Gastrin-releasing Peptide Receptor Differentially Regulates P21and P27in Neuroblastoma Cells

Neuroblastoma, the most common extra-cranial cancer in children, frequently presentsin an advanced stage and is treatment-resistant. Gastrin-releasing peptide（GRP）and its receptor（GRP-R）are highly expressed in undifferentiated and poorlydifferentiated aggressive Neuroblastoma. We previously reported that GRP the mammalianequivalent of bombesin, activates PI3K/AKT signaling pathway, promotes DNA synthesisand cell cycle progression in neuroblastoma cells. Reversely, GRP-R silencing shRNA（small hairpin RNA targeting GRP-R） results in cell cycle arrest. Overall, we haddemonstrated that enhanced expression of GRP/GRP-R correlate to tumor progression andGRP has critical mitogenic/oncogenic role in various cancers. In this study, we speculatedthat GRP/GRP-R promoted cell proliferation potentially through the regulation of cyclin-dependent kinase（CDK）inhibitors in neuroblastoma. We use human cell lines BE（2）-Cin Neuroblastoma. Through the Cell culture, Transfection this cell lines to BE（2）-C/shCON, BE（2）-C/shGRP-R and siRNA with Lipofectamine2000. And RT-PCR canconfirmed the expression of GRP after transfection. p21, p27and PTEN expression of proteinwas confirmed with Western blot analysis; p21, p27and PTEN intracellular localization wasdemonstrated by Cell fractionation; Immunofluorescence staining demonstrates theintracellular localization of p21, p27and PTEN and the expression was confirmed withWestern blot. In this study, We found GRP-R silencing differentially modulated p21and p27expression. GRP-R silencing can down-regulated p21expression in human neuroblastoma.p21intracellular localization was confirmed in nuclear; p27expression was up-regulated inGRP-R silencing in human neuroblastoma. p27intracellular localization was confirmed incytoplasm; PTEN expression was increased in GRP-R stably silenced cells.Immunofluorescence staining of PTEN was confirmed in cytoplasm. Surprisingly, we foundthat GRP/GRP-R differentially regulates p21and p27in neuroblastoma cells. Silencing GRP/GRP-R decreased the expression of p21, but increased the expression of p27inneuroblastoma cells. Furthermore, we found that the intracellular localization of p21and p27was in the nuclear and cytoplasm compartment, respectively. In addition, we found thatGRP/GRP-R silencing caused the increased the expression and accumulation of PTEN in thecytoplasm of neuroblastoma cells where it co-localized with p27; suggesting that p27promoted the function of PTEN as a tumor suppressor by stabilizing PTEN in the cytoplasm.Hence, understanding the regulation of GRP/GRP-R on CDK inhibitors and tumor suppressorPTEN will benefit in developing novel therapeutic drugs targeting GRP/GRP-R signaling. Todeeply explore more about the growth mechanism of tumor and provides more evidence fromthe cellular level understanding of tumor. For laid foundation development and screening ofthe new anti-tumor drugs.