Expression of the genomic organizer special AT-rich sequence binding protein-1 (SATB1) in bladder cancer cell lines
Maria A. Voznesensky, -, Shilpa Choudhary, -, Olga S. Voznesensky, -, Carol Pilbeam, PhD, MD, John A. Taylor, III, MD.
University of Connecticut, Farmington, CT, USA.
Background: SATB1 is involved in tissue specific gene expression through spatial reconfiguration of the chromatin structure. It has recently been reported that increased expression of SATB1 in humans correlates with upregulation of metastasis-associated genes and down regulation of tumor suppressor proteins, thereby promoting growth and metastasis. We explored the expression of SATB1 in bladder cancer cell lines.
Methods: Three human urothelial cell lines representing benign (UROtsa), malignant (HT-1376) and malignant/metastatic (HTB-5) were evaluated for SATB1 mRNA expression by quantitative real-time polymerase chain reaction (qPCR) and nuclear protein expression by Western Blot analysis. HTB-5 cells were stably transfected with 1 of 4 shRNA constructs for SATB1 or a non-silencing (NS) control to specifically knockdown SATB1 expression. RNA and potein levels were measured to assess efficiency of knockdown. Cell numbetrs were then measured from day 2-6.
Results: SATB1 mRNA and protein expression levels progressively increased from the benign to malignant to metastatic cell lines. mRNA relative quantification values were ~3-fold (p=0.01) and 28-fold (p=0.01) higher, respectively, for HT-1376 and HTB-5 lines compared to UROtsa. The nuclear protein expression levels, after normalization with β-actin, were ~2-fold and 5-fold higher in HT-1376 and HTB-5 cell lines, respectively, compared to UROtsa cells. One out of 4 shRNA constructs demonstrated an 80% efficiency relative to NS control as measured by qPCR and confirmed by dramatic decrease in protein by Western Blot. Cell counts over a period of 6 days confirmed a reduction in cell number at all time points relative to the NS control.
Conclusions: This is the first report documenting that SATB1 is detectable in human benign urothelial and bladder cancer cell lines. SATB1 expression was higher in the cancer cell lines relative to the benign cell line and correlated with the metastatic potential of the cancer lines. SATB1 expression was knocked down using siRNA methods which resulted in consistently lower cell numbers over a 6 day period.