Mesenchymal stem cell conditioned medium attenuates oxidative stress injury in hepatocytes partly by regulating the miR-486-5p/PIM1 axis and the TGF-β/Smad pathway

ABSTRACT This study investigated the role of microRNA (miRNA) miR-486-5p in oxidative stress injury in hepatocytes under the treatment of mesenchymal stem cell conditioned medium (MSC-CM). The oxidative stress injury in hepatocytes (L02) was induced by H2O2. Human umbilical cord blood MSC-CM (UCB-MSC-CM) was prepared. The effects of UCB-MSC-CM on the proliferation, apoptosis, and inflammatory response in L02 cells were detected by Cell Counting Kit-8 (CCK-8) assay, flow cytometry analysis, and enzyme-linked immunosorbent assay (ELISA). Subsequently, the target of miR-486-5p was predicted using bioinformatics analysis, and the possible signaling pathway addressed by miR-486-5p was explored using western blot. We found that miR-486-5p expression was elevated following oxidative stress injury and was reduced after UCB-MSC-CM treatment. UCB-MSC-CM protected L02 cells against H2O2-induced injury by downregulation of miR-486-5p. Proviral integration site for Moloney murine leukemia virus 1 (PIM1) was verified to be targeted by miR-486-5p. UCB-MSC-CM upregulated the expression of PIM1 reduced by H2O2 in L02 cells. Additionally, silencing PIM1 attenuated the protective effects of miR-486-5p downregulation against oxidative stress injury. We further demonstrated that UCB-MSC-CM inhibited the TGF-β/Smad signaling in H2O2-treated L02 cells by the miR-486-5p/PIM1 axis. Overall, UCB-MSC-CM attenuates oxidative stress injury in hepatocytes by downregulating miR-486-5p and upregulating PIM1, which may be related to the inhibition of TGF-β/Smad pathway.


Introduction
Oxidative stress injury is a major risk factor that leads to severe local and remote tissue injury and subsequent distant organ functional failure, including that of spinal cord, kidney, cardiac vessel, and liver [1][2][3][4]. Oxidative stress is a common pathophysiological basis of diverse liver diseases [5,6], and displays important roles in fatty liver, viral hepatitis, and liver fibrosis [7][8][9]. To date, no effective therapeutic strategies have been proven to modify the course of oxidative stress injury.
Increasing evidence has revealed the significant potentials of mesenchymal stem cells (MSCs) in repairing ischemic organ injury [10]. Studies have demonstrated that MSC conditioned medium (MSC-CM) is a promising therapeutic agent to promote cell proliferation and inhibit cell apoptosis and inflammation after acute organ damage [11][12][13]. CM is heterogeneous and contains various soluble factors. UCB-MSC-CM injection can stimulate hepatocyte regeneration and reduce hepatocyte apoptosis in animals with liver failure [14][15][16]. More importantly, a previous study demonstrated a favorable tendency toward survival caused by MSC-CM treatment [17]. Here, we explored the function of UCB-MSC-CM in oxidative stress-induced injury.
MicroRNAs (miRNAs), a group of short noncoding nucleotides that control mRNA translation at the posttranscriptional level, play critical roles in mediating physiological processes, including cell growth, apoptosis, and differentiation [18][19][20]. The regulatory effects of mature miRNAs are shown in numerous pathological processes [21]. UCB-MSC-CM has the potential of controlling miRNA expression, thus inducing alteration in cellular microenvironments [20]. Previous research showed the important functions of miR-486-5p in regulating hepatocellular carcinoma development. MiR-486-5p prevents cell viability, migration, and invasion in hepatocellular carcinoma by binding to phosphoinositide 3-kinase regulatory subunit 1 [22]. MiR-486-5p attenuates the malignancy of hepatocellular carcinoma by suppressing Casitas B-lineage lymphoma [23]. In addition, downregulation of miR-486-5p alleviates acute lung injury by inhibiting apoptosis through upregulating OTU-domain-containing 7B [24]. However, the biological role of miR-486-5p in hepatocyte injury is unclear.
Until now, little is known about the expression change of miRNAs in hepatocytes during the MSC-CM-mediated process. Here, miR-486-5p was shown to be downregulated after UCB-MSC-CM treatment. We hypothesized that UCB-MSC-CM could attenuate oxidative stress injury in hepatocytes by regulating miR-486-5p expression. This study was aimed to explore the role of miR-486-5p in cell proliferation, apoptosis, and inflammation in H 2 O 2 -treated hepatocytes in the presence of UCB-MSC-CM as well as the possible mechanisms involved, which may provide some theoretical insight into further exploration of molecular mechanisms related to the therapeutic effects of MSC-CM on hepatocyte injury.

