Partial Hepatectomy-Induced Upregulation of miR-1907 Accelerates Liver Regeneration by Activation Autophagy

Abstract

Liver regeneration after partial hepatectomy (PH) is a highly orchestrated biological process in which synchronized hepatocyte proliferation is induced after massive liver mass loss. Hepatocyte proliferation could be regulated by multiple signals, such as miRNAs and autophagy, but underlying mechanism remains unclear. Here a functional miRNA during liver regeneration was identified and its underlying mechanism was delineated in vitro and in vivo. We found that miR-1907 was highly upregulated during liver regeneration after 2/3 PH at various timepoints. The level of miR-1907 was also increased in normal liver cell line treated with HGF at different concentrations. Functionally, miR-1907 enhanced hepatocyte proliferation in vitro and in vivo, and the liver/body weight ratio in miR-1907-overexpressed mice was significantly higher in comparison to the control mice after 2/3 PH. Forced expression of miR-1907 promoted autophagy activation of hepatocyte. Importantly, autophagy inhibition significantly attenuated miR-1907-induced hepatocyte proliferation and the liver/body weight ratio. Finally, GSK3β, a suppressor of autophagy signaling, was identified as the direct target gene of miR-1907. Taken together, miR-1907 accelerates hepatocyte proliferation during liver regeneration by activating autophagy; thus pharmacological intervention regulating miR-1907/autophagy axis may be therapeutically beneficial in liver transplantation and liver failure by inducing liver regeneration.

Figure 1

miR-1907 expression is upregulated after HGF treatment and 2/3 PH. (a) Heatmap showed the aberrantly expressed miRNAs in CCL-9.1 cells following HGF (25ng/μl) treatment compared to control. (b) Primary mouse hepatocytes were isolated and treated with HGF, and qPCR analysis of miR-1907 expression in primary mouse hepatocytes was carried out with different concentration. (c) qPCR analysis of miR-1907 expression in liver tissues at different timepoints after 2/3 PH. ∗p<0.05.

Figure 2

miR-1907 promotes hepatocyte proliferation in vitro and in vivo. miR-1907 were overexpressed in CCL-9.1 (a) or in HL7702 (b) using miR-1907 mimics, and cell proliferation was assessed using the CCK-8 assay. miR-1907 were overexpressed in CCL-9.1 (c) or in HL7702 (d), and cell proliferation was assessed using EdU immunofluorescence staining. miR-1907 were overexpressed (e) or inhibited (f) in CCL-9.1, and cell proliferation was assessed using BrdU ELISA. (g) Immunohistochemistry analysis for PCNA showed the differences in hepatocyte proliferation between mice injected with miR-1907 and control. Original magnification ×200. Quantification of PCNA-positive cells at different timepoints after 2/3 PH was showed in (h). n=5. (i) Mice were injected with miR-1907 or control through the tail vein before and after 2/3 PH. The liver mass to body weight ratio was then calculated at different timepoint. ∗p<0.05.

Figure 3

miR-1907 increases the activation of hepatocyte autophagy. (a) Western blot analysis of LC-3B expression in liver tissues at different timepoints after 2/3 PH. CCL-9.1 cells (b) or HL7702 (c) cells were treated with miR-1907, and the autophagosome formation was visualized by assaying activated green puncta. Punctate staining is indicative for the redistribution of LC3 to autophagosomes. The average number of green puncta per cell with standard deviation for each group is presented. Scale bar, 50 μm. CCL-9.1 cells (d) or HL-7702 (e) cells were treated with miR-1907 and then western blot analysis of LC-3B protein expression to evaluate autophagy. CCL-9.1 cells (f) or HL7702 (g) cells were treated with miR-1907 and then western blot analysis of p62 protein level to evaluate autophagy. ∗p < 0.05.

Figure 4

miR-1907 activates hepatocyte autophagy by inhibiting GSK3 β. CCL-9.1 cell proliferation was assayed after miR-1907 treatment with or without 3-MA (1 μM). (b) Quantification of PCNA-positive cells in mice liver injected with miR-1907 with or without 3-MA (2mg/kg) at different timepoints before and after 2/3 PH. (c) Mice were injected with miR-1907 with or without 3-MA before and after 2/3 PH. The liver mass to body weight ratio was then calculated at different timepoint. (d) Schematic representation of the miR-1907 site in GSK3β 3′-UTR. (e) The 3′UTR reporter assay was carried out in CCL-9.1 cells overexpressed with miR-1907. pGL3-GSK3β-3′-UTR-WT or pGL3-GSK3β-3′-UTR-Mutation was cotransfected with pRL-TK. Luciferase assays were performed 48 h after transfection. Firefly luciferase activity was standardized to Renilla luciferase control. (f) Western blot analysis for endogenous GSK3β protein level after miR-1907 overexpression or inhibition in CCL-9.1 cells. (g) CCL-9.1 cells were treated with miR-1907 with or without 3-MA, and the autophagosome formation was visualized by assaying activated green puncta. Scale bar, 50 μm. ∗p < 0.05.