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Background and aims Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide, hence a major public health threat. Pleomorphic adenoma gene like-2 (PLAGL2) has been reported to play a role in tumorigenesis. However, its precise function in HCC remains poorly understood. Approach and results In this study, we demonstrated that PLAGL2 was up-regulated in HCC compared with that of adjacent nontumorous tissues and also correlated with overall survival times. We further showed that PLAGL2 promoted HCC cell proliferation, migration, and invasion both in vitro and in vivo. PLAGL2 expression was positively correlated with epidermal growth factor receptor (EGFR) expression. Mechanistically, this study demonstrated that PLAGL2 functions as a transcriptional regulator of EGFR and promotes HCC cell proliferation, migration, and invasion through the EGFR-AKT pathway. Moreover, hypoxia was found to significantly induce high expression of PLAGL2, which promoted hypoxia inducible factor 1/2 alpha subunit (HIF1/2A) expression through EGFR. Therefore, this study demonstrated that a PLAGL2-EGFR-HIF1/2A signaling loop promotes HCC progression. More importantly, PLAGL2 expression reduced hepatoma cells' response to the anti-EGFR drug erlotinib. PLAGL2 knockdown enhanced the response to erlotinib. Conclusions This study reveals the pivotal role of PLAGL2 in HCC cell proliferation, metastasis, and erlotinib insensitivity. This suggests that PLAGL2 can be a potential therapeutic target of HCC.

Transcription factor Sox2 is widely recognized for its critical roles in the nervous system, including the neural retina. Here, we aimed to reveal the function of Sox2 in the process of mouse postnatal development. After the suppression of Sox2 at P0, there was an increase number in bipolar cells but a decrease in amacrine cells. Inhibited Sox2 expression also led to decreased visual function. Furthermore, we found a distinctive type of retinal cells expressing the characteristic proteins of both bipolar cells and amacrine cells at P6, which may be an intermediate state in which amacrine cells were transforming into bipolar cells. Transcription factors associated with the development of bipolar cells and amacrine cells also support those changes. Our work indicated that inhibition of Sox2 could change cell fate by affecting transcription factors in the development of bipolar cells and amacrine cells, may provide new directions for the study and treatment of retinal genetic diseases and retinal dysplasia.

It is well known that GLP-1 activates GLP-1R to reduce body weight by inhibiting eating. GLP-1 is cleaved by the neutral endopeptidase (NEP) 24.11 into a pentapeptide GLP-1 (32-36) amide, which increases basal energy expenditure and inhibits weight gain in obese mice. It is well known that GLP-1 analogs can reduce weight by suppressing eating. However, there are few reports of reducing weight through the dual effects of inhibiting eating and increasing basic energy. Here, we report the peptide EGLP-1, a GLP-1 analogue, which can reduce food intake and increase basal energy expenditure. In C2C12 myotubes, EGLP-1 can increase both phosphorylation of acetyl CoA carboxylase (ACC) and the ratio between phosphorylation of ACC and the total expression of ACC (pACC/ACC). In diet-induced obese mice, EGLP-1 is more effective than exendin-4 in reducing body weight, reducing fat mass and improving hepatic steatosis. At the same time, EGLP-1 can improve hyperglycemia, reduce food intake, and improve insulin resistance, just like exendin-4. In addition, EGLP-1, not exendin-4, can improve physiological parameters associated with lipid metabolism and increase oxygen consumption by increasing uncoupling proteins 3 (UCP3) expression and pACC/ACC ratio in skeletal muscle. Taken together, this data showed that EGLP-1 is able to reduce body weight by reducing food intake and increasing basal energy expenditure, suggesting it may be more effective in treating diabetic and non-diabetic overweight or obese people than pure GLP-1R agonist exendin-4.

