SOX2OT inhibition enhances the choline O-Acetyltransferase gene expression in neuronal differentiation model of NT2 cells.

OBJECTIVE

This experimental study aimed to investigate the role of SOX2OT in NT2 cell neuronal differentiation to elucidate its specific function in this process, which remains unclear despite previous research highlighting its importance in central nervous system development.

MATERIAL AND METHODS

We utilized the NT2 cell line to create a constitutive SOX2OT knocked-down cell clone (SNT2) and a control clone (LNT2) through lentivirus-mediated shRNA delivery. All cell types underwent 3 weeks of neuronal differentiation induced by retinoic acid. Assessment involved morphological changes, Tubulin BIII immunofluorescence staining, and qRT-PCR for gene expression, including SOX2OT and neuronal markers (Nestin, CHAT, GFAP), compared to the control.

RESULTS

We demonstrated that stable SOX2OT inhibition did not hinder NT2 cell neuronal differentiation. SNT2 cells maintained their ability to morphologically differentiate into neurons, expressing Tubulin IIIB protein. Before differentiation, SNT2 cells showed a significant increase in CHAT expression (p-value = 0.01), persisting after 21 days (p-value = 0.037). While differentiation triggered a mild early decrease in SOX2OT expression in control cells, it increased in all cells during the third week. The neural differentiation lead to a general decrease in the gene expression of Nestin, and elevating trends in CHAT, and GFAP neuronal markers. By day 21, the expression of all genes in SNT2 cells significantly differed from NT2 controls (p-value = 0.037), suggesting SOX2OT's potential role in NT2 cell neuronal differentiation.

CONCLUSION

The results of this study showed that SOX2OT constitutive knocked down NT2 cells are still capable of differentiating into neuronal cells. However, SOX2OT inhibition can affect the expression of neuronal markers including choline O-Acetyltransferase which suggests its importance in the process of neuronal differentiation.

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