Role Of The MicroRNA-214/Bax Axis In The Progression Of Acute Liver Failure

Objective:Acute liver failure(ALF)is a severely fatal liver disease caused by acute liver cell death.Its occurrence and development are regulated by a series of factors.In recent years,it has been reported that microRNAs(microRNAs,miRNAs)play a key role in the development of various liver diseases,including ALF.Therefore,exploring the role and regulatory mechanism of miRNAs in ALF will help to clarify the process of ALF and has potential clinical value for finding new targets for the treatment of ALF.This study will explore the role and potential molecular mechanism of miR-214 in ALF through in vivo and in vitro experiments.Methods:(1)800 mg/kg D-galactosamine(D-GalN)and 10 μg/kg lipopolysaccharide(LPS)were used to induce ALF mouse model.Serum aspartate aminotransferase(AST)and alanine aminotransferase(ALT)levels were detected by automatic biochemical analyzer.Serum tumor necrosis factor alpha(TNF-α)and interleukin 6(IL-6)were detected by enzyme linked immunosorbent assay(ELISA).The protein expression level of Caspase-3 in liver tissue was examined by western blot.TUNEL staining was used to detect cell apoptosis in liver tissues.(2)Real-time quantitative PCR(RT-qPCR)technology was used to detect the expression levels of miR-214 and Bax mRNA in the liver tissues of ALF mice.Western blot was used to detect the protein expression of Bax in the liver tissues of ALF mice.(3)D-GalN(1 mg/ml)and TNF-α(100 ng/ml)were combined to treat the mouse embryonic liver cell line BNLCL2 to induce a hepatocyte injury model.RT-qPCR was used to detect the expression level of miR-214 and Bax mRNA.Western blot was used to detect the expression level of Bax protein to clarify the expression of miR-214 and Bax in the process of liver cell injury.(4)Online prediction software such as miRNA.org showed that Bax may be a potential target of miR-214.By constructing mouse Bax 3’UTR wild-type and mutant luciferase reporter vectors,the luciferase reporter assay was used to identify that miR-214 can directly bind to Bax 3’UTR.(5)BNLCL2 cells were transfected with miR-214 mimic and mimic control,and treated with D-GalN/TNF-α for 36 h.RT-qPCR and western blot were used to detect Bax mRNA and protein expression levels.Flow cytometry was used to detect hepatocyte apoptosis.RTqPCR and ELSIA were used to detect the expression levels of TNF-α and IL-6.(6)Bax overexpression vector was constructed and co-transfected into BNLCL2 cells with miR-214 mimic.The rescue experiment was conducted to further explore whether Bax overexpression can reverse the effect of miR-214 mimic on hepatocyte apoptosis and the secretion of inflammatory factors.Results:(1)Compared with the control group,the serum AST and ALT levels of the DGalN/LPS stimulated mice have increased gradually over time and reached the highest level at 7 h.Thus,D-GalN/LPS stimulation for 7 h was selected for follow-up research.The serum TNF-α and IL-6 levels of the mice in the control group and the D-GalN/LPS stimulated group were further detected by ELISA,and it was found that compared with the control group,the serum TNF-α and IL-6 levels of the mice in the D-GalN/LPS stimulated group were significantly increased.TUNEL staining analysis found that the apoptosis level of liver tissue of mice in the D-GalN/LPS stimulation group was significantly higher than that of the control group,and the protein expression level of Caspase-3 was also significantly higher than that of the control group.The above research results confirmed that D-GalN/LPS at this dose successfully induced the ALF model.(2)Compared with the control mice,the expression level of miR-124 in the liver tissue of the ALF model mice was significantly reduced,while the expression levels of Bax mRNA and protein were significantly increased.(3)The BNLCL2 cells were treated with D-GalN/TNF-α for 36 h to successfully establish a hepatocyte injury model.RT-qPCR detection revealed that,compared with the control group,miR-214 was under-expressed in the D-GalN/TNF-α-induced hepatocyte injury model,while Bax was highly expressed in the hepatocyte injury model.(4)The luciferase reporter assay proved that miR-214 directly binds to Bax 3’UTR,speculating that Bax may be a potential target of miR-214.(5)After miR-214 mimic was successfully transfected into BNLCL2 cells,it was found that the mRNA and protein expression levels of Bax were significantly reduced,indicating that Bax is the target gene of miR-214.Further study found that D-GalN/TNF-α induced liver cell apoptosis and the expression of TNF-α and IL-6 were significantly reduced after miR-214 overexpression,indicating that miR-214 can inhibit liver cell apoptosis and the secretion of inflammatory factors.(6)Rescue experiments showed that overexpression of Bax significantly reversed the effects of miR-214 mimic on hepatocyte apoptosis and the expression of TNF-α and IL-6,strengthening indicating that miR-214 can affect hepatocyte apoptosis and secretion of inflammatory factors through targeted regulation of Bax expression.Conclusion:miR-214 is downregulated in ALF mice and injured hepatocytes,and can inhibit hepatocyte apoptosis and secretion of inflammatory factors during ALF development through targeting Bax,thus indicating that miR-214 may be a potential target for ALF treatment.