P277 is a 24 amino-acids peptide, residues 437–460 of human heat shock protein 60 (HSP60). P277 or sequence repeated 6 × P277 was previously found showing potency preventive and therapeutic anti-diabetes functions in NOD mice, but aroused atherosclerosis due to the induction of anti-HSP65 autoantibodies as reported. To determine the intrinsic B epitope sequence, we screened P277 with pepscan method and then proved by detection of sera IgG from peptide fragments vaccinated mouse and rabbits. Results indicated HSP60 443–448 (ALLRCI) is potential intrinsic B epitope sequence of P277. We modified P277 by deleting the former three amino acids of ALLRCI (VP) or replacing these six with alanine (AP). The detection of serum lipid parameter in NOD mice and aorta endothelial damage levels in high-cholesterol diets fed rabbits demonstrated that VP induced higher anti-diabetes efficacy and caused less arteriosclerosis-liked diseases separately. With less TLR2/4 activation of dendritic cells and macrophages, VP treatment reduced Th1 related P277 specific pro-inflammatory cytokines production and increased regulatory immune responses both in vivo and in vitro. These results indicated that optimized VP peptide might serve as a promising candidate for mouse type 1 diabetes therapy.

Breast cancer stem cells (BCSCs), a small subset of breast cancer cells with stem cell-like properties, are essential in tumor formation, metastasis, resistance to anticancer therapies, and cancer recurrence. MicroRNAs (miRNAs) are involved in tumorigenicity by regulating specific oncogenes and tumor-suppressor genes, and their roles in BCSCs are becoming apparent. A novel, 3-dimensional (3D), semisolid culture system was established to culture MCF-7 spheroid cells with high percentage of BCSCs. The differences in miRNA expression among the MCF-7 parental cells, BCSC-enriched MCF-7 spheroid cells, and CD44+/CD24- MCF-7 cells were evaluated by miRNA microarray, and the high expression of miR-210 in MCF-7 spheroid cells and CD44+/CD24- MCF-7 cells was verified by quantitative RT-PCR. MCF-7 cells were cultured in a hypoxic chamber to detect the effect of hypoxia on miR-210 expression and the stemness of the cells. The 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl tetrazolium bromide (MTT), transwell, and sphere-formation assays were performed to detect the proliferation, migration, and self-renewal ability of miR-210-overexpressed MCF-7 cells and MCF-7 spheroid cells with miR-210 knocked down. The target of miR-210 was validated with a dual-luciferase reporter assay and Western blotting. In vivo xenograft assay and metastasis assay were performed to study the effects of miR-210 targeting E-cadherin on BCSCs growth and lung metastasis, and the tumors were assessed by immunohistochemistry and immunofluorescence. We developed a novel 3D, semisolid culture system to culture MCF-7 spheroid cells, which are enriched in BCSCs, and found, by performing miRNAs expression profiling, miR-210 was up-regulated in those cells compared with MCF-7 parental cells. High miR-210 expression was also detected in CD44+/CD24- MCF-7 cells and human CD44+/CD24- breast cancer cells, which was demonstrated to be partially due to the hypoxic microenvironment around BCSCs in MCF-7 spheroids or solid tumors. Ectopic expression of miR-210 in MCF-7 cells promoted their migration, invasion, proliferation, and self-renewal in both in vitro and in vivo studies. We further reported that miR-210 suppressed E-cadherin expression by targeting the open reading frame region of E-cadherin mRNA and by up-regulation of E-cadherin transcription repressor, Snail. Accordingly, E-cadherin overexpression compromises the migration, invasion, proliferation, and self-renewal ability of miR-210-overexpressed MCF-7 both in vitro and in vivo. These findings reveal a novel regulatory pathway centered on hypoxia-mediated miR-210 targeting of E-cadherin, which contributes to the properties and breast tumorigenesis of BCSCs.-Tang, T., Yang, Z., Zhu, Q., Wu, Y., Sun, K., Alahdal, M., Zhang, Y., Xing, Y., Shen, Y., Xia, T., Xi, T., Pan, Y., Jin, L. Up-regulation of miR-210 induced by a hypoxic microenvironment promotes breast cancer stem cells metastasis, proliferation, and self-renewal by targeting E-cadherin.

