Up-regulated MiR-200a Contributes To Liver Injury In Di-(2-ethylheyl)phthalate-exposed Mice

Research background and purpose:DEHP which is used as plasticizers in polymer products to make plastic flexible,best known as an environmental endocrine disruptors,is widely used in daily life.DEHP is easily soluble from plastic products,making people more and more frequently and universally exposed to it,causing damage to the nervous system,immune system,respiratory system,heart,liver,and the like.In recent years,with the deepening of epigenetic studies on the toxicity of endocrine disrupting chemicals,it has been confirmed that abnormally expressed miRNA plays a significant regulatory role in the toxic effects caused by endocrine disrupting chemicals.Meanwhile,the toxic effects of miRNA involvement in DEHP exposure are currently less reported.In this study,the expression of miR₂00a was up-regulated in DEEP-exposed male mice and AML 12 mouse hepatocytes.Based on this,we systematically analyzed the do,se-response relationship and molecular mechanism of DEHP-induced miR-200a expression;To investigate the important role of miR-200a in DEHP-induced hepatocyte injury in mice;to explore the apparent regulatory mechanism of increased expression of miR-200a in AML 12 cells induced by DEHP exposure.The successful completion of this subject is expected to provide new ideas and clues for the search for biomarkers in liver damage caused by DEHP exposure and the study of environmental endocrine disruptors.Research methods:1.Constructing a cell model of hepatocyte injury induced by DEHP exposure:AML 12 cells were chosen as subjects for continuous exposure to DEHP solution at concentrations of 0,0.01,0.1,and 1 μM for 72 hours.The MTT technique was adopted to measure DEHP exposure.The effect of apoptosis on AML12 cells was ascertained by Annexin-V FITC staining.The apoptosis rate of AML 12 cells was surveyed by flow cytometry.The expression of apoptosis-related proteins Xiap,Bc12 and Bax were monitored by Western blotting.2.The role of miR-200a in hepatocyte injury induced by DEHP exposure:AML12 cells were treated with different concentrations of DEHP solution for 72 hours,and the expression of miR-200a was monitored by Real-Time PCR.Transfection of mimic miR-200a and its control mimic control in AML12 cells for 72 hours,processing with Annexin-V FITC staining,then using flow cytometry to surveyed changes in apoptosis rate;transfection inhibitor control and inhibitor miR-200a in AML12 After 72 hours,the expression of the apoptosis-related protein Xiap was detected by Western blotting.In AML12 cells exposed to DEHP,the inhibitor control and inhibitor miR-200a were transfected respectively,and Annexin-V FITC staining was performed,using flow cytometry to monitors changes in the ratio of apoptosis.3.The role of miR-200a in liver injury induced by DEHP exposure:Weaning male C57B/L6 mice was treated with 2mg/kg/day DEHP,and 15 weeks later,the mice were executed and the expression of miR-200a was monitored by Real-Time PCR.The expression of apoptosis-related protein Xiap was identified by Western blotting.The miR-200a interference lentivirus injection was performed on this basis to detect changes in body weight and relative liver weight of mice.After isolation of serum,liver damage indicators ALT,AST,TG,LDL-C were detected.,HDL-C,TG expression changes;mouse liver tissue was fixed with 4%paraformaldehyde,embedded in paraffin,sliced,detected by HE staining Liver tissue damage;immunohistochemical staining was used to monitor the expression of apoptosis-related gene Caspase-3.4.DEHP up-regulated the epigenetic mechanism of miR-200a expression:AML12 cells were addressed in different concentrations of DEHP solution for 72 hours,and the expression of miR-192 was monitored by Real-Time PCR.The expression of Malatl,Neatl and Pvtl long non-coding RNA（1ncRNA）was detected by-Time PCR.The expression of P53 and Mdm2 in liver tissue was measured by Western blottingResearch results:1.After continuous exposure to DEHP for 72 hours in AML 12 cells,cell viability was significantly diminished by 0.01,0.1,1 pM DEHP,compared with the control group,indicating that the protein expression level of Xiap.2.The expression level of miR-200a was also significantly increased during the apoptosis of AML 12 cells induced by DEHP exposure;transfection of mimic miR-200a in AML12 cells,over-expression of miR-200a promoted apoptosis of AML12 cells;When the inhibitor miR-200a inhibits the expression of miR-200a,the protein expression level of Xiap in the cells was significantly increased;In a cell model in which DEHP exposed AML12 cells to apoptosis,inhibition of miR-200a expression by inhibitor miR-200a significantly improved apoptosis induced by DEHP exposure.3.Compared with the control group,the expression of apoptosis-related protein Xiap was significantly decreased in the liver tissue of the DEHP exposed group;the expression level of miR-200a was significantly increased;Compared with the Lv-Control group,the body weight of the Lv-Control-DEHP group did not change significantly,but the liver weight decreased significantly.Liver HE staining results showed that the Lv-Control-DEHP group had hepatic inflammation.The cell infiltration was obvious;the immunohistochemical results showed that the expression of Cas:pase-3 was significantly increased;the serum levels of AST and ALT were significantly increased;Compared with the Lv-Control-DEHP group,the relative liver weight of the Lv-miR-200a+DEHP miR-200a group recovered;the expression of AST and ALT decreased significantly;the expression of Caspase-3 also Significantly reduced,liver damage was significantly reversed.4.After continuous exposure to DEHP for 72 hours,the expression of miR-192 was increased by DEHP exposure.The results of Western blotting showed that DEHP exposure up-regulated the expression of P53 by down-regulating the expression of Mdm2 in AML 12 cells.DEHP exposure also inhibited the expression of IncRNA（Malatl,Neat1 and Pvt1）in AML12 cells.Analysis of conclusions:1.The continuous exposure of different doses of DEHP causes AML 12 cell apoptosis to be involved in up-regulation of miR-200a expression.2.Injection of miR-200a to interfere with lentivirus effectively interfered with liver damage in mice induced by DEHP exposure.3.Up-regulation of miR-200a expression by DEHP exposure may be associated with the expression of miR-192 and IncRNA.