ERa36 gene silencing promotes tau protein phosphorylation, inhibits cell proliferation, and induces apoptosis in human neuroblastoma SH-SY5Y cells

Abstract

Neuroblastoma is the most common cancer in infants and the third most common cancer in children after leukemia and brain cancer. The purpose of our study was to investigate the effects of estrogen receptor (ER)-a36 gene silencing on tau protein phosphorylation, cell proliferation, and cell apoptosis in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were treated with estrogen or left untreated, to investigate the effects of estrogen stimulation on ERa36 and the ERK/protein B kinase (AKT) signaling pathway. ERa36 mRNA expressions were detected by quantitative RT-PCR. A phosphatase kit was used to test protein phosphatase (PP)-2A activity before and after treatment. Western blot analysis was conducted to detect protein expression of ERa36; tau protein; phosphorylated- tau (p-tau) at site Thr231 [p-tau (Thr231)]; glycogen synthase kinase (GSK)3b and its specificity sites (Tyr216 and Ser9); Cyclin Dl; proliferating cell nuclear antigen (PCNA); B-cell lymphoma (Bcl)-2; and Bcl-2–associated X protein (Bax). A cell-counting kit (CCK)-8 assay was used to determine cell viability. Cell apoptosis and rate of tumor growth and volume were determined by Annexin V-FITC/PI staining and a xenotransplanted tumor model in nude mice. Results show that without estrogen stimulation, ERa36 was inactivated. When stimulated by estrogen, expression of ERa36, PP2A, p-GSK3b (Ser9)/total protein (t)-GSK3b, Cyclin Dl, PCNA, and Bcl-2 were up-regulated, and p-GSK3b (Tyr216)/ t-GSK3b expression was down-regulated, as was p-tau (Thr231) and Bax expression. The expression of p-ERK/ERK, p-AKT/AKT, p-methyl ethyl ketone (MEK)/MEK, and p-mammalian target of rapamycin (mTOR)/mTOR expression was up-regulated, suggesting that the ERK/AKT signaling pathway is activated. Cell proliferation was also accelerated, whereas apoptosis was inhibited with stimulation by estrogen. However, we found that the effects of silencing ERa36 on the expression of related intracellular factors had no association with estrogen. Our study demonstrates that ERa36 gene silencing can inhibit the activation of the ERK/AKT signaling pathway, increase tau protein phosphorylation, decrease cell vitality and tumorigenicity, and promote apoptosis of human neuroblastoma SH-SY5Y cells.—Wang, H.-B., Li, T., Ma, D.-Z., Zhi, H. ERa36 gene silencing promotes tau protein phosphorylation, inhibits cell proliferation, and induces apoptosis in human neuroblastoma SH-SY5Y cells.