RNA Binding Protetin HnRNPA2/B1 Regulates The Proliferation,migration And Apoptosis Of Enteric Neural Crest-derived Cells In Rats

Objective:Enteric nervous system（ENS）is composed of the neurons and glia cells in the intestinal wall.Its dysplasia may cause dysfunction or malformation of the gastrointestinal tract,but the specific regulation mechanism is not yet clear.In the previous study,we found that hnRNPA2/B1（heterogeneous nuclear ribonucleoprotein A2B1,hnRNPA2/B1）was highly expressed in patients with Hirschsprung’disease,and it was confirmed that there was epithelial-mesenchymal transition during the development of enteric nervous system.HnRNPA2/B1 is a member of the RNA binding proteins family and mainly affects the stability of mRNA and alternative splicing.HnRNPA2/B1 is differentially expressed in many kinds of malignant tumors and can induce epithelial-mesenchymal transition（EMT）in the development of malignant tumors,but there are no reports on the development of intestinal nervous system.How the ENS develops is that ENCSCs migrates,proliferates,differentiates and colonizes in the intestinal tract from head to tail.EMT is the basis of embryonic development and plays an important role in the differentiation of tissues and organs.ZEB family is an important EMT transcription factor,which promotes EMT by binding to the promoter region of E-cadherin.However,the relationship between HNRNPA2B1 and ZEB family and whether it can induce EMT is not clear.Therefore,for the first time in this study,real-time quantitative PCR（RT-PCR）,western blotting（WB）,cell proliferation（cck-8）,apoptosis（AV/PI flow assay）and cell migration（transwell）are used to confirm that hnRNPA2/B1 is involved in the regulation of ENS development in rats enteric neural crest stem cells（ENCSCs）.To explore the regulatory mechanism that how hnRNPA2/B1 induces EMT in the development of the enteric nervous system,RNA immunoprecipitation（RIP）is conducted.Methods:1.Isolation and culture of ENCSC_SThe female and male rats which were healthy,mature and unpregnant,were caged in the midnight at ratio of 3:1 and female vaginal smear was performed at8:00 am the next morning.Female rats whose sperms in their vaginas were observed more than 2/3 field of vision under the microscope or the vaginal plug was found to drop,were confirmed to be mated and pregnant.Wistar pregnant rats at E12 were selected to extract ENCSC_Sand be cultured.2.We constructed the rats hnRNPA2/B1 virus vector and designed the hnRNPA2/B1/si RNAs.Then we performed the qRT-PCR and WB to detect the most efficient of the knock-down for transfection experiments.3.We used RNAi to knock down the expression of hnRNPA2/B1 gene in the rats ENCSC_S.Next,qRT-PCR and WB were conducted to detected the expression of EMT markers E-cadherin and N-cadherin and EMT transcription factors ZEB1 or ZEB2 and mi R-200c in negative control group and the transfected group.4.The expression of hnRNPA2/B1 gene was knocked down by RNAi.Then the cell proliferation,apoptosis and cell migration of rats ENCSC_Swere detected respectively by CCK-8,AV/PI flow assay and transwell in negative control group and the transfected group.5.RIP assay was used to verify the binding of hnRNPA2/B1 to EMT transcription factors ZEB1 or ZEB2.6.statistical analysis:The experimental datas were expressed as mean±standard deviation and analyzed using Graph Pad Prism 8.0.Results:1.ENCSCs were successfully cultured in DMEM/F12 complete medium at E12.2.We designed two hnRNPA2/B1/sh RNA_Sand selected hnRNPA2/B1/si RNA1for the transient transfection by qRT-PCR and WB.3.qRT-PCR and WB confirmed consistently after knock-down of hnRNPA2/B1 by RNAi that the expression of ZEB1,ZEB2,N-cadherin was downregulated and E-cadherin,mi R-200c was upregulated in the transfected group compared to the negative control group.4.The expression of hnRNPA2/B1 was knocked down by RNAi and verified by qRT-PCR and WB:compared with the negative control group,the the rate of cell proliferation and migration was slower and apoptosis rate decreased in transfected group.5.It was confirmed that there is a direct binding between hnRNPA2/B1 and ZEB1/ZEB2 mRNA.Conclusion:Our study revealed that hnRNPA2/B1 affects the cell proliferation,apoptosis and cell migration of ENCSC_S。Moreover,hnRNPA2/B1 can be combined with ZEB1/ZEB2 mRNA directly and possibly regulating the EMT via regulating ZEB1/ZEB2 mRNA in ENCSC_S.It is suggested that hnRNPA2/B1 is involved in the regulation of enteric nervous system development.