The Gene Expression Changes Induced By Knockout Of Ptgs2 Gene In Hepatic Stellate Cell(HSCs)

Objective To investigate gene expression changes induced by knockout of Ptgs2 gene in hepatic stellate cellsMethods 1.The CRISPR/Cas9 technology was used to construct a rat HSC-T6 cell model that stably silenced the Ptgs2 gene.The experiment was divided into two groups:(1)negative control group(NC group),target cells plus negative control virus-infected cell group.(2)LV-Ptgs2-sgRNA group(KO group),target cells plus LV-Ptgs2-sgRNA lentivirus-infected cell group.2.The expression of Ptgs2 gene in KO group and NC group was detected by Western Blot method.3.Affmetrix gene expression profiling chip was used for analysis,and Trizol method was used to extract HSC Total RNA in the RNA,fluorescently label the RNA,chip hybridize,and wash the slides before applying GenePix40 OOB scanner acquires the fluorescence signal image,analyzes and processes the original image Screen differentially expressed genes.4.The differential genes were introduced into the IPA(Ingenuity Pathway Analysis)system for biological information analysis,looking for classic pathways,diseases and functions,and upstream and downstream genes related to the Ptgs2 gene.Results 1.HSC-T6 cells stably silenced the Ptgs2 gene were constructed,and the protein expression level of the Ptgs2 gene was stably down-regulated.2.A total of 3152 differential genes were screened using gene expression profiling chip technology,of which There were 1,574 differentially regulated genes and 1,578 differentially expressed genes.3.The signal pathway enrichment analysis of differentially expressed genes through the IPA system revealed that the signal pathways that are significantly involved in the differentially expressed genes are cholesterol synthesis pathway,degradation of chondroitin sulfate,angiotensin signal pathway,choline biosynthesis,and TNFR1 signal pathway.Oxidative phosphorylation and the antioxidant effect of vitamin C.Disease and function analysis showed significant suppression of morbidity and mortality.Preliminary speculations that Cby1,Dhcr7,Txnip,Krt14,Trexl,MAPK1,MYC,GTPBP4 and other key genes may be diagnostic and therapeutic targets for liver fibrosis.Conclusion 1.After the knockout of Ptgs2,there were a large number of differentially expressed genes in HSCs cells,including 1574 up-regulated genes and 1578 down-regulated genes.2.Through IPA analysis,it is preliminarily speculated that Cby1,Dhcr7,Txnip,Krt14,MAPK1,MYC,GTPBP4 and other key genes may be the diagnostic and therapeutic targets of liver fibrosis,and the specific regulatory mechanism needs to be further studied.