Jing Wang

Telomerase reverse transcriptase subunit (TERT) is a key factor involved in telomere maintenance and genome stability, and the decline in its expression is closely related to cellular senescence. In this study, we established TERT monoallelic knockout (TERT+/−) and TERT overexpression (TERT-Over) cell lines in porcine iliac artery endothelial cells (PIEC) using CRISPR/Cas9 and PiggyBac systems to compare the effects of TERT monoallelic knockout versus overexpression on cellular biology. TERT expression and telomere length were assessed via qPCR and Western blot analysis. Cellular proliferation and senescence were evaluated using CCK-8 assays, cell cycle analysis, and SA-β-gal staining. Furthermore, the expression of key genes involved in cell proliferation, metabolism, and related signaling pathways was quantified using q-PCR. The results showed that the TERT mRNA level and telomere length decreased in TERT+/− cells. Meanwhile, we also observed that TERT+/− cells exhibited G1 phase arrest in the cell cycle, with suppressed proliferation and increased SA-β-gal-positive cells. This was accompanied by downregulation of cell cycle and proliferation-related genes, including c-Myc, the E2F family, and Ki-67, as well as downregulation of cell metabolism-related genes, including HIF1α, HK2, GLUT1, the SMAD family, FOXO1, and ATF4. In addition, cytochrome C was downregulated, suggesting activation of mitochondrial apoptotic signaling. Together, these findings indicate impaired proliferative and metabolic activity and are consistent with cellular senescence associated with telomere shortening. In TERT-overexpressing cells, the TERT gene expression and telomere length increase, cell proliferation accelerates, and the survival rate significantly increases under H₂O₂ treatment. This indicated that the overexpression of TERT can enhance resistance to oxidative stress, thus showing a kind of anti-aging phenotype. In conclusion, TERT monoallelic knockout induces cellular senescence-associated phenotypes in porcine endothelial cells, whereas TERT overexpression enhances proliferation and resistance to oxidative stress under the experimental conditions used in this study. The two porcine cell models established here may provide useful experimental materials for studying aging-related mechanisms and evaluating anti-aging interventions in large animals. Further studies are needed to directly determine their effects on cellular replicative lifespan.

Triple-negative breast cancer (TNBC) poses significant challenges due to its high aggressiveness, poor prognosis, and the lack of effective targeted therapies. Paclitaxel (PTX) is a chemotherapeutic agent commonly used in the treatment of TNBC; however, its efficacy is often compromised by drug resistance mediated by autophagy. This study investigated the synergistic effects of the autophagy inhibitor 3-methyladenine (3-MA) and PTX in a TNBC nude mouse model. Monitoring tumor volume and employing HE staining, immunofluorescence, and transmission electron microscopy revealed that PTX monotherapy induced tumor autophagy, characterized by the accumulation of LC3B/VPS34 proteins and an increase in autophagosomes. However, the co-administration of 3-MA reversed this process, significantly decreasing the tumor growth rate. Immunofluorescence and qPCR demonstrated that the combination group had fewer Ki-67-positive cells and more Caspase-3-positive cells, along with upregulated expression of autophagy-related genes and Caspase-family apoptosis genes. Consequently, this study suggests that inhibiting autophagy with 3-MA disrupts the autophagy-mediated protective mechanism of tumor cells, promoting the activation of apoptotic signals and enhancing the antitumor activity of PTX. These findings may offer new molecular mechanistic insights and potential therapeutic strategies for overcoming PTX resistance in TNBC.

Recombination-activating genes (RAGs) play a crucial role in the V(D)J recombination process and the development of immune cells. The development of the immune system and its mechanisms in pigs exhibit greater similarity to those of humans compared to other animals, thus rendering pigs a valuable tool for biomedical research. In this study, we utilized CRISPR/Cas9 gene editing and somatic cell nuclear transfer technology to generate RAG2 knockout (KO) pigs. Furthermore, we evaluated the impact of RAG2 KO on the immune organs and immune cell development through morphological observations, blood analysis and flow cytometry technology. RAG2 KO cell lines were used as donors for cloning. The reconstructed embryos were transplanted into 4 surrogate sows, and after 116 days of gestation, 2 sows gave birth to 12 live piglets, all of which were confirmed to be RAG2 KO. The thymus and spleen sizes of RAG2 KO pigs were significantly smaller than those of wild-type (WT) pigs. Hematoxylin-eosin staining results revealed that the thymus and spleen tissue structures of RAG2 KO pigs were disorganized and lacked the characteristic structures, indicating that RAG2 KO leads to dysplasia of the thymus and spleen. Hematological analysis demonstrated that the total number of white blood cells and lymphocytes in the circulation of RAG2 KO pigs was significantly lower, while the number of eosinophils was higher. Flow cytometry results indicated that the proportions of mature T and B lymphocytes were significantly reduced compared to WT pigs. These findings successfully verified the immunodeficiency phenotype of RAG2 KO pigs. This study may provide experimental animals for the development of tumor models and humanized animals.