Culture of UCB-MSCs
Umbilical cord blood-derived MSCs (UCB-MSCs) were obtained from the umbilical cord of a pregnant woman who underwent a cesarean section and signed informed consent at China-Japan Union Hospital of Jilin University. This study was approved by the Ethics Committee of China-Japan Union Hospital of Jilin University (approval number: 2,021,081,012). Mononuclear cells, collected by Ficoll-Paque density gradient centrifugation (GE Healthcare, Pittsburgh, PA, USA), were incubated in Dulbecco's modified Eagle medium (Gibco; Thermo Fisher Scientific, MA, USA) containing antibiotics (100 U/mL of penicillin and 100 U/mL of streptomycin) and 10% fetal bovine serum (Gibco) in a humidified incubator at 37°C with 5% CO 2 . The medium was refreshed every 2-3 days. The UCB-MSCs between passage 3 and 6 were used in this study.

Preparation of UCB-MSC-CM
When the culture reached 70-80% confluency, UCB-MSCs were washed three times with phosphate buffered saline and the medium was replaced with a fresh complete medium. Subsequently, UCB-MSC-CM was centrifuged at 2500 rpm for 20 minutes to remove any cell debris and passed through a 0.22 μm of filter. The resulting UCB-MSC-CM was then diluted with fresh high-glucose Dulbecco's modified Eagle medium to achieve a final concentration of 50% [25].

Cell surface antigen phenotyping
The characteristics of cultured UCB-MSCs at passage 3 were identified by flow cytometry. As previously published [26], cells were treated with 1 mL of trypsin-Ethylene Diamine Tetraacetic Acid at 37°C for 5-10 minutes. Cells were then resuspended in 10 mL of phosphate buffered saline and centrifuged at 1,000 × g for 5 minutes. After that, cells were resuspended in flow cytometry buffer (phosphate buffered saline containing 2 mM of Ethylene Diamine Tetraacetic Acid and 10% blocking reagent) at 1 × 10 6 cells/mL. Next, 50-100 µL of cell suspension was added to a 1.5 mL of tube and incubated with 2 µL of fluorescent antibodies (CD29, CD34, CD45, CD105; BD Pharmingen, United States) and the homotypic controls on ice for 45 minutes. Next, cells were washed with flow cytometry buffer, fixed in 10% formalin, and stained with 50-100 µL of 0.2% viability dye solution. After incubation at room temperature for 15 minutes, cells were washed with flow cytometry buffer twice and filtered through a 70 µm of cell strainer. The positive rate of antigen was analyzed by a flow cytometry system (Guava easyCyte8HT, EMD Millipore, Billerica, MA).

