Antisense Oligonucleotide-Mediated XIST Knockdown Modulates Endogenous Pluripotency-Associated Transcription Factor Expression in Porcine Female Somatic Cells

The long non-coding RNA XIST is a key regulator of X-chromosome inactivation (XCI) and contributes to epigenetic regulation during cell reprogramming. However, whether XIST suppression influences endogenous pluripotency-associated transcriptional programs in porcine somatic cells remains unclear. In this study, we investigated the effects of XIST knockdown on endogenous pluripotency-associated transcription factors (pTFs) in IBRS-2 cells. Chemically modified antisense oligonucleotides (ASOs) targeting XIST were designed and transfected into IBRS-2 cells. XIST expression was efficiently suppressed for up to 120 h after ASO treatment. RT-qPCR analysis showed that XIST knockdown induced sustained upregulation of KLF4 and LIF expression, prolonged intermittent activation of NANOG, and transient activation of G6PD. In contrast, OCT4, SOX2, and c-MYC were significantly upregulated only during the early stage after transfection. KLF4 immunofluorescence was detectable in a subset of ASO-2-treated cells. These findings demonstrate that ASO-mediated XIST knockdown partially facilitates the activation of a subset of endogenous pluripotency-associated genes in porcine female somatic cells. This study provides new insights into the role of XIST as an epigenetic regulator during porcine somatic reprogramming and suggests that optimized XIST interference may represent a potential strategy for enhancing endogenous pluripotency-associated gene activation without relying on exogenous transcription factor delivery.