Chimeric RNA was considered a special marker of cancer. However, recent studies have demonstrated that chimeric RNAs also exist in non-cancerous cells and tissues. Here, we analyzed and predicted jointly 49 chimeric RNAs by Star-Fusion and FusionMap. One chimeric RNA, we named TNNI2-ACTA1, and its eight transcript variants were identified by reverse transcriptase–polymerase chain reaction. The overexpression of TNNI2-ACTA1 V1 inhibited the proliferation of porcine skeletal muscle satellite cells through down-regulating the mRNA expression levels of cell cycle–related genes cyclinD1. However, as parental genes, there is no such effect in the TNNI2 and ACTA1. To explore the underlying mechanism for this phenomenon, we used RNA-seq to profile the transcriptomes of PSCs with overexpression. Compared with the negative control group, 1,592 differentially expressed genes (DEGs) were upregulated and 1,077 DEGs downregulated in TNNI2 group; 1,226 DEGs were upregulated and 902 DEGs downregulated in ACTA1 group; and 13 DEGs were upregulated and 16 DEGs downregulated in TNNI2-ACTA1 V1 group, respectively. Compared with the parental gene groups, three specific genes were enriched in the TNNI2-ACTA1 V1 group (NCOA3, Radixin, and DDR2). These three genes may be the key to TNNI2-ACTA1 V1 regulating cell proliferation. Taken together, our study explores the role of chimeric RNAs in normal tissues. In addition, our study as the first research provides the foundation for the mechanism of chimeric RNAs regulating porcine skeletal muscle growth.

cis-Splicing of adjacent genes (cis-SAGe) has been involved in multiple physiological and pathological processes in humans. However, to the best of our knowledge, there is no report of cis-SAGe in adipogenic regulation. In this study, a cis-SAGe product, BCL2L2–PABPN1 (BP), was characterized in fat tissue of pigs with RT-PCR and RACE method. BP is an in-frame fusion product composed of 333 aa and all the functional domains of both parents. BP is highly conserved among species and rich in splicing variants. BP was found to promote proliferation and inhibit differentiation of primary porcine preadipocytes. A total of 3074/44 differentially expressed mRNAs (DEmRs)/known miRNAs (DEmiRs) were identified in porcine preadipocytes overexpressing BP through RNA-Seq analysis. Both DEmRs and target genes of DEmiRs were involved in various fat-related pathways with MAPK and PI3K-Akt being the top enriched. PPP2CB, EGFR, Wnt5A and EHHADH were hub genes among the fat-related pathways identified. Moreover, ssc-miR-339-3p was found to be critical for BP regulating adipogenesis through integrated analysis of mRNA and miRNA data. The results highlight the role of cis-SAGe in adipogenesis and contribute to further revealing the mechanisms underlying fat deposition, which will be conductive to human obesity control.

The porcine epidemic diarrhea virus, PEDV, which causes diarrhea, vomiting and death in piglets, causes huge economic losses. Therefore, understanding how to induce mucosal immune responses in piglets is essential in the mechanism and application against PEDV infection with mucosal immunity. A method of treatment in our research was used to make an oral vaccine that packaged the inactive PEDV with microencapsulation, which consisted of sodium alginate and chitosan, and adapted the condition of the gut in mice. The in vitro release experiment of microcapsules showed that inactive PEDV was not only easily released in saline and acid solutions but also had an excellent storage tolerance, and was suitable for use as an oral vaccine. Interestingly, both experimental groups with different doses of inactive virus enhanced the secretion of specific antibodies in the serum and intestinal mucus, which caused the effective neutralization against PEDV in the Vero cell by both IgG and IgA, respectively. Moreover, the microencapsulation could stimulate the differentiation of CD11b+ and CD11c+ dendritic cells, which means that the microencapsulation was also identified as an oral adjuvant to help phagocytosis of dendritic cells in mice. Flow cytometry revealed that the B220+ and CD23+ of the B cells could significantly increase antibody production with the stimulation from the antigens’ PEDV groups, and the microencapsulation could also increase the cell viability of B cells, stimulating the secretion of antibodies such as IgG and IgA in mice. In addition, the microencapsulation promoted the expression of anti-inflammatory cytokines, such as IL-10 and TGF-β. Moreover, proinflammatory cytokines, such as IL-1, TNF-α, and IL-17, were inhibited by alginate and chitosan in the microencapsulation groups compared with the inactivated PEDV group. Taken together, our results demonstrate that the microparticle could play the role of mucosal adjuvant, and release inactivated PEDV in the gut, which can effectively stimulate mucosal and systemic immune responses in mice.

