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miR-124-3p and miR-140-3p.2 act as negative regulators of Beclin1 and LC3 expression in the liver of rat model with hepatic impact injury

Yulin Niu, Yinglian Zhang, Kun Li, Chengyi Sun, Chungen Xing

Objective: To investigate the effect of autophagy on hepatic impact injury in rats and its molecular mechanism inovlved.

Methods: Sixty healthy male Sprague-Dawley (SD) rats (clean grade) were randomly divided into two groups, the normal group and the model group (n=30 in each group). An impact injury model of rat liver was established by impacting rat liver with 200 kPa impact using BIM-V biological impactor. The enzyme activity of ALT and AST in blood serum was measured by rate method; the changes of TNF-α, IL-8 and IL-2 levels were detected by ELISA; morphological changes were observed by microscope after routine HE staining; the formation of autophagosomes was observed by laser scanning confocal microscopy; protein expression levels were detected by Western blotting and immunohistochemical methods, and the relative expression levels of genes were determined by RT-PCR; Bioinformatics software was used to analysis the regulate miRNAs of microtubule-associated protein l light chain 3 (LC3) and Beclin1; a double-fluorescent reporter gene vector was constructed to detect the interactions between genes.

Results: The serum levels of ALT, AST, TNF, IL-8 and IL-2 in the model group were markedly higher than those in the normal group, and the difference was statistically significant (p<0.01). The morphology of the liver tissue in the normal group was normal, versus that in the model group was damaged to different degrees, for example, the sheet necrosis occurred in the surrounding area of the central veins of hepatic lobules where a large number of necrotic cells were observed. The expression of LC3 and Beclin1 proteins of liver tissues in the model group was significantly increased (p<0.05). MiR-124-3p negatively regulated the Beclin1 expression and miR-140-3p.2 negatively regulated the LC3 expression.

Conclusion: Autophagy plays an important role in the hepatic impact injury in rats, and miR-124-3p and miR-140-3p.2 may act as negative regulators of Beclin1 and LC3 expressions, respectively.