Hindawi BioMed Research International Volume 2021, Article ID 6685605, 13 pages https://doi.org/10.1155/2021/6685605 Research Article Human Umbilical Cord Mesenchymal Stem Cells Ameliorate Hepatic Stellate Cell Activation and Liver Fibrosis by Upregulating MicroRNA-455-3p through Suppression of p21-Activated Kinase-2 Qing Zhou ,1,2 Tengfei Gu ,3 Yong Zhang ,2 Hongda Li ,2 Xuemei Zhuansun ,4 Sanrong Xu ,2 and Yuting Kuang 1 1Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou 215006, China 2Department of General Surgery, Affiliated Hospital of Jiangsu University, Zhenjiang 212001, China 3Department of Anesthesiology, People’s Hospital of Lianshui County, Jiangsu Province, Lianshui 223400, China 4Laboratory Animal Research Center, Medical College of Soochow University, Suzhou 215123, China Correspondence should be addressed to Sanrong Xu; [email protected] and Yuting Kuang; [email protected] Received 2 November 2020; Revised 2 February 2021; Accepted 6 February 2021; Published 25 February 2021 Academic Editor: Kai-Chiang Yang Copyright © 2021 Qing Zhou et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mesenchymal stem cells (MSCs) were shown to have potential therapeutic effects for treatment of liver fibrosis, and dysregulated expression of microRNAs (miRNAs) played a pivotal role in the pathogenesis of liver fibrosis by regulating their downstream target genes. However, the mechanism by which MSCs affect the progression of liver fibrosis by regulating miRNA expression remains unclear. Here, we investigated whether human umbilical cord MSCs (HUC-MSCs) attenuated hepatic fibrosis by regulating miR-455-3p and its target gene. Significantly upregulated miRNA (miR-455-3p) was screened out by GEO datasets analysis and coculture HUC-MSCs with hepatic stellate cell (HSC) LX-2 cells. p21-activated kinase-2 (PAK2) was forecasted to be the target gene of miR-455-3p by bioinformatics analyses and confirmed by luciferase reporter assay. HUC-MSCs were transplanted into fi fi mice with carbon tetrachloride- (CCl4-) induced liver brosis, the result showed that HUC-MSC transplantation signi cantly fi ameliorated the severity of CCl4-induced liver brosis, attenuated collagen deposition, improved liver function by reducing the expression of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, upregulated miR-455-3p, and suppressed PAK2 expression of liver tissue in mice. Taken together, our study suggests that HUC-MSCs inhibit the activation of fi HSCs and mouse CCl4-induced liver brosis by upregulation of miR-455-3p through targeting PAK2. 1. Introduction immunosuppression limit its application [4]. Therefore, there is an urgent need to find a new alternative treatment strategy. Liver fibrosis and cirrhosis are common pathological processes Mesenchymal stem cells (MSCs) are multipotent stem of many chronic liver diseases, including chronic hepatitis B cells, which can be isolated from bone marrow, adipose tissue, and C, alcoholic steatohepatitis, nonalcoholic steatohepatitis, umbilical cord, and other tissues [5, 6]. There have been dem- and autoimmune hepatitis, which can eventually lead to liver onstrated that MSCs have an inflammatory tendency and can failure or liver cancer. Hepatic stellate cells (HSCs) are widely homing to the injured site to play an anti-inflammatory role accepted to be the fibrogenic cell type in the liver [1]. The char- [7, 8]. Recently, accumulating evidences suggest that exoge- acteristic of fibrosis is accompanied by the activation and pro- nous MSC treatment could ameliorate HSC activation and liferation of HSCs and increased production of excessive liver fibrosis, differentiate into hepatic cells, and promote the extracellular matrix [2, 3]. Currently, there are still few strate- recovery of liver function [9, 10]. gies to reverse liver fibrosis except orthotopic liver transplanta- MicroRNAs (miRNAs), a class of endogenous noncoding tion, but organ donor shortage, complications of surgery, and RNAs, approximately 22 nucleotides long, function as nega- 2 BioMed Research International tively regulate protein translation by binding to the 3′ treated with TGFβ1 (Sigma, St. Louis, MO, USA) at a untranslated region (3′ UTR) of mRNAs of their target gene concentration of 10 ng/mL for 48 hours. [11]. Recent studies have shown that miRNAs regulate the 2.3. HUC-MSCs/LX-2 Cocultures. Fully activated LX-2 cells activation of HSCs and the progress and development of liver were cultured alone or cocultured with HUC-MSCs using fibrosis. For example, miR-125b promotes activation of transwell inserts (24 mm diameter, 0.4-μm pore size, Corn- HSCs and liver fibrosis by activating the ras homolog gene ing, NY, USA) to assess the effects of HUC-MSCs on LX-2 family, member A (RhoA) signaling [12], miR-26b-5p cells. All coculture experiments were performed with LX-2 inhibits liver fibrogenesis by targeting platelet derived growth cells in the lower chamber and HUC-MSCs in the upper factor receptor-β (PDGFR-β) [13], and miR-455-3p allevi- compartment using DMEM medium with 10% FBS. LX-2 ates HSC activation and prevents liver fibrosis by suppressing cells were collected after culturing for 48 hours. heat shock factor 1 (HSF1) expression [14]. However, the underlying mechanisms of MSCs alleviating HSC activation 2.4. Cell Proliferation Assays. The proliferation of LX-2 cells by regulating miRNA expression remains unknown. was measured using a Cell Counting Kit-8 (CCK8) assay fi In this study, we used two miRNA pro le data (GSE33857 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). fi and GSE77271) about liver brosis which were downloaded In brief, LX-2 cells were seeded in the lower chamber of 6- fi from publicly available GEO datasets and screened out ve com- well plate using transwell inserts at a density of 5×104 cells ff mon di erentially expressed miRNAs (DEMs) (miR-342-3p, per well, while in the coculture group the same number of miR-125a-5p, miR-455-3p, miR-574-5p, and miR-193a-3p) HUC-MSCs was seeded in the upper compartment. After which were upregulated or downregulated in experimental ani- culturing for 48 hours, the upper department was removed, fi fi mals or human liver brosis samples. The result was veri ed by and the CCK8 reagent was added to the lower chamber con- quantitative real-time reverse transcription-polymerase chain taining LX-2 cells and incubated for additional 2 hours reaction (qRT-PCR) in activated HSCs induced by transform- according to the manufacturer’s protocol. Optical densities β β ing growth factor 1(TGF 1). Subsequently, we cocultured were measured using a microplate reader (SpectraMax M5; activated HSCs with human umbilical cord mesenchymal stem Molecular Devices, Sunnyvale, CA, USA) at a wavelength of cells (HUC-MSCs) and found that miR-455-3p expression was 450 nm. the most significant difference compared with the HSC group cultured alone. These findings were subsequently verified in 2.5. miR-455-3p Transfection. miR-455-3p mimics, inhibi- mouse models of liver fibrosis treated with HUC-MSCs. tors, and their negative controls (NC) were purchased from Finally, our results demonstrated that HUC-MSC treatment GenePharma (Shanghai, China). LX-2 cells were transfected ameliorated liver fibrosis by upregulating miR-455-3p through with Lipofectamine™ 3000 reagent (Invitrogen, Carlsbad, suppression of p21-activated kinase-2 (PAK2). CA, USA) according to the manufacturer’s instructions. ° Then, the cells were maintained at 37 C in a humidified atmosphere containing 5% CO for 48 hours. 2. Materials and Methods 2 2.6. Luciferase Reporter Assay. The 3′ UTR sequence of 2.1. Isolation, Culture, and Expansion of HUC-MSCs. HUC- human PAK2 that contained the putative wild-type (WT) MSCs were isolated as described previously [15]. Umbilical or mutant (MUT) miR-455-3p binding site was inserted into cords were donated by mothers who provided written the pGL3 luciferase vector (Promega, Madison, WI, USA). informed consent for research purposes. The collection and For the luciferase reporter experiments, LX-2 cells were isolation procedure were both approved by the Ethics Com- cotransfected with the above constructed 3′ UTR luciferase mittee of Soochow University (Suzhou, China). Cells were ™ ’ fi ’ vector and miR-455-3p mimics or NC using Lipofectamine cultured in Dulbecco s modi ed Eagle s medium (DMEM) 3000 reagent. Cells were harvested 48 hours after transfection supplemented with 10% fetal bovine serum (FBS) (Gibco, for dual-luciferase reporter assay by a fluorescence detector Grand Island, NY, USA), 1 mmol/L GlutaMAX™ (Gibco), ° (Promega). The relative firefly luciferase activity was normal- and 100 IU/mL penicillin/streptomycin (Gibco) at 37 Cina fi ized to Renilla luciferase activity. 5% CO2-humidi ed atmosphere with medium changes every 2-3 days and were subcultured by using 0.25% trypsin-EDTA 2.7. Animal Models. Animal experiments were performed (Thermo Fisher Scientific, Waltham, MA, USA) upon reach- according to the guidelines of Institutional Animal Care and ing 80%-90% confluence. The ability to differentiate into Use Committee of Soochow University (Suzhou, China). Male osteoblasts and adipocytes and the surface antigen profile of C57BL/6 mice of 8-10 weeks age were purchased from Shang- HUC-MSCs were confirmed as previously reported [15, 16]. hai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Liver fibrosis mice were induced by carbon tetrachloride μ 2.2. HSC Culture and Treatment. The human HSC line LX-2 (CCl4) (20% in olive oil, 5 L/g) intraperitoneal administration cells were purchased from the Cell Center of Shanghai Insti- twice a week for 12 weeks, while mice in the control group tutes for Biological Sciences (Shanghai, China). Cells were were treated with olive oil. Each experimental group contained cultured in DMEM supplemented with 10% FBS, Gluta- 6 mice. Mice were divided into three groups: control group, ™ MAX , and 100 IU/mL penicillin/streptomycin.