Background Lactobacillus casei had four possible probiotic roles, such as antimicrobial antagonism, competitional adhesion, immunoregulation and inhibition of bacterial toxin. The complex environment of gut microbe blocked to explore the antimicrobial scheme through genetics, metabolomics, proteomics, and signal transduction wth Lactobacillus casei. The genome editing Lactobacillus and germ-free animal provided the similar studying model of cell signal pathways to fully elucidate the probiotic mechanisms in animal. Results To delineate the metabolic reactions of nucleotides from L. casei associated with mechanisms of inhibiting pathogens and immunoregulation, we report the PyrR gene which was an important regulon to promote the pyrimidine biosynthesis, and PyrR deficient L. casei strain was successfully constructed with CRISPR-Cas9D10A tool. There were some changes with the basic biological characterization, such as decreasing the growth, auxotroph and morphological damage in the PyrR deficient strain. The metabolic profiles of supernatant between PyrR deficient and wild strain revealed a complementary regulation from other metabolic pathways within decreasing pyrimidine biosynthesis. Besides, the PyrR deficient strain significantly lost the character of inhibiting the growth of pathogens. We further identify PyrR regulating pyrimidine biosynthesis, which further improved the internalization and colocalization with the development of macrophages. Conclusions Evidence shows that PyrR gene is an active key for regulating pyrimidine biosynthesis in L. casei supernatant against a wide range of pathogens. The complementary regulation for the PyrR deficient could no inhibit the growth of pathogens, and immune regulation in the macrophages from mice. Thus, the deletion of PyrR in L. casei lost the probiotic character of antimicrobial antagonism and immunoregulation.

Chimeric RNA is a crucial target for tumor diagnosis and drug therapy, also having its unique biological role in normal tissues. TNNI2-ACTA1-V1 (TA-V1), a chimeric RNA discovered by our laboratory in porcine muscle tissue, can inhibit the proliferation of Porcine Skeletal Muscle Satellite Cells (PSCs). The regulatory mechanism of TA-V1 in PSCs remains unclear, but we speculate that NCOA3, DDR2 and RDX may be the target genes of TA-V1. In this study, we explored the effects of NCOA3, DDR2 and RDX on cell viability and cell proliferation by CCK-8 assay, EdU staining and flow cytometry. Furthermore, the regulatory pathway of proliferation in PSCs mediated by TA-V1 through NCOA3 or CyclinD1 was elucidated by co-transfection and co-immunoprecipitation (Co-IP). The results revealed that overexpression of NCOA3 significantly increased cell viability and the expression level of CyclinD1, and also promotes cell proliferation by changing cells from the G1 phase to the S phase. In addition, inhibiting the expression of NCOA3 substantially reduced cell viability and inhibited cell proliferation. Overexpression of DDR2 and RDX had no significant effect on cell viability and proliferation. Co-transfection experiments showed that NCOA3 could rescue the proliferation inhibition of PSCs caused by TA-V1. Co-IP assay indicated that TA-V1 directly interacts with NCOA3. Our study explores the hypothesis that TA-V1 directly regulates NCOA3, indirectly regulating CyclinD1, thereby regulating PSCs proliferation. We provide new putative mechanisms of porcine skeletal muscle growth and lay the foundation for the study of chimeric RNA in normal tissues.