| Objectives CircRNA expression profiles were detected in serum of patients with anti-tuberculosis drug-induced liver injury(ADLI)and patients without liver injury as well as in cell models simulating clinically induced liver cell injury by using gene microarray technology.The common differential expression of circMARS and circITPR1 was verified in the population,and its potential as diagnostic markers for ADLI was analyzed.The bioinformatics analysis and mechanism of circMARS were studied to provide a theoretical basis for the diagnosis and treatment of ADLI.Methods 1 Screening,validation and diagnostic significance of differentially expressed circRNA in ADLI: The basic information and blood samples of patients with anti-tuberculosis treated at Tangshan Tuberculosis Hospital from July 2015 to July 2018 were collected.CircRNA microarray detection was performed on serum samples of 16 patients with liver injury and 16 patients without liver injury.In the cell model,hepatocyte injury model was established by combining two drugs(isoniazid + rifampicin)and three drugs(isoniazid + rifampicin + pyrazinamide)on human normal liver cell line(HL7702).CircRNA microarray technology was performed in two drug combination group,three drug combination group and normal control group.The two mircoarray data were analyzed for common differential expression.From the circRNAs with common differential expression,one up-regulated and one down-regulated circRNAs were verified by fluorescence quantitative polymerase chain reaction(qPCR)in 150 patients with ADLI and 150 patients without ADLI.T-test and chi-square test were used to evaluate the statistical difference of population data and expression levels about circRNA,ALT and AST between the two groups.The pearson linear correlation coefficient was used to analyze the linear relationship between circRNAs and ALT,AST.The diagnostic value of circMARS and circITPR1 for ADLI were analyzed by using ROC(receiver operating characteristic curve).2 Study on the mechanism of circMARS in ADLI: Firstly,among the above two circRNAs,circMARS with up-regulated differential expression was selected to study the mechanism.Through the miRanda-3.3 and TargetScan7.1 software to predict circMARS targeted miRNAs and miRNA target genes.The role of circITPR1 in miRNA sponges function is not clear,then we will study in the future.Through KEGG pathway and GO category analysis,circMARS — mir-6808-5p/-6874-3p/-3157-5p — KMT2 C — EGFR functional axis was selected.Secondly,the ADLI cell model was established and circMARS expression was knocked down by small interfering RNA.The hepatocyte status was determined by CCK8,ALT and AST.Then,qPCR was used to detect the expression levels of circMARS,miR-6808-5p,miR-6874-3p,miR-3157-5p and lysine methyltransferase 2C(KMT2C)genes in cells of each group.The protein expression level of epidermal growth factor receptor(EGFR)was detected by ELISA method.Finally,by collecting peripheral blood of 35 groups of tuberculosis patients before and after the occurrence of ADLI,qPCR technology was used to detect the changes of circMARS,miR-6808-5p,miR-6874-3p and miR-3157-5p expression in serum,and ELISA method was used to detect the expression changes of EGFR protein in serum of the patients.Results 1 CircRNA microarray results showed that there were 6661 differentially expressed circRNAs in the serum of ADLI patients,among which 272 were up-regulated and 6389 were down-regulated.There were 8187 differentially expressed circRNAs in the model of ADLI,among which 1800 circRNAs were up-regulated and 6377 circRNAs were down-regulated.A total of 113 circRNAs with totally differentially expressed circRNAs were obtained from the intersection of the two times chip detection results,among which 7 circRNAs were up-regulated and 106 circRNAs were down-regulated.2 Combined with circRNA abundances level,the up-regulated circMARS and down-regulated circITPR1 were selected for qPCR verification,and the expression was consistent with the chip results.CircMARS and circITPR1 were positively correlated with ADLI.According to ROC curve analysis,the area under the curve(AUC)of circMARS and circITPR1 diagnosis ADLI were 0.80 and 0.76,the sensitivity were 70.04% and 52.06%,the specificity were 77.25% and 78.19%.Combined with the ROC analysis of circMARS and circITPR1,the AUC was 0.88,the sensitivity and specificity were 75.34% and 82.06%(all P<0.05).3 Bioinformatics analysis showed that circMARS can be used as a miRNA sponge to target miR-6808-5p,miR-6874-3p and miR-3157-5p,and these three miRNAs can enrich into lysine degradation signaling pathway and histone methyltransferase activity biological function,and inhibit KMT2 C to regulate EGFR activity.4 The results of cell experiments showed that circMARS and KMT2 C gene expressions in the combination of two drugs and three drugs were higher than those in the normal group,while the expressions of miR-6808-5p,miR-6874-3p and miR-3157-5p were decreased(all P<0.05).CircMARS knockdown by siRNA resulted in increased miRNAs expression and decreased KMT2 C gene expression(all P<0.05).ALT and AST contents in the cell culture supernatant were increased,and the cell survival rate was further decreased in si-circMARS groups(all P<0.05).5 After the occurrence of ADLI in patients receiving anti-tuberculosis treatment,the relative expression levels of circMARS in the serum were significantly higher than those before the occurrence of ADLI,and the relative expression levels of miR-6808-5p,miR-6874-3p and miR-3157-5p were lower than those before the occurrence of ADLI(all P<0.05).The expression level of EGFR protein in the serum was significantly increased when ADLI occurred(P<0.05).Conclusions 1 CircRNA expression profile in serum of ADLI patients was partially changed,suggesting that circRNA may be involved in the development of ADLI.2 The expression of circMARS in the serum of ADLI patients was up-regulated and the expression of circITPR1 was down-regulated.There was a correlation between their expression levels and ADLI.Combined with circMARS and circITPR1,it could be a promising diagnostic marker in ADLI.3 CircMARS may participate in the compensatory and regenerative function of liver injury through the circMARS — miR-6808-5p/-6874-3p/-3157-5p — KMT2 C — EGFR functional axis.Figure 12;Table 10;Reference 102... |
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