PRAK Negative Regulation Of PCK2 To Promote The Invasion And Metastasis In Human Melanoma A375 Cells

ObjectiveTo study the p38 regulates/activates kinase(p38 regulates/activates kinase,PRAK)negatively regulates the effect of the mitochondrial phosphoenolpyruvate carboxykinase(PCK2)on the proliferation,invasion,and metastasis in human melanoma A375 cells,we construct the tumor model that overexpression or silences the expression of the PCK2 kinase in the A375 cell line in vitro.MethodsFirstly,we use the Illumina high-throughput sequencing platform(HiSeq)to sequence the mouse-derived melanoma B16-F10 cells and Prak-/-B16-F10 cells,and then we use the DESeq2 differential analysis method to screen the differentially expressed genes after PRAK knockout,the GO and KEGG databases analysis to perform functionally significant enrichment analysis of differential genes.Meanwhile,we use the real-time PCR(qRT-PCR)method to validate the expression of 6 selected differentially expressed genes in wild type or PRAK knock out mouse melanoma cells(B16-F10,or Prak-/-B16-F10),human melanoma cells(A375),human colon cancer cells(HCT116),human breast cancer cells(MDA-MB-231).Furthermore,we design and construct siRNA against Pck2 and Pck2 overexpression plasmid,and transient transfection the plasmids into A375 cells,then detect the proliferation ability of the cells through the CCK-8 analysis,detect the invasion ability of the cells by transwell invasion analysis,and detect the migration ability of the cells by scratch test analysis.ResultsBy analyzing the results of RNA-seq,a total of 1,000 differentially expressed genes were obtained,of which 828 were up-regulated genes(82.8%)and 172 were down-regulated genes(17.2%).The biological processes of GO enrichment analysis of the differentially expressed gene mainly include cell motility,cell adhesion,cell development,etc.The cellular components of GO enrichment analysis of the differentially expressed gene mainly include cell membrane,stress fiber,cytoplasmic perikaryotic region,etc.The molecular function of GO enrichment analysis of the differentially expressed gene mainly include glycoprotein binding,cytoskeleton proteins binding,and fibronectin binding,etc.KEGG enrichment analysis showed that differentially expressed genes were enriched in 10 signal pathways such as MAPK,p53 and glycolysis/gluconeogenesis.According to the results of the differential expression genes,6 genes related to tumor proliferation,invasion and metastasis were selected for the following study,they are up-regulated expression genes:Id3,Cst6 and St3gal1,and down-regulated expression genes:Timp3,Ccng2 and Pck2.The results of qRT-PCR analysis of B16-F10 and Prak-/-B16-F10 cells showed that the mRNA expression of the above six genes was consistent with the RNA-seq results.The expression of the above 6 genes in A375 cells,HCT116 cells or MDA-MB-231 cells was also detected with or without different doses of PRAK inhibitor(XZP-0353)adminstration,then the Pck2 gene was selected for the following research.We found that Pck2 overexpression significantly inhibits the invasion and migration of A375 cells.However,when interfering with Pck2 expression by siRNA,the invasion and migration capabilities of A375 cells are significantly improved.In addition,Pck2 overexpression and silent expression have little effect on A35 cell proliferation.Furthermore,the ability of Pck2 inhibits A375 cell invasion and metastasis can be significantly improved when the PRAK kinase inhibitor(XZP-0353)is added.ConclusionsHere,we have found that Pck2 overexpression can significantly inhibit the proliferation,invasion,and migration of A375 cells.PRAK can negatively regulate Pck2 expression,and then promote the invasion and metastasis of A375 cells.Pck2 overexpression or Pck2 knock down have no influence on the proliferation of A375 cells.