Transmissible gastroenteritis virus (TGEV) causes severe diarrhea and high mortality in newborn piglets. It is well established that porcine intestinal epithelium is the target of the TGEV infection, however the mechanism that TGEV invades the host epithelium remains largely unknown. Aminopeptidase N (APN) is a known receptor of TGEV. This study discovered that the extracellular receptor binding domain 1 pertaining to epidermal growth receptor (EGFR) interact with TGEV spike protein. APN and EGFR synergistically promote TGEV invasion. TGEV promotes APN and EGFR clustering early in infection. Furthermore APN and EGFR synergistically stimulate PI3K/AKT as well as MEK/ERK1/2 endocytosis signaling pathways. TGEV entry is via clathrin and caveolin mediated endocytosis in IPEC-J2 cells. TGEV binds with EGFR, and subsequently promotes EGFR internalization by a clathrin-mediated endocytosis pathway. These results show that EGFR is a co-factor of TGEV, and that it plays a synergistic role with APN early in TGEV infection., Highlights • EGFR acts as a co-factor for TGEV entry. • EGFR plays a synergistic role with APN early in TGEV infection. • TGEV internalization through the clathrin-mediated endocytosis pathway.

Background/Aims: Pancreatic cancer remains one of the deadliest human malignancies, the lethality of which may be attributed to the presence of pancreatic cancer stem cells (PCSCs), a small subpopulation of cells existing within pancreatic tumor with high carcinogenesis. Therefore, it is crucial to establish an efficient enrichment and culture system of PCSCs and identify the key genes involved in the regulation of PCSCs. The three-dimensional (3D) liquid suspension mammosphere culture system has been established for enrichment and culture of PCSCs in vitro as the cell spheres are likely to originate from individual cell clone, but it has been challenged because the cell spheroids could be a result of cell aggregation. Methods: We optimized the existing culture system by adding methylcellulose to create a 3D semi-solid system which prevented the non-specific aggregation. Then we identified the CSC properties of Panc-1 spheroid cells cultured by this system by detecting the genes associated with stemness and by evaluation of the tumorigenicity in vitro and in vivo through invasion, migration and xenograft experiments methods. Subsequently, we performed high-throughput sequencing (HTS) of the Panc-1 spheroid cells. Results: We confirmed the PCSCs properties and high malignancy of the Panc-1 spheroid cells enriched by our novel 3D semi-solid system both in vitro and in vivo. Hundreds of mRNA, microRNA (miRNA) and dozens of long non-coding RNA (LncRNA) were identified to be differentially regulated in PCSCs-like Panc-1 spheroid cells compared with their parental cells by HTS. Conclusions: Our results demonstrate an efficient enrichment and culture system for Panc-1 spheroid cells with the PCSCs properties. The differentially expressed genes and their targets identified by the HTS of the Panc-1 spheroid cells can serve as new potential biomarkers for pancreatic cancer diagnosis and targeted therapy.

Progressive degeneration of retinal ganglion cells (RGCs) will cause a blinding disease. Most of the study is focusing on the RGCs itself. In this study, we demonstrate a decline of the presynaptic rod bipolar cells (RBCs) response precedes RGCs loss and a decrease of protein kinase Cα (PKCα) protein expression in RBCs dendrites, using whole-cell voltage-clamp, electroretinography (ERG) measurements, immunostaining and co-immunoprecipitation. We present evidence showing that N-methyl D-aspartate receptor subtype 2B (NR2B)/protein interacting with C kinase 1 (PICK1)-dependent degradation of PKCα protein in RBCs contributes to RBCs functional loss. Mechanistically, NR2B forms a complex with PKCα and PICK1 to promote the degradation of PKCα in a phosphorylation- and proteasome-dependent manner. Similar deficits in PKCα expression and response sensitivity were observed in acute ocular hypertension and optic never crush models. In conclusion, we find that three separate experimental models of neurodegeneration, often used to specifically target RGCs, disrupt RBCs function prior to the loss of RGCs. Our findings provide useful information for developing new diagnostic tools and treatments for retinal ganglion cells degeneration disease.

