Nuclear Protein PCNP Mediates Proliferation And Apoptosis Of Human Neuroblastoma Cells

BackgroundNeuroblastoma(NB)is an embryonal tumor,mostly in the cerebral hemisphere,and is the most common extracranial tumor in children,is the most common tumor in infants.Nearly half of neuroblastomas occur in infants younger than 2 years old.Neuroblastomas account for about 6-10% of children’s tumors,and 15% of childhood cancer deaths.Neuroblastoma is a highly malignant and undifferentiated neurogenic tumor.No matter in vitro or in vivo,neuroblastoma cells there are nerve cells and glial cells differentiation,therefore neuroblastoma cell is the study of tumor differentiation and apoptosis and its mechanism of excellent cell model.Many neuroblastoma cell lines are used at home and abroad,such as SH-SY5 Y,SK-N-SH,SK-SN-BE2,LA-N-5,etc.we adopt neuroblastoma SH-SY5 Y,SK-N-SH cells,they can support the plasmid replication,so cloning genes can be efficient expression of iconic material can be expressed neurons,the cell morphology and some physiological function are similar to normal neurons.SH-SY5 Y,SK-N-SH cells are the ideal model of the cell to research on nerve cell function.PCNP(PEST-containing nuclear protein)is a newly discovered nucleoprotein which containing PEST sequences.PCNP was first reported by Japanese scientists in 2004 as the substrate for the ubiquitin ligase NIRF(Np95/ICBP90-like RING figer protein).PCNP and NIRF together constitute a new signal transduction pathway,which is closely related to cell cycle regulation and cell proliferation.PCNP mainly exists in the nucleus,and this new type of ring protein is a ubiquitin protein binding enzyme,which is involved in the ubiquitination pathway of protein degradation.At present,there are few relevant reports on PCNP,but there are many researches on PESTstructure.The PEST motif is a peptide sequence which is rich in proline(P),glutamic acid(E),serine(S),and threonine(T).PEST domin has a regulatory role in cell cycle and proliferation,and is related to the stability of chromosomes,the occurrence of tumors,and immune system diseases.Therefore,we hypothesized and verified that PCNP was also related to cell growth and apoptosis,and this topic made a preliminary discussion on the effect of PCNP on the growth and apoptosis of human neuroblastoma cells.PurposeTo explore the effects of PCNP gene overexpression and silencing on human neuroblastoma cell proliferation,migration,invasion and tumor formation,and to find mechanisms that may lead to these changes.MethodsTransfecting the PCNP overexpression and the interfering plasmid into SH-SY5 Y cells and SK-N-SH cells of human neuroblastoma,and use G418 and puromycin to screen for stable strains.Cell proliferation was detected by MTS and EDU method.Apoptosis was detected by TUNEL staining.The cell migration and invasion ability were detected by wound healing,transwell,invasion and soft agar assay.The expression of MAPK and PI3K/AKT/mTOR signaling pathway was detected by Western blot.PCNP overexpression and silencing human neuroblastoma cells were injected into the nude mice to detect the effect of PCNP on the human neuroblastoma cells tumor capacity.Results(1)Successfully constructed PCNP overexpression and silent stable strain;(2)MTS and EDU results showed that PCNP overexpression inhibited cell proliferation and silencing promoted cell proliferation;(3)Apoptosis was detected by Tunel staining method,the results showed that PCNP overexpressionpromoted apoptosis and silencing inhibited apoptosis.(4)Cell migration ability was detected by wound healing and transwell,the results showed that the cell migration ability was weakened after PCNP over-expression,and the cell migration ability was enhanced after silence.(5)Cell invasion ability was detected by invasion and soft agar assay,the results showed that the cell invasion ability was weakened after PCNP over-expression,and the cell invasion ability was enhanced after silence.(6)Our results showed that PCNP over-expression remarkably increased the apoptotic index,protein expressions of Cleaved Caspase-3,8,9,as well as Bax/Bcl-2 and Bad/Bcl-xl ratios,suggesting the activation of mitochondria-mediated pathway.(7)Our results indicated that PCNP over-expression could induce apoptosis by triggering the phosphorylations of p38,JNK,and ERK 1/2 in both SH-SY5 Y and SK-N-SH cells.However,PCNP knockdown could promote the growth,migration,and invasion of neuroblastoma cells by decreasing the phosphorylations of p38,JNK,and ERK 1/2.These results together suggest that PCNP can regulate the growth process of human neuroblastoma cells via the MAPK signaling pathway.Our results showed that PCNP over-expression significantly induced apoptosis by inhibiting the phosphorylations of PI3 K,AKT,and mTOR.Nevertheless,PCNP knockdown promoted the growth,migration,and invasion of neuroblastoma cells via increasing the phosphorylations of PI3 K,AKT,and mTOR.These data reveal that PCNP can regulate the growth,migration,and invasion of human neuroblastoma cells through the PI3K/Akt/mTOR signaling pathway.(8)PCNP over-expression significantly decreased the growth of neuroblastoma xenograft tumors,whereas PCNP knockdown notably promoted tumor growth.Conclusions1、PCNP inhibited the proliferation of human neuroblastoma sh-sy5 y cells and sk-n-sh cells.2、PCNP promotes apoptosis of neuroblastoma cells.3、PCNP inhibits the migration and invasion of neuroblastoma cells.4 、PCNP can regulate the growth process of human neuroblastoma cells via the MAPK signaling pathway,can regulate the growth,migration,and invasion of human neuroblastoma cells through the PI3K/Akt/mTOR signaling pathway.5、PCNP inhibits the formation of neuroblastoma xenograft tumors.