MicroRNA-21 Contributes to Liver Regeneration by Targeting PTEN

Abstract

Background: Multiple microRNAs (miRNAs, miRs), including miR-21, have been documented to be critical regulators of liver regeneration, but the mechanism underlying their roles in hepatocyte proliferation and cell cycle progression is still far from understood.

Material/methods: miR-21 levels were determined using qRT-PCRs in mouse livers at 48 h after 70% partial hepatectomy (PH-48 h). Cell proliferation was determined by use of a cell-counting kit-8 (CCK-8), EdU incorporation staining, and flow cytometry. Phosphatase and tensin homolog (PTEN) expressions were determined using qRT-PCR and Western blot analysis. PTEN siRNA was used to perform the rescue experiment.

Results: A marked upregulation of miR-21 was observed in mouse livers at 48 h after 70% partial hepatectomy (PH-48 h) compared to 0 h after PH (PH-0 h). Overexpression of miR-21 was associated with increased proliferation and a rapid G1-to-S phase transition of the cell cycle in BNL CL.2 normal liver cells in vitro. In addition, we showed that PTEN expression was inversely correlated with miR-21 in BNL CL.2 cells and demonstrated that PTEN expression is lower in mouse livers at PH-48 h. Moreover, the presence of PTEN siRNA significantly abolished the suppressive effect of miR-21 inhibitor on hepatocyte proliferation.

Conclusions: miR-21 overexpression contributes to liver regeneration and hepatocyte proliferation by targeting PTEN. Upregulation of miR-21 might be a useful therapeutic strategy to promote liver regeneration.

Figure 1

miR-21 is upregulated during the proliferative phase of liver regeneration. qRT-PCR analysis demonstrated a marked upregulation of miR-21 in mouse regenerating livers at 48 h after PH (PH-48 h, n=5) versus that at 0 h after PH (PH-0 h, n=5). ^* P<0.05.

Figure 2

The efficiency of miR-21 mimic or inhibitor transfection in hepatocytes. qRT-PCR analysis confirmed that miR-21 mimic (A) increased and miR-21 inhibitor (B) reduced miR-21 level in BNL CL.2 cells (n=5). ^** P<0.01.

Figure 3

miR-21 regulation exhibits no effect on the cell size of hepatocytes. Dil (red) fluorescent staining showed that miR-21 mimic or inhibitor did not affect the size of BNL CL.2 cells in vitro. Nuclei were counterstained with DAPI (blue) (n=5). Scale bar=50 μm.

Figure 4

miR-21 accelerates the proliferation and G1/S phase transition of the cell cycle in hepatocytes. CCK-8 cell proliferation assay (A) and EdU (green) incorporation assay (B) demonstrated that miR-21 overexpression promoted and miR-21 inhibition reduced the proliferation of BNL CL.2 cells in vitro. Nuclei were counterstained with DAPI (blue) (n=10 and n=5, respectively). Scale bar=50 μm. (C) Flow cytometry showed that miR-21 mimic induced a G1/S phase transition of the cell cycle in BNL CL.2 cells, and miR-21 inhibitor resulted in G1 phase arrest (n=5). ^* P<0.05; ^** P<0.01.

Figure 5

PTEN expression is inversely correlated with miR-21 both in vitro and in vivo. qRT-PCR (A) and Western blot (B) analysis showed that miR-21 negatively regulated PTEN expression at both mRNA and protein levels in BNL CL.2 cells in vitro (n=5 and n=3, respectively). A reduction of PTEN expression was also found at mRNA (C) and protein (D) levels in mouse livers at PH-48 h versus those at PH-0 h (n=5 and n=5, respectively). ^* P<0.05; ^** P<0.01.

Figure 6

Silencing PTEN reverses the proliferation-suppressing effect of miR-21 inhibitor in hepatocytes. (A) qRT-PCR analysis verified that PTEN siRNA (si-01 or si-02) reduced PTEN expression at mRNA level in BNL CL.2 cells (n=5). (B) EdU (green) staining demonstrated that the presence of PTEN siRNA (si-01 or si-02) can abolish the reducing effect of miR-21 inhibitor on the proliferation of BNL CL.2 cells. Nuclei were counterstained with DAPI (blue) (n=5). Scale bar=50 μm. ^** P<0.01.