Construction of TERT Monoallelic Knockout and TERT Overexpression of Porcine Cell Lines and Study of the Cellular Biological Characteristics

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Abstract
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.
Funding Information
  • Major Science and Technology Project, Yunnan Province (202102AA100054)
  • National Natural Science Foundation of China (82560518)

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