Inhibitory Effects Of Activin A On The Proliferation Of Neuro-2a Cells And Its Mechanism

Activin A,one member of transforming growth factor β(TGF-β)superfamily,is located on the long arm(q)of chromosome 2 and it is an immunomodulatory factor with two-way regulatory functions.Activin A is widely distributed in the histology and it is expressed in a variety of tissues and organs,such as brain tissue and uterus.The controversy that the role of activin A plays on the tumor still exists.In the recent years,the researches have found that activin A plays the dual roles of promotion and suppression,which is extremely likely to be the marker of a tumor screening and a potential therapeutic target in the occurrence and development of the tumor.The research has found that activin A has a dual role in carcinogenesis and suppression in tumor progression.However,it remains unclear whether Neuro-2a cells can compound activin A.It has not been studied that whether activin A can inhibit the proliferation,induce apoptosis and promote the migration of Neuro-2a cells,while it still lack of definite inclusion.Therefore,firstly this research used Neuro-2a cells incubated in vitro to investigate the effects of activin A on the proliferation,apoptosis and morphology of Neuro-2a cells,and then confirmed the mechanism of activin A inhibiting the proliferation of Neuro-2a cells,which will lay the foundation for looking for new therapeutic targets and the methods for neuronal tumors.1.Neuro-2a cells autocrine activin ALPS and NGF were used to stimulate Neuro-2a cells.ELISA was used to detect the level of activin A in the supernatant of Neuro-2a cells,the results showed that Neuro-2a cells secreted activin A,and LPS promoted the secretion,while NGF had no apparent effect.2.The effects of activin A on proliferation and apoptosis of Neuro-2a cellsThe expressions of activin receptor and its signal molecules in Neuro-2a cells were detected by RT-PCR,and the results showed that Neuro-2a cells not only expressed ActRIIA and ActRIIB mRNA,but also expressed ARIP1,2 and Smad2,3,4 mRNA.The experiment continued to investigate the proliferation capabilities of Neuro-2a cells by MTT and BrdU.It showed that activin A inhibited the proliferation of Neuro-2a cells,which had statistical significance.Then,when the Neuro-2a cells stained with Hoechst,it found that the nucleus of Neuro-2a cells stimulated by activin A was intact and had no nuclear fragmentation.Furthermore,Neuro-2a cells were stained with AnnexinV-FITC/7-AAD to determine whether activin A can promote apoptosis of Neuro-2a cells.The results showed that compared with the control group cells,there was no significant change in apoptosis rate of Neuro-2a cells stimulated by activin A.3.The changes that activin A on the morphology of Neuro-2a cells and the secretion of 5-HTThe morphological changes of Neuro-2a cells were observed by Gimesa staining,the results showed that Neuro-2a cells stimulated by activin A showed irregular morphology obviously.At the same time,Neuro-2a cells had mature-like neuronal extensions.Differentiated and mature neurons have the ability to produce neurotransmitters.Therefore,changes in the levels of neurotransmitter 5-HT were analyzed by ELISA.The results showed that activin A promoted the secretion of 5-HT.4.The effects of activin A on the expressions of activin receptors and signal molecules in Neuro-2a cells and the effects of overexpression of Smad3,ARIP1 and ARIP2 on the proliferation of Neuro-2a cells.Extracting the mRNA of Neuro-2a cells stimulated by activin A for 4h and 12 h.RT-PCR was used to examine the expressions of activin receptors and signal molecules.The results showed that when Neuro-2a cells were stimulated by activin A for 4h,the expressions of ARIP1,Smad3,4,ActRIIA and ActRIIB mRNA were all increased,while ARIP2 and Smad2 had no significant change.When Neuro-2a cells were stimulated by activin A for 12 h,the expressions of Smad3,4,ActRIIA and ActRIIB mRNA were all increased,and others did not change significantly.In a word,the results showed that the expressions of Smad3 and ARIP1 changed.Therefore,experiments were continued to transfect Neuro-2a cells to further analyze overexpression of Smad3 and ARIP1,2 on the proliferation of Neuro-2a cells.Neuro-2a cells were transfected with control empty plasmid pcDNA3(PC),expression plasmids pcDNA3-ARIP1(ARIP1)or pcDNA3-ARIP2(ARIP2),pcDNA3-Smad3(Smad3)and pcDNA3(NC),the RT-PCR method was used to identify the expressions of transfection efficiency.The results of MTT assay showed that overexpression of ARIP1 could inhibit viabilities of Neuro-2a cells,while overexpression of ARIP2 and Smad3 had no significant effect.At the same time,the results of BrdU showed that overexpression of ARIP1 inhibited the proliferation of Neuro-2a cells,while overexpression of ARIP2 and Smad3 had no significant effect.The results suggested that activin A may reduce viabilities of Neuro-2a cells through the way of autocrine or paracrine,while activin A inhibited the proliferation of Neuro-2a cells,and its role is related with the induction of Neuro-2a cells differentiation into mature neurons.The expressions of Smad3 and ARIP1 increased when Neuro-2a cells were stimulated by activin A,overexpression of ARIP1 inhibited the proliferation of Neuro-2a cells,while overexpression of ARIP2 and Smad3 had no effect,it suggested that activin A inhibited the proliferation of Neuro-2a cells,which may be related to signal transduction protein ARIP1,but not directly related to Smad3 signaling pathway.