 (BDB2). BDB1 is caused by mutations in the receptor tyrosine kinase gene ROR2, which maps to chromosome 9q22, whereas BDB2 is caused by point mutations in the bone morphogenetic protein antagonist NOGGIN. Here, we report a three-generation Chinese family with dominant inheritance of the BDB1 limb phenotype. Sequence analysis identified a novel heterozygous base deletion (c.1396–1398delAA) in the gene ROR2 in all affected family members. This new deletion is expected to produce a truncated Ror2 protein with a new polypeptide of 57 amino acids at the C-terminal.     

Mutation and expression analysis of the IDH1, IDH2, DNMT3A, and MYD88 genes in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of death around the world. Its genetic mechanism was intensively investigated in the past decades with findings of a number of canonical oncogenes and tumor-suppressor genes such as APC, KRAS, and TP53. Recent genome-wide association and sequencing studies have identified a series of promising oncogenes including IDH1, IDH2, DNMT3A, and MYD88 in hematologic malignancies. However, whether these genes are involved in CRC remains unknown. In this study, we screened the hotspot mutations of these four genes in 305 CRC samples from Han Chinese by direct sequencing. mRNA expression levels of these genes were quantified by quantitative real-time PCR (RT-qPCR) in paired cancerous and paracancerous tissues. Association analyses between mRNA expression levels and different cancerous stages were performed. Except for one patient harboring IDH1 mutation p.I99M, we identified no previously reported hotspot mutations in colorectal cancer tissues. mRNA expression levels of IDH1, DNMT3A, and MYD88, but not IDH2, were significantly decreased in the cancerous tissues comparing with the paired paracancerous normal tissues. Taken together, the hotspot mutations of IDH1, IDH2, DNMT3A, and MYD88 gene were absent in CRC. Aberrant mRNA expression of IDH1, DNMT3A, and MYD88 gene might be actively involved in the development of CRC.     

Let-7g induces granulosa cell apoptosis by targeting MAP3K1 in the porcine ovary

Follicular atresia mainly results from apoptosis of granulosa cells (GCs). Our previous microRNA array data indicated that the miRNA let-7g level increases significantly during porcine ovary follicular atresia. It is uncertain if GCs apoptosis is mediated by microRNA let-7g. In this study, the expression levels of the apoptosis-associated genes CASP3, BAX and BIM were significantly upregulated when let-7g mimic was transfected into porcine GCs, and the anti-apoptotic genes BCL-2 and MCL-1 were significantly downregulated. The apoptosis rate was measured by flow cytometry, and our results indicated that let-7g significantly enhanced GCs apoptosis. In further studies, we found that overexpression of let-7g induced the expression of FoxO1 in GCs and led to nuclear accumulation of dephosphorylated FoxO1. In addition, the effect of let-7g on FoxO1 expression and dephosphorylation resulted from repression of the expression of the MAP3K1 gene in porcine GCs. The site on MAP3K1 mRNA targeted by let-7g was confirmed by luciferase reporter assay. The anti-apoptotic effect of MAP3K1 was validated by silencing MAP3K1 using small interfering RNA technology. In conclusion, our data indicate that let-7g induces porcine GCs apoptosis by inhibiting the MAP3K1 gene, which promotes FoxO1 expression and dephosphorylation with nuclear accumulation.     

Bio-informatics based analysis of genes implicated in alcohol mediated liver injury

Alcohol induced liver injury has been studied extensively. Using literature search and bioinformatics tools, the present study characterizes the genes involved in alcohol induced liver injury. The cellular and metabolic processes in which genes involved in alcohol induced liver injury are implicated are also discussed. The genes related to alcohol induced liver injury are also involved in affecting certain molecular functions and metabolism of drugs, besides being associated with diseases. In conclusion, the changes in regulation of genes implicated in alcohol induced liver injury apart from causing alcohol mediated hepatic dysfunction may affect other vital processes in the body.