Hepatic cell culture and processing
Human normal liver cell line L02 (the American Type Culture Collection; MD, USA) was maintained in Dulbecco's modified Eagle medium containing antibiotics (100 U/mL of penicillin and 100 U/mL of streptomycin) and 10% fetal bovine serum (Gibco) in a humidified incubator at 37°C with 5% CO 2 . When the culture reached 80%, 1 mM of H 2 O 2 (Sigma-Aldrich) was used to treat cells for 4 h to induce oxidative stress injury. Subsequently, cells were divided into 3 groups: the control group, the H 2 O 2 group (treatment with 1 mM of H 2 O 2 for 4 h), and the H 2 O 2 + UCB-MSC-CM group (treatment with 30% UCB-MSC-CM for 6, 24, and 48 h after H 2 O 2 stimulation). Cell Counting Kit-8 (CCK-8) assay was used to detect the optimal time of UCB-MSC-CM, which was determined to be 24 h.
In detail, the transfected L02 cells were divided into 7 groups: the control group, the H 2 O 2 group (treatment with 0. 8

Cell Counting Kit-8 (CCK-8)
As previously described [29], the transfected L02 cells were seeded in 96-well plates at 5 × 10 3 cells/well. CCK-8 reagent (10 µL; Dojin Laboratories, Japan) was added to each well at 24 h, and then incubated for another 4 h at 37°C. Cell viability was detected by measuring the optical density (OD) value at 450 nm using a Microplate Reader (Bio-Rad, USA).

Flow cytometry
Annexin V-fluoresceine isothiocyanate (FITC)/ Prodium Iodide (PI) double-labeled staining kit (Sigma-Aldrich) was used to detect apoptosis. The procedure was performed as previously described [30]. The transfected L02 cells were treated with 0.25% Ethylene Diamine Tetraacetic Acid-free trypsin, followed by centrifugation at 2000 rpm for 15 minutes. Cells were then resuspended in pre-cooled phosphate buffered saline and centrifuged again at 2000 rpm for 15 minutes. After washing, cells were resuspended in 300 μL of binding buffer, and then stained with 10 μL of Annexin V-FITC and 5 μL of PI at room temperature for 10 minutes in the dark. Finally, cell apoptosis was assessed using a BD FACS Calibur Flow Cytometer (Beckman Coulter, USA). Data analysis was performed using Guava Incyte (EMD Millipore, USA).

Luciferase reporter assay
The wild type (Wt) or mutant (Mut) sequence in the PIM1 3ʹUTR containing the predicted binding site for miR-486-5p was synthesized to generate the fragment of PIM1-wild type (PIM1-Wt) and fragment of PIM1-mutant (PIM1-Mut). The targeted fragments were inserted into the pmirGLO vector (Promega, Madison, WI, USA). Then the recombinant plasmids (PIM1-Wt and PIM1-Mut) were transfected with miR-486-5p mimics or NC mimics into 293 T cells, respectively. After 48 h, the luciferase activity was examined using the Dual-luciferase Reporter Assay System (Promega) [29].

Statistical analysis
The experimental data are shown as the mean ± standard deviation. Statistical analysis was performed using SPSS20.0 statistics program (IBM, USA). For data conforming to normal distribution and homogeneity of variance, the paired t-test was employed to compare data within a group, while the unpaired t-test was used for comparisons between two groups. One-way analysis of variance followed by Tukey's post hoc test was adopted for comparison among multiple groups. P < 0.05 was statistically significant.

Results
We hypothesized that UCB-MSC-CM could attenuate oxidative stress injury in hepatocytes by regulating miR-486-5p expression. This study was aimed to explore the role of miR-486-5p in cell proliferation, apoptosis, and inflammation in H 2 O 2 -treated hepatocytes in the presence of UCB-MSC-CM as well as the possible mechanisms involved. We examined the proliferation, apoptosis, and inflammatory response in L02 cells. Our results showed that miR-486-5p expression was elevated in L02 cells following oxidative stress injury and was reduced after UCB-MSC-CM treatment. UCB-MSC-CM attenuates oxidative stress injury in hepatocytes by inhibiting miR-486-5p and upregulating PIM1, which may be related to the inhibition of TGF-β/Smad pathway.

