Effect Of RIPK3 Protein On Expression Of Damage Repair Related Gene Of SH-SY5Y

Objective: Receptor-interacting protein kinase 3(RIPK3)an important member of the Receptor-interacting protein kinases with distinctive serine / threonine enzyme activity.RIPK3 widely expressed in various tissues of human,activated by stimulating factors in inflammation,injury,infection in vitro and in vivo via tumor necrosis factor1-α(TNF1-α)-RIPK1 signaling pathway.Then activate downstream signaling pathways including apoptosis,necroptosis,mitochondrial reactive oxygen species(ROS)generation and NF kappa B activation.RIPK3 participate in the process of cell adaptation to the external environment by regulating cell proliferation and death.Studies have shown that RIPK3 plays an important role in the nervous system,by regulating the damage of hippocampal neurons induced by TNF-α.While suppressing RIPK3 activation could remarkably reduce the hematoma volume of intracerebral hemorrhage(ICH)mouse,reduce vascular injury of neuron,prevent neuronal death and improve the prognosis of ICH model mice.However,in recent years,research on RIPK3 were mainly concentrated on revealing its role in programmed cell necrosis process,most of the objective is generally rather than the specific tissue and system,especially the role of RIPK3 in the nervous system and its mechanism is still beyond the research and exploration.Methods:1.Stably overexpress RIPK3 protein in SH-SY5 Y cells2.Construct and amplify of the plasmid carrying over expression of RIPK3 gene and the control empty plasmid.Set the SH-SY5 Y cells transfected with RIPK3 gene as the experimental group,while transfected with the empty plasmid as control group.48 h after transfection,cells of both groups were observed by laser scanning confocal microscopy to confirm the expression of green fluorescent protein(GFP)carried by plasmid,Western blot was used to detect weather exogenous RIPK3 is stably over expression in the cell3.Absorbance value of two groups of cells was determined by MTT experiment at 12,20,28,36,44 hours,calculated and obtained cell proliferation of two groups of cells in the corresponding time points and to validate whether exogenous RIPK3 in SH-SY5 Y cells has biological activity.4.Extracted RNA of both groups of cell.Comprehensive test took place to ensure that the RNA accord with the requirement of experiment,obtained purified mRNA of two groups of cell for transcriptome sequencing technology(RNAseq),obtain the complete transcriptome information.The results were input into Pathway Analysis Ingenuity(IPA)database to analyze the RIPK3 downstream signal pathway and the key molecules.5.Extracted total RNA of two groups of cells,reverse transcription for cDNA.Droplet Digital PCR(ddPCR)was used to verify the gene transcription level changes of genes in the core position of RNAseq results.Results:After transfection,the cells showed green fluorescence under laser scanning confocal microscope,the expression plasmid of GFP protein was expressed in the cells.Blot Western showed that the endogenous RIPK3(molecular weight of 56kD)was normal in the experimental group,and the expression level was the same as that of the control group.At the same time,the expression of exogenous RIPK3-GFP fusion protein(molecular weight 85Kd)was stably expressed in the experimental group.Cell proliferation activity was detected at 12,20,28,36 and 44 h,respectively,in the MTT test.The cell proliferation activity of experimental group is lower than that of the control group(n = 5 and t = 6.717,509.4,14.900,11.914,23.903,P<0.01),suggesting that exogenous RIPK3-GFP protein had biological activity and inhibitor proliferation of SH-SY5 Y cells.The cell transcription group had a significant change as RIPK3 expression level increased.RNAseq showed that the cells of two groups were significantly different at the RNA level.Data analysis and strict screening of the sequencing results of IPA shown that the signal pathway of the partial transcription level was obviously changed.Including: HIF1-α and its relative genes such as UBC,VHL,TCEB1,VEGFA;ZFP36 and its relative genes such as VEGF,DCP2,BDNF,TNF;mTOR and relative genes such as SREBP,S6 K,Eif4B,eEF2 K,ULK.DdPCR was used to detect changes of expression levels of the key signaling molecules of above signal pathway: HIF1-α,ZFP36 and mTOR,to repeat the results of the RNAseq.The results showed that changes of the expression levels of HIF1-α,ZFP36 and mTOR were the same with the RNAseq results.Conclusions:In this paper,changes of the expression level of the gene transcription in SH-SY5 Y cells were detected and analyzed after the expression level of RIPK3 were upregulated.And the potential effect of RIPK3 signal pathway was confirmed.