Inhibition of miR-34a-5p alleviates hypoxia-reoxygenation injury by enhancing autophagy in steatotic hepatocytes

Abstract

Hypoxia-reoxygenation (H/R) injury in steatotic hepatocytes has been implicated in liver dysfunction after liver transplantation. MicroRNAs (miRs) play important roles in regulating several cell biology mechanisms related to H/R injury. However, the role of miRs in regulating H/R injury in steatotic hepatocytes is still unclear. We established an in vitro model for studying H/R injury in steatotic hepatocytes and identified miR-34a-5p as a miR that was substantially upregulated in steatotic hepatocytes under H/R challenge. MiR-34a-5p expression was modified by transfecting miR-34a-5p mimic and inhibitor into H/R-challenged steatotic hepatocytes. We found that inhibition of miR-34a-5p alleviated H/R-induced apoptosis and promoted post-H/R proliferation in steatotic hepatocytes. Whereas, overexpression of miR-34a-5p augmented H/R-induced apoptosis and prohibited post-H/R proliferation. By examining autophagy, our data demonstrated that miR-34a-5p suppressed autophagy in H/R-challenged steatotic hepatocytes, induction of autophagy partially rescued the exaggeration of H/R injury induced by miR-34a-5p mimic, while inhibition of autophagy impaired the protection of the miR-34a-5p inhibitor against H/R injury. In conclusion, miR-34a-5p is crucial in exaggerating H/R injury, likely by suppressing autophagy in steatotic hepatocytes. Inhibition of miR-34a may be a promising strategy to protect steatotic hepatocytes against H/R-injury.

Keywords: Autophagy; Hypoxia/Reoxygenation injury; MiR-34a-5p; Steatosis.

Fig. 1.

Establishment of an in vitro model for studying H/R injury in fatty liver. (A) Experimental design. L02 cells were fed with FFA to induce steatosis then challenged with hypoxic condition (0.1% oxygen) for 6 h followed by reoxygenation for up to 24 h. (B,C) Steatosis in L02 cells. Oil Red O-stained FFA-treated and BSA-treated L02 cells were observed under microscopy (B) and optical density was measured (C). (D) Cell viability. Cell viability was measured by MTT assay after 24 h induction of steatosis. (E) mRNA levels of Hif-2α in hypoxia-challenged steatotic hepatocyte. Total mRNA were extracted from FFA-treated L02 cells after 6 h hypoxic culture or normoxic culture, then subjected real-time PCR to measure Hif-2α mRNA levels. (F) Apoptosis in H/R-challenged steatotic hepatocytes. Proteins were extracted from FFA-treated and BSA-treated L02 cells after H/R challenge, then subjected to immunoblotting to detect PARP and its cleaved form. (G) Cell viability of H/R-challenged steatotic hepatocytes. MTT assay was performed to measure cell viability of FFA-treated and BSA-treated L02 cells after H/R challenge. Data represent three independent experiments. ***P<0.001; **P<0.05.

Fig. 2.

MiR-34a-5p expression is substantially unregulated in H/R-challenged steatotic hepatocytes. (A) Levels of miRs in H/R-challenged steatotic hepatocyte. Total miRs were extracted from FFA-treated L02 cells after 6 h hypoxic culture followed by 2 h reoxygenation, then levels of miR-550a-5p, miR-133a-3p, miR-212-5p, miR-501-3p, and miR-34a-5p were measured by real-time PCR. (B) Levels of miR-34a-5p in H/R-challenged steatotic hepatocyte. Total miRs were extracted from FFA-treated L02 cells after 6 h hypoxic culture followed by reoxygenation at indicated time points, then levels of miR-34a-5p were measured. (C) Levels of miR-34a-5p in H/R-challenged steatotic hepatocyte and normal hepatocytes. Levels of miR-34a-5p were measured in BAS-treated and FFA-treated L02 cells after 6 h hypoxic culture followed by 6 h reoxygenation. Data represent three independent experiments. ***P<0.001.

Fig. 3.

MiR-34a-5p augments H/R-induced apoptosis in steatotic hepatocytes. (A) Levels of miR-34a-5p in steatotic hepatocyte after transfection. At 24 h after transfection, total miRs were extracted from miR-34a-5p inhibitor, mimic, and negative control miR-transfected steatotic L02 cells, then levels of miR-34a-5p were measured by real-time PCR. (B-E) Apoptosis in transfected steatotic L02 cells under H/R challenge. Transfected steatotic L02 cells were challenged with 6 h hypoxia followed by 6 h reoxygenation. Cells were fixed and subjected to Hoechst 33342 staining (B), quantification of PI and Annexin V-stained cells by FACS (C,D), and immunoblotting of BCL-2 and cleaved caspase-3 (E) to detect apoptosis. Data represent three independent experiments. ***P<0.001.

Fig. 4.

MiR-34a-5p prohibits proliferation in steatotic hepatocytes after H/R injury. (A,B) Edu labeling proliferation assays. MiR-34a-5p mimic, miR-34a-5p inhibitor, and negative control miR transfected steatotic L02 cells were subjected to 6 h hypoxia followed by reoxygenation for 48 h. Cells were labeled with EdU (A) and quantified by flow cytometry (B). (C) Time-course cell viability measured by CCK-8 assays. MiR-34a-5p mimic, miR-34a-5p inhibitor, and negative control miR transfected steatotic L02 cells were collected at indicated time points after reoxygenation, and cell viability was measure by subjected to CCK-8 assay. Data represent three independent experiments. ***P<0.001.

Fig. 5.

MiR-34a-5p suppresses autophagy in steatotic hepatocytes under H/R condition. (A) Electronic microscopic images of H/R-challenged steatotic hepatocytes. After miR-34a-5p mimic, miR-34a-5p inhibitor, and negative control mi-RNA transfection, steatotic L02 cells were subjected to 6 h hypoxia followed by 6 h reoxygenation. Cells were fixed for electronic microscopy scanning; red arrows indicate autophagosomes in cells. (B) Immunofluorescence staining of LC3 and p62 in H/R-challenged steatotic hepatocytes. Transfected steatotic L02 cells were subjected to H/R stimulation. Cells were fixed and stained with LC3 and p63 antibody followed with Alexa Fluor 488-conjugated and Rhodamine-conjugated secondary antibodies, respectively. Immunofluorescence staining was observed under fluorescence microscope. (C,D) Immunoblotting of LC3 and p62 in H/R-challenged steatotic hepatocytes. Proteins were extracted from transfected steatotic L02 cells after H/R challenge, then subjected to immunoblotting of LC3-I, LC3-II, and p62 (C). The ratio of LC3-II/LC3-I was quantified by measuring band intensity using ImageJ software (D). Data represent three independent experiments. ***P<0.001.

Fig. 6.

MiR-34a-5p regulates H/R injury in steatotic hepatocytes by altering autophagy. (A,B) 5 µM rapamycin (RAPA) was added to cell media to induce autophagy during H/R challenge. MTT assay was performed to measure cell viability of steatotic hepatocytes after 6 h hypoxic culture followed by 6 h reoxygenation (A). Time-course cell viability measured by CCK-8 assays (B). (C,D) 10 µM chloroquine (QC) was added to cell media to inhibit autophagy during H/R challenge. MTT assay was performed to measure cell viability of steatotic hepatocytes after 6 h hypoxic culture followed by 6 h reoxygenation (C). Time-course cell viability measured by CCK-8 assays (D). Data represent three independent experiments. ***P<0.001, versus Control+PBS; ^###P<0.001, versus Mimic+PBS or Inhibitor+PBS.