Transcriptomics and Phenotypic Analysis of gpr56 Knockout in Zebrafish.

The adhesion G-protein-coupled receptor is a seven-transmembrane receptor protein with a complex structure. Impaired GPR56 has been found to cause developmental damage to the human brain, resulting in intellectual disability and motor dysfunction. To date, studies on gpr56 deficiency in zebrafish have been limited to the nervous system, and there have been no reports of its systemic effects on juvenile fish at developmental stages. In order to explore the function of gpr56 in zebrafish, the CRISPR/Cas9 gene-editing system was used to construct a gpr56-knockout zebrafish. Subsequently, the differentially expressed genes (DEGs) at the transcriptional level between the 3 days post fertilization (dpf) homozygotes of the gpr56 mutation and the wildtype zebrafish were analyzed via RNA-seq. The results of the clustering analysis, quantitative PCR (qPCR), and in situ hybridization demonstrated that the expression of innate immunity-related genes in the mutant was disordered, and multiple genes encoding digestive enzymes of the pancreatic exocrine glands were significantly downregulated in the mutant. Motor ability tests demonstrated that the gpr56-/- zebrafish were more active, and this change was more pronounced in the presence of cold and additional stimuli. In conclusion, our results revealed the effect of gpr56 deletion on the gene expression of juvenile zebrafish and found that the gpr56 mutant was extremely active, providing an important clue for studying the mechanism of gpr56 in the development of juvenile zebrafish.

Transmembrane protein 121 as a novel inhibitor of cervical cancer metastasis.

Transmembrane protein 121 (TMEM121) is isolated from the chicken heart using subtraction hybridisation. A previous study by the authors indicated that TMEM121 is highly expressed in adult mouse hearts and acts as an inhibitor of pathological cardiac hypertrophy. In the present study, the association between TMEM121 and cancer was investigated using bioinformatics tools, including Tumour Immune Estimation Resource (TIMER) 2.0, cBioPortal, LinkedOmics analysis, Kaplan-Meier plotter and UALCAN analysis. The expression, genetic variation, gene interaction network and co-expression pattern of TMEM121 in tumours were analysed. The results revealed that TMEM121 was expressed in various tumours and significantly downregulated in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) when compared with its expression in paracancerous tissues, whereas the methylation level of its promoter was increased in tumour tissues. Additionally, associations between TMEM121 and the PI3K/AKT signalling pathway, as well as the expression of cancer-related molecules, were detected. The aforementioned bioinformatics analysis suggests that TMEM121 may be involved in the development of cervical cancer. Therefore, gain-of-function and loss-of-function experiments in HeLa cells were conducted to verify the role of TMEM121 in cervical cancer. The assay using Cell Counting Kit-8 (CCK-8) revealed that the cell viability of HeLa cells with TMEM121 overexpression was significantly reduced. High TMEM121 expression inhibited HeLa cell migration, as indicated by the decrease in the cell scratch healing rate. The western blot assay revealed that TMEM121 overexpression downregulated the expression of B-cell lymphoma 2 (BCL-2), cyclin D1, cyclin E2 and phosphorylated (p)-AKT, while upregulating that of p27, E-cadherin and p-p38. When TMEM121 was knocked down, retinoblastoma protein (RB), p53, p27, E-cadherin, p-JNK and p-p38 were inhibited, but cyclin E1 was promoted. By combining bioinformatics and experimental biology in the present study, the results demonstrated for the first time, to the best of our knowledge, that TMEM121 may be a novel inhibitor of cervical cancer that is linked to multiple signalling pathways, paving the way for the development of novel diagnostic and therapeutic strategies.