Posted on April 24, 2021
Supplementary Materialsmolce-42-2-135-suppl. the nucleus. Overexpression of OCT4B19kDa promoted colony formation of glioblastoma cells when produced in soft agar culture conditions. Clinical data analysis revealed that patients with gliomas that expressed OCT4B at high levels experienced a poorer prognosis than patients with gliomas that expressed OCT4B at low levels. Thus, OCT4B19kDa may play a crucial role in regulating malignancy cell survival and adaption in a rigid environment. (Verhaak et al., 2010). Despite many efforts to develop effective treatment strategies, surgery followed by concurrent treatment of temozolomide (TMZ) and ionizing radiation (IR) is the only standard therapy currently available. The presence of the blood-brain barrier and heterogeneous cell populations in the tumor bulk are key obstacles against varying treatments (Eun et al., 2017; Lathia et al., 2015). However, these features do not fully account for the high frequency of recurrence and resistance against standard therapies. Glioblastoma stem cells (GSCs) are a small populace of glioblastoma cells that exhibit self-renewal capabilities, prolonged proliferation, and tumor initiation (Lathia et al., 2015). GSCs have been YHO-13177 reported to be responsible for the resistance to TMZ and IR therapies and consequent tumor recurrence as well as a poor prognosis in patients with GBM (Kim et al., 2015). OCT4, also known as POU5F1, is a transcription factor involved in stem cell pluripotency. The OCT4 gene is located on chromosome 6 and comprises of 7 exons (Takeda et al., 1992). This gene encodes three isoforms (OCT4A, OCT4B, and OCT4B1) as a result of option splicing (Wang and Dai, 2010). OCT4A translates into one protein (360 amino acids), whereas OCT4B and OCT4B1 can translate up to three proteins (265, 190, and 164 amino acids, respectively) through differential usage of translational initiation sites (Gao et al., 2010). Currently, many studies have exhibited that aberrant expression of OCT4B has been detected in various human malignancies including gastric malignancy (Asadi et al., 2011), colorectal malignancy (Gazouli et al., 2012), bladder malignancy (Asadzadeh et al., 2012), and cervical malignancy (Li et al., 2015). OCT4B also renders cells resistant to apoptotic cell YHO-13177 death and heat shock or genotoxic stresses (Gao et al., 2012; Wang et al., 2009). OCT4A and OCT4B are localized in different subcellular regions: OCT4A is usually localized to the nucleus and functions as a transcription factor, whereas OCT4B is mainly located in cytoplasm (Lee et al., 2006). Therefore, the precise expression pattern and biological functions of OCT4B isoforms remain largely unknown. In the YHO-13177 present study, we delineate the expression pattern of the OCT4A and OCT4B isoforms in human glioblastoma cells and reveal a novel biological function of OCT4B, which is predominantly expressed in human glioblastoma cells. MATERIALS AND METHODS Cells and culture conditions Human glioblastoma cell lines U87MG (wt, mut, mut, wt, mut, wt, del), T98G (mut, mut, del), A172 (wt, del, wt, 0.05 (*), 0.01 (**) or 0.001 (***) were considered statistically significant for different experiments as indicated in the figure legends. Data are offered as means standard error of the mean (SEM). RESULTS Structure of OCT4 variants and their expression pattern in human glioblastoma cells The OCT4 gene consists of 7 exons, and OCT4A, OCT4B, and OCT4B1 are generated by option splicing (Fig. 1A). Only has exon 1, indicating that OCT4A has Grem1 a different N-terminal region compared with OCT4B and OCT4B1. OCT4B and OCT4B1 have comparable transcript structures except of exon 2c, but the function of exon 2c remains uncharacterized. The gene encodes a single protein consisting of 360 amino acids, whereas the and genes enable the generation of three proteins consisting of 164, 190, and 265 amino acids via differential usage of translational start sites (Fig. 1A). Open in a separate windows Fig. 1 Expression of OCT4 variants in human glioblastoma cells(A) A schematic diagram showing mRNAs and proteins expressed from your human gene. (B) A qRT-PCR analysis showing mRNA expression levels of human OCT4 isoforms including in normal human astrocytes (NHA), glioblastoma stem cells (528NS, 84NS, 19, YHO-13177 and MD13) and glioblastoma cells (LN18, LN229, T98G, U87MG, A1207, and A172). (C) Relative OCT4B19kDa protein expression levels in different cell types explained in (b). The transmission intensity of western blot bands was quantified using NIH ImageJ. First, we examined the expression of OCT4 isoforms in several human main GSCs and glioblastoma cell lines. Quantitative RT-PCR analysis showed that mRNA was abundantly expressed in human GSCs and glioblastoma cells (Fig. 1B). Western blot analysis revealed that OCT4A was predominantly upregulated in induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs)(Supplementary Fig. S1A). Human GSCs and glioblastoma cells expressed only the 190-amino acid version of OCT4B (OCT4B19kDa) (Supplementary Fig. S1A; the 16-kDa protein is not shown in these data). OCT4B19kDa expression was markedly increased in.