Expression, localization, and function of P4HB in the spermatogenesis of Chinese mitten crab (Eriocheir sinensis).

Background: The sperm of Chinese mitten crab (Eriocheir sinensis) have special noncondensed nuclei. The formation and stability of the special nuclei are closely related to the correct folding of proteins during spermatogenesis. P4HB plays a key role in protein folding, but its expression and role in the spermatogenesis of E. sinensis are unclear. Objective: To investigate the expression and distribution characteristics of P4HB in the spermatogenesis of E. sinensis as well as its possible role. Methods: The testis tissues of adult and juvenile E. sinensis were used as materials. We utilized a variety of techniques, including homology modeling, phylogenetic analysis, RT-qPCR, western blotting, and immunofluorescence staining to predict the protein structure and sequence homology of P4HB, analyze its expression in the testis tissues, and localize and semi-quantitatively assess its expression in different male germ cells. Results: The sequence of P4HB protein in E. sinensis shared a high similarity of 58.09% with the human protein disulfide isomerase, and the phylogenetic tree analysis indicated that the protein sequence was highly conserved among crustaceans, arthropods, and other animals species. P4HB was found to be expressed in both juvenile and adult E. sinensis testis tissues, with different localization patterns observed all over the developmental stages of male germ cells. It was higher expressed in the spermatogonia, spermatocytes, and stage I spermatids, followed by the mature sperm than in the stage II and III spermatids. The subcellular localization analysis revealed that P4HB was predominantly expressed in the cytoplasm, cell membrane, and extracellular matrix in the spermatogonia, spermatocytes, stage I and stage II spermatids, with some present in specific regions of the nuclei in the spermatogonia. In contrast, P4HB was mainly localized in the nuclei of stage III spermatids and sperm, with little expression observed in the cytoplasm. Conclusion: P4HB was expressed in the testis tissues of both adult and juvenile E. sinensis, but the expression and localization were different in male germ cells at various developmental stages. The observed differences in the expression and localization of P4HB may be an essential factor in maintaining the cell morphology and structure of diverse male germ cells in E. sinensis. Additionally, P4HB expressed in the nuclei of spermatogonia, late spermatids, and sperm may play an indispensable role in maintaining the stability of the noncondensed spermatozoal nuclei in E. sinensis.

RNA binding proteins involved in regulation of protein synthesis to initiate biogenesis of secondary tumor in hepatocellular carcinoma in mice.

BACKGROUND: The tumor microenvironment (TM) in close contact with cancer cells is highly related to tumor growth and cancer metastasis. This study is to explore the biogenesis mechanism of a secondary hepatocellular carcinoma (HCC) based on the function of RNA binding proteins (RBPs)-encoding genes in the physiological microenvironment (PM). METHODS: The healthy and HCC mice were used to isolate the PM, pre-tumor microenvironment (PTM), and TM. The samples were analyzed using the technology of RNA-seq and bioinformatics. The differentially expressed RBPs-encoding genes (DERs) and differentially expressed DERs-associated genes (DEDs) were screened to undergo GO and KEGG analysis. RESULTS: 18 DERs and DEDs were identified in the PTM vs. PM, 87 in the TM vs. PTM, and 87 in the TM vs. PM. Those DERs and DEDs participated in the regulation of gene expression at the levels of chromatin conformation, gene activation and silencing, splicing and degradation of mRNA, biogenesis of piRNA and miRNA, ribosome assemble, and translation of proteins. CONCLUSION: The genes encoding RBPs and the relevant genes are involved in the transformation from PM to PTM, then constructing the TM by regulating protein synthesis. This regulation included whole process of biological genetic information transmission from chromatin conformation to gene activation and silencing to mRNA splicing to ribosome assemble to translation of proteins and degradation of mRNA. The abnormality of those functions in the organic microenvironments promoted the metastasis of HCC and initiated the biogenesis of a secondary HCC in a PM when the PM encountered the invasion of cancer cells.

Pretumor microenvironment of hepatocellular carcinoma: Cancerization or anticancerization?

BACKGROUND: Tumor microenvironment (TM) has been deeply concerned. However, the pretumor microenvironment (PTM) was poorly understood. The purpose in this study was to explore the possible pathophysiological features of PTM before hepatocellular carcinoma (HCC) appearance. METHODS: Mouse livers with no swelling but with tumors present elsewhere in the body after subcutaneous injection of H22 in the fore underarm were considered a PTM, HCC tumors presenting far away from the PTM were regarded as a TM, and the healthy livers of mice without injection of H22 were regarded as a physiological microenvironment (PM). The transcriptomes of samples were generated using RNA-seq and validated using RT-qPCR. RESULTS: Overall, 4483 differentially expressed genes (DEGs) were found in the TM compared with the PTM (TM/PTM), but only 194 were altered in the PTM compared with the PM (PTM/PM). Among those 194 DEGs, 104 displayed upregulation and 90 downregulation. Some of these DEGs could promote the ability to resist cancerization or facilitate cancer metastasis, while others indicated liver impairment. The DEGs were involved in 16 relevant pathways. Additionally, the frequency of alternative splicing (AS) in the DEGs in various samples was positively related to the expression of those DEGs. CONCLUSIONS: The PTM initiatively armed itself to combat cancerization when its indications appeared although the PTM did not manifest any tissue swelling. However, the cancer cells were negatively influencing immunity to prevent clearance and positively promoting transformation to construct a suitable environment. During transformation by cancer cells, some genes with acquired AS participated in the construction of the PTM. This alteration created an invadable space and an appropriate environment for cancer cells.