Breast cancer is the most prevalent cancer in women worldwide, which remains incurable once metastatic. Breast cancer stem cells (BCSCs) are a small subset of breast cancer cells which are essential in tumor formation, metastasis, and drug resistance. microRNAs (miRNAs) play important roles in the breast cancer cells and BCSCs by regulating specific genes. In this study, we found that miR-29a was up-regulated in BCSCs, in aggressive breast cancer cell line and in breast cancer tissues. We also confirmed suppressor of variegation 4–20 homolog 2 (SUV420H2), which is a histone methyltransferase that specifically trimethylates Lys-20 of histone H4 (H4K20), as the target of miR-29a. Both miR-29a overexpression and SUV420H2 knockdown in breast cancer cells promoted their migration and invasion in vitro and in vivo. Furthermore, we discovered that SUV420H2-targeting miR-29a attenuated the repression of connective tissue growth factor (CTGF) and growth response protein-1 (EGR1) by H4K20 trimethylation and promoted the EMT progress of breast cancer cells. Taken together, our findings reveal that miR-29a plays critical roles in the EMT and metastasis of breast cancer cells through targeting SUV420H2. These findings may provide new insights into novel molecular therapeutic targets for breast cancer.

Mycoplasma hyopneumoniae (Mhp) is the primary etiological agent responsible for swine enzootic pneumonia (EP), a disease that cause tremendous economic losses all over the swine industry. Dendritic cells (DCs), the most effective antigen-presenting cells, are widely distributed beneath respiratory epithelium. DCs uptake and present antigens to T cells, to initiate protective immune responses or generate immune-mediated pathology in different infections. In this study, we investigated the changes in the different DCs subpopulations, T cells and SIgA positive cells counts in porcine nasal cavity after long time Mhp infection. We further evaluated the role of porcine DCs in Mhp exposure. Our results showed that the number of SLA-II-DR+SWC3a+DCs, SLA-II-DR+CD11b+ DCs, T cells, SIgA positive cells in nasal cavity were decreased after Mhp 28 days infection in vivo experiment. The antigen presenting ability of DCs were inhibited by Mhp exposure. DCs couldn't activate T-cell proliferation by down-regulating the antigen presenting molecule CD1a expression and promoting high level of IL-10 production. Further more, the expression levels of IL-12 and IFN-γ in DCs were decreased, suggesting that DCs favour for Th2 immune response development after Mhp exposure in vitro. Taken together, Mhp infection impairs the immune function which allows the persistence of Mhp and cause predispose pigs to secondary infections. The decline of DCs presentation ability is the reason why dysfunction and persistence in Mhp infection. These findings are benefit for exploring the pathogenic mechanisms of Mhp in pigs.

Sanhuang Xiexin Tang (SXT), a classic prescription, has been clinically used to cure diabetes for thousands of years, but its mechanism remains unclear. Here, a systematic in-depth research was performed to unravel how it worked by the signaling pathway and metabonomics analysis. Our studies were conducted using high-fat diets (HFD) and streptozocin (STZ)-induced type 2 diabetes mellitus (T2DM) rats. The blood glucose was measured by a glucose-meter. Protein contents were determined by western blotting or ELISA and mRNA expression was identified by RT-PCR analysis. The pathological status of pancreas was assessed by histopathological analysis. Furthermore, Ultra Performance Liquid Chromatography-Quadrupole-Time of Flight/Mass Spectrometry (UPLC-Q-TOF/MS) coupled with multivariate statistical analysis was performed to discover potential biomarkers and the associated pathways. Hyperglycaemia, insulin resistance, dyslipidemia and inflammation in T2DM rats were significantly ameliorated after 7-week oral administration of SXT. The expressions of phosphatidylinositol-3-kinase (PI-3K), protein kinase B (Akt), glucose transporters-4 (GLUT4) Mrna, and p-PI-3K, p-Akt, GLUT4 protein involved in the PI-3K/Akt signaling pathway of T2DM were markedly up-regulated. Further investigation indicated that the perturbance of metabolic profiling in T2DM rats was obviously reversed by SXT and 38 potential biomarkers were screened and identified. Our study might help clarify the mechanism of SXT and provide some evidences for its clinical application in the future.

