Overexpression of miR-125a-5p Inhibits Hepatocyte Proliferation through the STAT3 Regulation In Vivo and In Vitro

Abstract

microRNAs (miRNAs) are critically involved in liver regeneration (LR). miR-125a-5p (miR-125a) is a tumor-suppressing miRNA, but its role in LR has not been studied. Our previous studies have proved that miR-125a was related to LR at the initiation phase, while the mechanism hepatocyte proliferation triggered by miR-125a in LR has been rarely evaluated. Herein, we mainly studied the molecular mechanism of miR-125a in triggering hepatocyte proliferation and the proliferation stage of LR. Firstly, a striking reduction of miR-125a was found at 24 h as well as 30 h following partial hepatectomy (PH) in rat liver tissue by miRNAs expression profiles as well as qRT-PCR analysis. Furthermore, in vitro, upregulation of miR-125a decreased proliferation as well as G₁/S conversion, which promoted hepatocytes apoptosis. STAT3 was the target of miR-125a. In vivo, upregulation of miR-125a by tail vein injection of agomir inhibited LR index. Upregulation of miR-125a inhibited LR index and hepatocytes proliferation by STAT3/p-STAT3/JUN/BCL2 axis. In summary, these current discoveries indicated that miR-125a inhibited hepatocytes proliferation as well as LR by targeting STAT3 and via acting on the STAT3/p-STAT3/JUN/BCL2 axis.

Keywords: STAT3; cell cycle; liver regeneration; miR-125a; proliferation.

Figure 1

Detection of miR-125a in liver tissue and BRL-3A cells. (A) Some abnormally expressed miRNAs are presented by red-green map. (B) miR-125a level in liver tissue was verified by qRT-PCR. (C) miR-125a level was examined in BRL-3A through qRT-PCR. (D). PCNA level was examined through qRT-PCR in BRL-3A cells. Data are shown as mean ±SEM, * p < 0.05, ** p < 0.01.

Figure 2

The effect of miR-125a on hepatocytes proliferation (A). miR-125a levels were examined through qRT-PCR following transfection of miR-125a mimics (miR-125a-m), and its control NC mimics in BRL-3A cells. (B). Cell viability was examined through MTT. (C). The role of miR-125a in BRL-3A cell cycles was detected by FACScan. (D). Cell proliferation was examined through EdU (red) assay. Data are shown as mean ±SEM, * p < 0.05, ** p < 0.01.

Figure 3

The effect of miR-125a on hepatocytes apoptosis. (A).The apoptotic rate was examined through FACScan the after treatment of miR-125a mimics and NC mimics, respectively. J4 (the number of early apoptosis cells) as well as J2 (the number of late apoptosis cells) areas are represented as apoptotic cells. (B). Apoptosis rate is represented by a histogram. Data are shown as mean ± SEM, * p < 0.05.

Figure 4

Detection of STAT3 level in vitro and in vivo. (A) STAT3 level was examined through qRT-PCR in hepatocytes after treatment of miR-125a reagents. (B) STAT3 level were examined through WB in BRL-3A after treatment of miR-125a reagents. (C) STAT3 level were examined trough qRT-PCR at 12/24/30 h post-PH (PH-12/24/30 h) in liver tissues. (D) miR-125a as well as STAT3 level were examined through WB at 12/24/30 h post-PH (PH-12/24/30 h) in liver tissues. Data are shown as mean ±SEM, * p < 0.05.

Figure 5

STAT3 was a target gene of miR-125a. (A) Luciferase activity was detected by luciferase report assay after co-transfection of STAT3- WT/Mu -UTR as well as miR-125a mimics and NC mimics in BRL-3A. (B) The binding site of miR-125a and STAT3- WT/Mu -UTR. Data are shown as mean ± SEM, * p < 0.05.

Figure 6

miR-125a inhibited hepatocytes proliferation by STAT3/P-STAT3/JUN/BCL2 axis. (A) STAT3, JUN, BCL2, PCNA and CASPASE3 were detected by qRT-PCR in BRL-3A cells after the treatment of miR-125a reagents. (B). STAT3, JUN, BCL2, PCNA and Cleaved-CASPASE3 were detected by WB in BRL-3A cells after treatment of miR-125a reagents, the samples derive from the same experiment and that gels/blots were processed in parallel. Data are shown as mean ± SEM, * p < 0.05.

Figure 7

Efficiency detection of miR-125a agomir in mouse liver. (A) Cy5-labeled cells were found in miR-125a agomir and its control NC groups by fluorescence microscope. (B) miR-125a level was examined through qRT-PCR analysis following injection of miR-125a agomir compared with controls. Data are shown as mean ± SEM. Nuclei were stained with DAPI (blue). Scale bar, 100 μm, ** p < 0.01.

Figure 8

miR-125a inhibited LR through STAT3/p-STAT3/JUN/BCL2 axis in vivo. (A) Liver index (liver weight/body weight ratio) was measured at 48 h post-PH after transfection of miR-125a agomir/NC through tail vein injection. (B) Representative IHC staining of PCNA (brown) on liver sections obtained from transfection of miR-125a agomir/NC through tail vein injection mice at 48 h post PH; Scale bars: 100 μm. (C) STAT3, JUN, BCL2, PCNA as well as CASPASE3 were detected by qRT-PCR in liver tissue after transfection of miR-125a agomir/NC through tail vein injection at 48 h post-PH. (D) STAT3, p-STAT3, JUN, BCL2, PCNA as well as Cleaved-CASPASE3 were detected by WB in liver tissue after transfection of miR-125a agomir/NC through tail vein injection at 48 h post-PH, the samples derive from the same experiment and that gels/blots were processed in parallel. Data were shown as mean ± SEM, * p < 0.05.