Characterization and function of PTEN-induced putative kinase 1 (PINK1) in process of Zinc alleviates hepatic lipid deposition of yellow catfish (Pelteobagrus fulvidraco).

PTEN-induced putative kinase 1 (PINK1) is a key regulator of mitophagy, however, the relevant information remains poorly understood on aquatic animals. Here, a PINK1 gene was cloned, characterized and functionally studied in yellow catfish. PINK1 encoded a protein containing 570 amino acids, 2 functional domains. High fat (15.66%) fed fish showed a downregulation trend of liver PINK1 expression than that of normal fat (10.14%) group, and was reversed by the addition of Zn. In the in vitro study, high fat (HF) can increase lipid deposition and decrease by addition Zn (HFZ) in hepatocytes, whereas above phenomena reversed by overexpression/interference of PINK1, respectively. In addition, the addition of Zn can significantly affect mitochondrial activity, increase mitophagy, and improve the antioxidant activity of hepatocytes. Together, these findings illustrated that yellow catfish PINK1 is conserve, and it participated in mitochondria control of fish. These findings indicate Zn could alleviate high fat-induced hepatic lipid deposition of fish by activating PINK1-mediated mitophagy and provide basis for further exploring new approach for decreasing lipid deposition in fish products during aquaculture.

Screening and identification of miR-181a-5p in oral squamous cell carcinoma and functional verification in vivo and in vitro.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a common malignant tumor associated with poor prognosis. MicroRNAs (miRNAs) play crucial regulatory roles in the cancer development. However, the role of miRNAs in OSCC development and progression is not well understood. METHODS: We sought to establish a dynamic Chinese hamster OSCC animal model, construct miRNA differential expression profiles of its occurrence and development, predict its targets, and perform functional analysis and validation in vitro. RESULTS: Using expression and functional analyses, the key candidate miRNA (miR-181a-5p) was selected for further functional research, and the expression of miR-181a-5p in OSCC tissues and cell lines was detected. Subsequently, transfection technology and a nude mouse tumorigenic model were used to explore potential molecular mechanisms. miR-181a-5p was significantly downregulated in human OSCC specimens and cell lines, and decreased miR-181a-5p expression was observed in multiple stages of the Chinese hamster OSCC animal model. Moreover, upregulated miR-181a-5p significantly inhibited OSCC cell proliferation, colony formation, invasion, and migration; blocked the cell cycle; and promoted apoptosis. BCL2 was identified as a target of miR-181a-5p. BCL2 may interact with apoptosis- (BAX), invasion- and migration- (TIMP1, MMP2, and MMP9), and cell cycle-related genes (KI67, E2F1, CYCLIND1, and CDK6) to further regulate biological behavior. Tumor xenograft analysis indicated that tumor growth was significantly inhibited in the high miR-181a-5p expression group. CONCLUSION: Our findings indicate that miR-181a-5p can be used as a potential biomarker and provide a novel animal model for mechanistic research on oral cancer.

Identification of antioxidant peptides after digestion and absorption of isinglass by serum peptidomics and cellular antioxidant activity analysis.

Isinglass, a dried product of the swim bladder, has been widely used in traditional Chinese medicine. This study attempts to identify natural antioxidant peptides after digestion and absorption of isinglass in vivo. The antioxidant effects of dietary isinglass were demonstrated by evaluating the activities of SOD, CAT and MDA contents in the mouse liver. Four novel antioxidant-related peptides (RLLWENGNLL, GSKAENPTNPGP, SPVPDLVPGSF and VPDLVPGSF) were screened based on serum peptidomics and amino acid composition. Furthermore, pretreating with four peptides significantly increased the cell viability, and SOD and CAT activities of AML12 cells with H2O2-mediated oxidative damage, meanwhile, significantly reduced the ROS level, MDA content and apoptosis rate and attenuated DNA damage. Therefore, it was concluded that pretreatment of the identified peptides had a protective effect on oxidatively damaged cells. This result can aid in the recognition of active peptides from isinglass consumption for potential application in nutraceuticals or functional ingredients in food.

Construction of Fzd6Q152E mice through CRISPR/Cas9 technology and their reproduction and identification.

BACKGROUND: The CRISPR/Cas9 system is widely used for genome editing in human, rat and mouse cells. In this study, we established Fzd6 mutant mice using CRISPR/Cas9 technology, and obtained Fzd6 homozygous mutant (Fzd6Q152E) mice through breeding. Fzd6 plays a role in depression, but there are few related reports. We used this model to investigate the mechanism of Fzd6 involved in depression, and build a solid foundation for subsequent in-depth studies. METHODS AND RESULTS: The target of Fzd6 mutation was obtained by CRISPR/Cas9 technology and hippocampal tissue was collected for Nissl staining and histological analysis. Blood was collected for enzyme linked immunosorbent assay (ELISA); The gene expression of Fzd6 and the related genes expression in wnt pathway was quantified by quantitative real-time PCR (qRT-PCR), and then expression of Fzd6 and proteins in the Wnt pathway were identified by western blotting. ELISA results showed that the expression levels of brain derived neurotrophic factor (BDNF), 5-hydroxytryptamine (5-HT), and Noradrenaline (NE) in serum were significantly decreased in Fzd6Q152E mice, whereas the mRNA expression of Lrp5, Lrp6, and Dkk2 is increased. The western blotting revealed that the expression of Fzd6 and Lrp6 is decreased, although the expression of Dkk2 and Gsk-3ß increased. CONCLUSION: Our study successfully established homozygous Fzd6 mutant mice model. The relationship between Fzd6-Wnt and depression was preliminarily clarified, which provides an ideal animal model for subsequent research on diseases induced by the Fzd6 mutation.