A major source of β cell generation is pancreatic progenitor-like cell differentiation. Multiple studies have confirmed that stem cell metabolism plays important roles in self-renewal and proliferation. In the absence of glucose, glutamine provides the energy for cell division and growth. Furthermore, α-ketoglutarate (αKG), a precursor for glutamine synthesis, is sufficient for enabling glutamine-independent cell proliferation. We have demonstrated that αKG contributes to the large-scale proliferation of pancreatic progenitor-like cells that can provide an ample amount of clinically relevant β cells. We compared the mRNA expression of a subset of genes, the abundance of ATP, reactive oxide species, mitochondrial number, and the colony-forming frequency between mouse pancreatic CD133+ and CD133− cells. We employed Real-Time PCR, immunostaining and passage assays to investigate self-renewal and proliferation of pancreatic progenitor-like cells in a 3D culture system in the presence and absence of αKG. The energy metabolism of CD133+ cells was more prone to oxidative phosphorylation. However, in the 3D culture system, when αKG was supplemented to the culture medium, the proliferation of the pancreatic progenitor-like cells was significantly elevated. We confirmed that the presence of αKG correlated with the up-regulation of Ten-Eleven Translocation (Tet). αKG can promote the proliferation of pancreatic progenitor-like cells via the up-regulation of Tet.

Background/Aims: Abnormal glucose metabolism and lipid metabolism are two key issues in Type 1 diabetes mellitus (T1DM). Insulin can control carbohydrate metabolism adequately, but cannot regulate lipid metabolism well in patients with T1DM. Glucagon-like peptide-1 receptor agonists (GLP-1RAs) have cured type 2 diabetes mellitus in clinical trials and have improved T1DM glycemic control in preclinical studies. However, previous studies have not reported whether GLP-1 can lower the serum concentration of non-esterified fatty acids (NEFAs). In this study, we examine whether GLP-1 can affect serum NEFA levels. Methods: The bioactivity of EGLP-1 (a novel GLP-1 analog) in vitro was analyzed in CG-HEK293 cells and with high-performance liquid chromatography. An intraperitoneal glucose tolerance test (IPGTT) was used to analyze the acute and sustained hypoglycemic effects of EGLP-1 in normal C57BL/6J mice. Streptozotocin-induced hyperglycemic mice were used to study the effects of EGLP-1 on blood glucose and NEFAs as well as its mechanism. Results: EGLP-1 activated GLP-1R and resisted dipeptidyl peptidase-IV digestion in vitro. Additionally, EGLP-1 had an insulinotropic action in vivo that lasted for approximately 6 h. In Streptozotocin-induced hyperglycemic mice, EGLP-1 improved hyperglycemia, inhibited food intake, and increased β-cell area. Serum physiological indexes showed that insulin and C-peptide levels were increased, while NEFA and triacylglycerol concentrations were decreased. Western blot analysis revealed that EGLP-1 significantly reduced phosphorylated-hormone sensitive lipase (pHSL) levels in white adipose tissue. Conclusions: EGLP-1 can improve hyperglycemia by increasing islet β-cell area and improving β-cell function, possibly due to reduced NEFA content in serum by lowering pHSL levels.

Background Mycoplasma hyopneumoniae (Mhp) causes porcine enzootic pneumonia, a disease that cause major economic losses in the pig industry. Dendritic cells (DCs), the most effective antigen-presenting cells, are widely distributed beneath respiratory epithelium, DCs uptake and present antigens to T cells, to initiate protective immune responses in different infections. In this study, we investigated the role of porcine DCs in vaccine Mhp-168 exposure. Results The antigen presenting ability of DCs were improved by vaccine Mhp-168 exposure. DCs could activate T-cell proliferation by up-regulating the antigen presenting molecule MHCII expression and co-stimulatory molecule CD80/86. However, the up-regulation of IL-10 and accompany with down-regulation of IFN-γ gene level may account for the limitation of attenuated Mhp-168 strain use as vaccine alone. Conclusion These findings are benefit for exploring the protection mechanisms and the possible limitations of this attenuated Mhp-168 vaccine.