Characteristics and differentiation of UCB-MSCs
As indicated by flow cytometry analysis in Figure 1 (a), antigen profiling of the UCB-derived MSCs showed the high expression of positive stromal markers (CD105 and CD29), as well as the absence of negative hematopoietic markers (CD34 and CD45), suggesting that UCB-MSCs share common immunophenotypes with MSCs. Furthermore, the characteristics of UCB-MSCs were confirmed by osteogenic and adipogenic differentiation assays.
For adipogenic differentiation, the intracellular lipid droplets stained by Oil Red O could be observed (Figure 1(b)). Osteogenic differentiation caused calcium deposits, as shown by Alizarin Red staining (Figure 1(c)). These findings indicated that UBC-derived MSCs are characterized by the capacities of stem cells.

UCB-MSC-CM protects L02 cells against H 2 O 2 -induced injury by suppressing miR-486-5p
RT-qPCR indicated that miR-486-5p expression was elevated in the H 2 O 2 group while downregulated in the UCB-MSC-CM group (Figure 2

Discussion
Increasing studies have revealed the close relationship between abnormal expression of miRNAs and hepatocyte injury [34,35]. UCB-MSC-CM plays a crucial role in repairing damaged cells by regulating the expression of miRNAs and changing the cell microenvironment [20]. In this work, the protective effects of UCB-MSC-CM against oxidative stress injury in hepatocytes was verified. Our study  showed that UCB-MSC-CM protected hepatocytes against oxidative injury by mediating proliferation, apoptosis, inflammation in a miR-486-5pdependent manner. Evidence demonstrates the significant roles of miRNAs regulated by UCB-MSC-CM in injury in myocardium [36], brain [37], and kidney [38]. A previous study indicates that silencing miR-486-5p alleviates acute lung injury by inhibiting apoptosis and inflammation via targeting OTU domain-containing protein 7B [24]. MiR-486-5p derived from exosomes of MSCs suppresses cardiomyocyte apoptosis under hypoxic impairment by activating the PTEN/PI3K/AKT pathway [39]. The present study revealed that downregulation of miR-486-5p promoted hepatocyte proliferation and inhibited H 2 O 2 -induced apoptosis and inflammation, suggesting the protective role of silencing miR-486-5p against oxidative stress injury in hepatocytes.
In the present study, PIM1 was predicted as a target of miR-486-5p using bioinformatics analysis. PIM1 has been found to participate in the progression of a variety of diseases. For example, PIM1 acts as an oncogenic gene in lung adenocarcinoma and promotes tumor growth by activating the c-mesenchymal to epithelial transition factor signaling pathway [40]. PIM1 promotes cell invasion, epithelial to mesenchymal transition process, and cancer cell stemness in IL-6-treated breast cancer cells [41]. PIM1 prevents oxidative stress and apoptosis in cardiomyocytes after exposure to hypoxia by promoting cell autophagy [42]. Additionally, PIM1 inhibits cellular senescence in cardiomyocytes by inhibiting TGF-β/Smad pathway [33]. In this study, PIM1 was identified to be a functional target of miR-486-5p and was downregulated in H 2 O 2 -treated hepatocytes. UCB-MSC -CM attenuated the damaging effects of H 2 O 2 by upregulating PIM1 expression. Moreover, PIM1 knockdown reversed the protective effects of miR-486-5p downregulation on oxidative stress injury in H 2 O 2 -treated hepatocytes.

Conclusion
In summary, this study shows that UCB-MSC-CM has significant potential to alleviate H 2 O 2 -induced oxidative stress injury and demonstrates for the first time that miR-486-5p is involved in the molecular mechanisms underlying the therapeutic effects of UCB-MSC-CM on oxidative stress injury in hepatocytes by controlling PIM1 and TGF-β/Smad3 signaling. There are still limitations in this study.
Future study endeavors are required to investigate the precise regulatory mechanisms of how miR-486-5p exert its functions in the MSC-CM-mediated protection of hepatocytes against oxidative stress. More importantly, in vivo experiments are needed to validate the results of in vitro studies.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This study was supported by Key Scientific and Technological Research and Development Project of Jilin Province (No. 20180201055YY).

Data availability statement
The datasets used during the current study are available from the corresponding author on reasonable request.