Serum proteomic analysis reveals possible mechanism underlying physiological hemostasis of swim bladder.

The hemostatic effect of isinglass (dried swim bladder) in traditional Chinese medicine is well known. But its mechanism of action remains unclear. Here, mice were gavaged with the dried swim bladder of the chu's croaker (Nibea coibor). The hemostatic effect of swim bladder was investigated, tandem mass tag (TMT)-based quantitative proteomics analysis was performed to screen differentially abundant proteins associated with hemostasis in mouse serum. Results indicated that isinglass significantly shorten bleeding time and promoted coagulation after acute trauma (cut out mouse tail). In total, 57 differentially expressed proteins were identified in the sera between control and swim bladder group, of which 31 were up-regulated and 26 were down-regulated in swim bladder group. KEGG pathway enrichment analysis further demonstrated that the Neutrophil extracellular trap formation pathway was significantly affected. Combined with RT-qPCR verification, our findings further suggested that five candidate proteins in the pathway may be involved in the onset of hemostasis after swim bladder gavage, indicating their important role during the hemostasis process promoting by swim bladder. SIGNIFICANCE: Serum proteomics after swim bladder gavage described differentially enriched proteins related to hemostasis, and enriched pathways were validated. This study revealed the possible pathways involved in the hemostatic effect of swim bladder, which may provide a new effector target for the development of new hemostatic drugs.

Animal model and bioinformatics analyses suggest the TIMP1/MMP9 axis as a potential biomarker in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a common malignant tumor of the head and neck. However, the molecular mechanism underlying its development and progression is yet unclear. Genes that are differentially expressed, that is, differentially expressed genes (DEGs), between normal and diseased tissues are believed to be involved in disease development and progression. To identify the DEGs in OSCC and explore their role in occurrence and progression, we established a Chinese hamster OSCC model, determined the DEG, screened the identified DEGs, and performed Gene Ontology (GO) and KEGG enrichment analyses. A protein-protein interaction (PPI) network was generated to screen potential candidate genes. We then analyzed the expression, tumor stage and prognosis of candidate genes using the Gene Expression Profiling Interactive Analysis (GEPIA) database. Finally, we verified the candidate DEGs by quantitative real-time PCR and Gene Expression Omnibus analysis. The results showed 194 significantly DEGs, 140 enriched GO terms, and 8 KEGG pathways, which suggested that OSCC was closely related to the immune system, cell migration, and extracellular matrix. GEPIA and PPI network analysis revealed that SPP1, TNC, and ACTA1 were significantly related to tumor staging; SPP1, tissue inhibitors of matrix metallopeptidases (MMPs) 1 (TIMP1), and ACTA1 were closely related to prognosis. The scores for the top five highest degree genes were close, and the TIMP1/MMP9 axis appeared to be at the center of the PPI network, indicating that expression changes in the TIMP1/MMP9 axis and related genes may be involved in tumor invasion and metastasis. These findings provide novel insights into the mechanism of oral cancer.

MicroRNA-504 functions as a tumor suppressor in oral squamous cell carcinoma through inhibiting cell proliferation, migration and invasion by targeting CDK6.

MicroRNA (miRNA), as tumor suppressor or oncogene, is involved in the regulation of tumor development. However, the role of miRNA in human oral squamous cell carcinoma (OSCC) is less well understood. In our previous studies, we have successfully established a Chinese hamster oral squamous cell carcinoma animal model and constructed a miRNA differential expression profile, and screened out the abnormal expressed gene (miR-504). The purpose of this study was to investigate the regulatory mechanism of miR-504 in human OSCC cells and to elucidate its new target genes. CCK-8 assay, colony formation assay, transwell migration and invasion assay were used to test the cell proliferation, cell growth, cell migration and cell invasion abilities of the miR-504 mimics group, respectively. Flow cytometry was used to detect the apoptotic ability. In order to verify the direct target of miR-504, we used the dual luciferase reporting system for detection. The expressions of RNA and proteins were detected by qRT-PCR and western blotting. The results of our study showed that miR-504 expression was down-regulated in OSCC animal tissue samples. In human OSCC cell lines, miR-504 over-expression significantly inhibited cell proliferation, migration and invasion. The dual luciferase reporting system confirmed that CDK6 was a direct target of miR-504 and that miR-504 expression inhibited CDK6 expression. In addition, the over-expression of miR-504 in OSCC cells could significantly inhibit the expression of cell cycle-related proteins (E2F1, Cyclin D1), but the expression of p21 was significantly increased. The results of this study suggest that miR-504 may be a new therapeutic target for OSCC.

Identification and profiling of microRNAs expressed in oral buccal mucosa squamous cell carcinoma of Chinese hamster.

MicroRNAs are known to play essential role in the gene expression regulation in cancer. In our research, next-generation sequencing technology was applied to explore the abnormal miRNA expression of oral squamous cell carcinoma (OSCC) in Chinese hamster. A total of 3 novel miRNAs (Novel-117, Novel-118, and Novel-135) and 11 known miRNAs (crg-miR-130b-3p, crg-miR-142-5p, crg-miR-21-3p, crg-miR-21-5p, crg-miR-542-3p, crg-miR-486-3p, crg-miR-499-5p, crg-miR-504, crg-miR-34c-5p, crg-miR-34b-5p and crg-miR-34c-3p) were identified. We conducted functional analysis, finding that 340 biological processes, 47 cell components, 46 molecular functions were associated with OSCC. Meanwhile the gene expression of Caspase-9, Caspase-3, Bax, and Bcl-2 were determined by qRT-PCR and the protein expression of PTEN and p-AKT by immunohistochemistry. Our research proposed further insights to the profiles of these miRNAs and provided a basis for investigating the regulatory mechanisms involved in oral cancer research.