Tissue expression and promoter activity analysis of the porcine TNFSF11 gene.

Tumour necrosis factor (TNF) superfamily member 11 (TNFSF11), also known as RANKL, plays a crucial role in regulating several physiological and pathological activities. Additionally, it is a vital factor in bone physiology, and the sex hormone progesterone regulates the expansion of stem cells and the proliferation of mammary epithelial cells. It is essential for animal growth and reproductive physiological processes. This study aimed to evaluate the tissue-specific expression characteristics and promoter activity of the TNFSF11 gene in pigs. As a result, the study examined the presence of TNFSF11 expression in the tissues of Xiangsu pigs at 0.6 and 12 months of age. Moreover, the core promoter region of TNFSF11 was also identified by utilizing a combination of bioinformatic prediction and dual-luciferase activity tests. Finally, the effect of transcription factors on the transcriptional activity of the core promoter region was determined using site-directed mutagenesis. TNFSF11 was uniformly expressed in all tissues; however, its expression in muscles was comparatively low. The core promoter region of TNFSF11 was located in the -555 to -1 region. The prediction of the transcription start site of TNFSF11 gene-2000 ∼ + 500bp showed that there was a CpG site in 17 ∼ + 487bp. Analysis of mutations in the transcription factor binding sites revealed that mutations in the Stat5b, Myog, Trl, and EN1 binding sites had significant effects on the transcriptional activity of the TNFSF11 gene, particularly following the EN1 binding site mutation (P < 0.001). This study provides insights into both the tissue-specific expression patterns of TNFSF11 in the tissues of Xiangsu pigs and the potential regulatory effects of transcription factors on its promoter activity. These results may be helpful for future research aimed at clarifying the expression and role of the porcine TNFSF11 gene.

Association between polymorphisms in NOBOX and litter size traits in Xiangsu pigs.

The newborn ovary homeobox gene (NOBOX) regulates ovarian and early oocyte development, and thus plays an essential role in reproduction. In this study, the mRNA expression level and single nucleotide polymorphism (SNP) of NOBOX in various tissues of Xiangsu pigs were studied to explore the relationship between its polymorphism and litter size traits. Also, bioinformatics was used to evaluate the effects of missense substitutions on protein structure and function. The results revealed that NOBOX is preferentially expressed in the ovary. Six mutations were detected in the NOBOX sequence, including g.1624 T>C, g.1858 G>A, g.2770 G>A, g.2821 A>G, g.5659 A>G, and g.6025 T>A, of which g.1858 G>A was a missense mutation. However, only g.1858 G>A, g.5659 A>G, and g.6025 T>A were significantly associated with litter size traits (p < 0.05). Further prediction of the effect of the missense mutation g.1858 G>A on protein function revealed that p.V82M is a non-conservative mutation that significantly reduces protein stability and thus alters protein function. Overall, these findings suggest that NOBOX polymorphism is closely related to the litter size of Xiangsu pigs, which may provide new insights into pig breeding.

Photoactivated DNA Nanodrugs Damage Mitochondria to Improve Gene Therapy for Reversing Chemoresistance.

Multidrug resistance (MDR) is a major cause of chemotherapy failure in oncology, and gene therapy is an excellent measure to reverse MDR. However, conventional gene therapy only modulates the expression of MDR-associated proteins but hardly affects their existing function, thus limiting the efficiency of tumor treatment. Herein, we designed a photoactivated DNA nanodrug (MCD@TMPyP4@DOX) to improve tumor chemosensitivity through the downregulation of MDR-related genes and mitochondria-targeted photodynamic therapy (PDT). The self-assembled DNA nanodrug encodes the mucin 1 (MUC1) aptamer and the cytochrome C (CytC) aptamer to facilitate its selective targeting to the mitochondria in tumor cells; the encoded P-gp DNAzyme can specifically cleave the substrate and silence MDR1 mRNA with the help of Mg2+ cofactors. Under near-infrared (NIR) light irradiation, PDT generates reactive oxygen species (ROS) that precisely damage the mitochondria of tumor cells and break single-stranded DNA (ssDNA) to activate MCD@TMPyP4@DOX self-disassembly for release of DOX and DNAzyme. We have demonstrated that this multifunctional DNA nanodrug has high drug delivery capacity and biosafety. It enables downregulation of P-gp expression while reducing the ATP on which P-gp pumps out drugs, improving the latency of gene therapy and synergistically reducing DOX efflux to sensitize tumor chemotherapy. We envision that this gene-modulating DNA nanodrug based on damaging mitochondria is expected to provide an important perspective for sensitizing tumor chemotherapy.