Abstract

Introduction: Transmembrane protein 121 (TMEM121) is a conserved integral membrane protein highly expressed in the rat adrenal sub-capsular niche, where it co-localises with the proliferation marker Ki67 and is implicated in stem/progenitor cell self-renewal and tissue homeostasis. Thy-1⁺positive cells (Thy1cells), isolated from rat adrenal cortex, exhibit mesenchymal stem cell characteristics early in culture but undergo spontaneous immortalisation with prolonged passaging. The dynamics of TMEM121 expression across this transition remain uncharacterised. Materials and Methods: Thy1cells were isolated by Magnetic-Activated Cell Sorting (MACS) and monitored over nine months. TMEM121 mRNA expression was assessed by RT-qPCR using the ΔΔCt method with β-Actin as reference. Protein expression was evaluated by dual immunocytofluorescence using anti-TMEM121 and anti-Thy1 antibodies, with fluorescence area (μm²) and signal intensity quantified in >100 cells/condition using Fiji/ImageJ. Data are presented as mean ± SEM; group differences were analysed by unpaired Student’s t-test. Results: Thy1cells displayed slow, fibroblast-like colony growth in early culture, then underwent a marked phenotypic shift between months 4 and 6, with reduced substrate adherence and accelerated proliferation, indicating immortalisation. RT-qPCR showed a significant reduction in TMEM121 mRNA in late-passage (9-month) cells (1.1±0.3-fold) versus early-passage (2-month) cells (1.9±0.2-fold). Immunocytofluorescence confirmed a significant (p < 0.001) decline in protein expression, with 46.3% reduction in expression area and 63.6% reduction in signal intensity in late-passage cells. Discussion: The progressive loss of TMEM121 parallels the transition from a homeostatic, niche-dependent state to unrestrained proliferation, mirroring its downregulation in human malignancies where it correlates with poor prognosis and dysregulation of the AKT1/p27/cyclin axis. Its context-dependent role—tumour-suppressive in some cancers yet oncogenic in others—suggests TMEM121 operates as a microenvironment-sensitive regulator whose silencing may confer a competitive proliferative advantage in vitro. Conclusion: TMEM121 is significantly downregulated at both mRNA and protein levels during spontaneous immortalisation of Thy1cells, identifying it as a candidate marker of the stem-like homeostatic state and a potential target for further investigation in adrenocortical pathology.