As a member of the PIKs family, PIK3C3 participates in autophagy and plays a central role in liver function. Several studies demonstrated that the complete suppression of PIK3C3 in mammals can cause hepatomegaly and hepatosteatosis. However, the function of PIK3C3 overexpression on the liver and other organs is still unknown. In this study, we successfully generated PIK3C3 transgenic pigs through somatic cell nuclear transfer (SCNT) by designing a specific vector for the overexpression of PIK3C3. Plasmid identification was performed through enzyme digestion and transfected into the fetal fibroblasts derived from Diannan miniature pigs. After 2 weeks of culturing, six positive colonies obtained from a total of 14 cell colonies were identified through PCR. One positive cell line was selected as the donor cell line for SCNT for the construction of PIK3C3transgenic pigs. Thirty single blastocysts were collected and identified as PIK3C3 transgenic-positive blastocysts. Two surrogates became pregnant after transferring the reconstructed embryos into four surrogates. Fetal fibroblasts of PIK3C3-positive fetuses identified through PCR were used as donor cells for SCNT to generate PIK3C3 transgenic pigs. To further explore the function of PIK3C3 overexpression, genotyping and phenotyping of the fetuses and piglets obtained were performed by PCR, immunohistochemical, HE, and apoptosis staining. The results showed that inflammatory infiltration and vacuolar formation in hepatocytes and apoptotic cells, and the mRNA expression of NF-κB, TGF-β1, TLR4, TNF-α, and IL-6 significantly increased in the livers of PIK3C3 transgenic pigs when compared with wild-type (WT) pigs. Immunofluorescence staining showed that LC3B and LAMP-1-positive cells increased in the livers of PIK3C3 transgenic pigs. In the EBSS-induced autophagy of the porcine fibroblast cells (PFCs), the accumulated LC3II protein was cleared faster in PIK3C3 transgenic (PFCs) thanWT (PFCs). In conclusion, PIK3C3 overexpression promoted autophagy in the liver and associated molecular mechanisms related to the activation of ULK1, AMBR1, DRAM1, and MTOR, causing liver damage in pigs. Therefore, the construction of PIK3C3 transgenic pigs may provide a new experimental animal resource for liver diseases.

Multiple genetic modification is necessary for successful xenotransplantation from pigs. However, multiple-genetically modified cells usually suffer from various drug selections and long-term in vitro culture, which have a poor performance for somatic cell nuclear transfer (SCNT) to produce genetically modified pigs. We used to generate GTKO/hCD55/hCD59 triple-gene modified pigs by using drug-selective cell lines for SCNT, but the majority of cloned pigs were transgenic-negative individuals. In this study, to improve the production efficiency of multiple genetically modified pigs, we performed the recloning process by using transgenic porcine fetal fibroblast cells. As a result, two fetuses expressing hCD55 and hCD59 were obtained from 12 live-cloned fetuses, and one carrying high transgene expression was selected as a source of donor cells for recloning. Then we obtained 12 cloned piglets, all GTKO and carrying hCD55 and hCD59. Both hCD55 and hCD59 were expressed in fibroblast cells, but the expression levels of hCD55 and hCD59 were different among these piglets. Furthermore, piglet P5# had the highest expression of hCD55 and hCD59 in fibroblast cells than other piglets. Correspondingly, fibroblast cells of piglet P5# had significantly higher resistance against human serum-mediated cytolysis than those of piglet P11#. In conclusion, our results firstly provide support for improving efficiency of generating multiple genetically modified pig by recloning.