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@#Objective To knockout interferon alpha/beta receptor subunit 1(IFNAR1) gene in human colorectal adenocarcinoma cells Caco-2 using clustered regularly interspaced short palinmic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system to construct IFNAR1 knockout Caco-2 cell line.Methods The single guide RNA(sgRNA)sequence was designed to specifically recognize the exon region of IFNAR1 gene using CRISPR/Cas9 technology,and the LentiCRISPRv2-IFNAR1-sgRNA recombinant plasmid was constructed.Caco-2 cells were infected with the plasmid packaged by lentivirus and screened by puromycin resistance.The obtained monoclonal cell lines were cultured by limited dilution method,which were verified for the effect of IFNAR1 gene knockout by target gene sequencing and Western blot,and detected for the mRNA levels of CXC chemokine ligand 10(CXCL10)and interferon-stimulatd gene 20(ISG20)in IFNAR1knockout cells by adding exogenous IFNβ.Results Sequencing results of plasmid LentiCRISPRv2-IFNAR1-sgRNA showed that the insertion sites were all located at the sticky end of BsmBⅠenzyme digestion.Two IFNAR1 knockout monoclonal cell lines were obtained.The sequencing results showed that Caco-2-IFNAR1-KO1 had 5 bp deletion in the sixth exon of IFNAR1,and Caco-2-IFNAR1-KO2 had 18 bp deletion and 1 bp insertion in the seventh exon.Compared with wild-type Caco-2 cells,Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells showed no expression of IFNAR1 protein.Compared with no IFNβ stimulation,the mRNA levels of CXCL10 gene(t = 0.566 and 1.268 respectively,P>0.05)and ISG20 gene(t =1.522 and 1.733 respectively,P>0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells stimulated by 50 ng/mL IFNβ showed no significant increase.While compared with those of wild-type Caco-2 cells,the mRNA levels of CXCL10gene(t = 6.763 and 6.777 respectively,P<0.05)and ISG20 gene(t = 5.664 and 5.65 respectively,P<0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells decreased significantly under the stimulation of 50 ng/mL exogenous IFNβ.Conclusion Caco-2 cell line with IFNAR1 knockout was successfully constructed by using CRISPR/Cas9 technology,and the downstream molecules activated by IFNAR(interferon alpha/beta receptor)in this cell line were obviously inhibited,which provided a powerful tool for further exploration of the innate immune response and replication packaging mechanism of Caco-2 cells after virus infection.
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Objective:To explore the expression of signal sequence receptor subunit 1 (SSR1) and its prognostic value in hepatocellular carcinoma.Methods:Search the expression data and relevant clinical data of SSR1 in hepatocellular carcinoma patients from the Cancer Genome Atlas (TCGA) database to June 20, 2021, and download relevant public data. The expression levels of SSR1 in 334 cases of hepatocellular carcinoma with complete information and data were analyzed retrospectively. The expression difference of SSR1 gene between hepatocellular carcinoma and adjacent tissues was analyzed by Wilcoxon signed rank test. Patients with hepatocellular carcinoma were divided into high expression group and low expression group based on the median value of SSR1 expression level (14.660). χ 2 test was conducted to analyze the relationship between SSR1 expression and clinicopathological features. Cox regression and Log-rank survival test were used to analyze the relationship between SSR1 gene expression, clinicopathological features and overall survival rate in patients with hepatocellular carcinoma. Univariate and multivariate Cox regression analysis were used to determine the factors affecting prognosis. Gene set enrichment analysis (GSEA) was used to predict the possible regulatory pathways. Result:Bioinformatics analysis based on TCGA database showed that the expression level of SSR1 in hepatocellular carcinoma (16.320±7.231) was significantly higher than that in normal liver tissue (7.473±1.410). The difference between groups was statistically significant ( t=8.621, P<0.001).The overall survival rate of patients with high SSR1 gene expression group was lower than that of patients with high SSR1 gene expression group (χ 2=10.1, P<0.001). The high expression of SSR1 gene was related to sex (χ 2=4.392, P=0.036), Stage (χ 2=6.264, P=0.012), T stage (χ 2=4.561, P=0.033) and Grade classification (χ 2=14.015, P<0.001). Multivariate Cox regression analysis showed that patients with high expression of SSR1 gene got worse risk of death ( HR=1.030, 95% CI:1.002-1.060, P=0.036), and SSR1 gene expression was an independent predictor of hepatocellular carcinoma. Gene set enrichment analysis showed that the high expression of SSR1 was related to ubiquitination, cell cycle, RNA degradation, mTOR signal pathway, Wnt signal pathway and MAPK signal pathway. Conclusion:SSR1 gene is significantly up-regulated in hepatocellular carcinoma, which is related to gender, Stage, T stage and Grade classification. Ubiquitination, cell cycle, RNA degradation, mTOR signal pathway, Wnt signal pathway and MAPK signal pathway may be the key pathways for SSR1 to promote the occurrence and development of hepatocellular carcinoma.
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Objective To explore the effects of chronic levodopa treatment on characteristics of N-methyl-D-aspartate(NMDA) receptor subunit 1(NR1) on the neurons of striatum in rat models of Parkinsonism related motor complications.Methods Rat models(n=25)of Parkinsonism related motor complications were established and were randomly divided into solvent treatment group(n=5,intraperitoneal injection with vitamin C),levodopa chronic treatment group(n=10,intraperitoneal injection with levodopa for 23 d) and MK-801 treatment group(n=10,intraperitoneal injection with MK-801 on d 23 after intraperitoneal injection with levodopa for 22 d).Another 5 rats were served as controls(sham-operation group,n=5).Locomotion changes of rats in MK-801 treatment group were recorded,and NR1 phosphorylation in the other three groups was detected by Western blotting. Results After chronic treatment with levodopa methylester for 1,8,15 and 22 d,rats in MK-801 treatment group displayed shortened rotational duration and increased peak rotation.Compared with d 22,MK-801 both prolonged rotational duration and reduced peak rotation(P0.05).The expression of NR1,phosphorylated NR1S890,NR1S896 and NR1S897 in striatal membranes in levodopa chronic treatment group was increased compared with that of solvent treatment group(P