MicroRNA-15a inhibits hepatic stellate cell activation and proliferation via targeting SRY-box transcription factor 9.

Accumulating research have indicated that microRNAs are associated with the progression of hepatic fibrosis (HF). Nevertheless, the biological role and function of microRNA (miR)-15a in HF are still unknown. Our data revealed that miR-15a expression was decreased in TGF-β1-treated LX-2 cells and CCl4-induced mouse model. Additionally, miR-15a could directly target the 3'‑untranslated region of SRY-box transcription factor 9 (SOX9) to inhibit its expression. miR-15a overexpression attenuated the viability and invasion, but enhanced apoptosis in LX-2 cells. However, miR-15a knockdown had the opposite effects. Interestingly, SOX9 overexpression reversed the changes in cell viability, invasion and apoptosis mediated by miR-15a overexpression. Moreover, the miR-15a overexpression-mediated collagen I and alpha smooth muscle actin (a-SMA) downregulation were reversed by SOX9 overexpression. Overall, miR-15a could inhibit LX-2 cell viability and HF pathogenesis by targeting SOX9 in vitro and in vivo.

Introduction

Hepatic fibrosis (HF), a common outcome of chronic liver diseases, is a leading cause of morbidity and mortality in the world [1-3]. The development of fibrosis is associated with the sustained wound-healing process in response to liver injury, characterized by the excessive deposition of extracellular matrix (ECM) components [4,5]. HF has become a major threat for public health, since it may lead to cirrhosis, hepatocellular carcinoma and eventually death [6]. Numerous studies have suggested that the pathogenesis of HF is mainly driven by activated hepatic stellate cells (HSCs), which are the main non-parenchymal and collagen-producing cells distributed in liver tissues [7]. Therefore, inhibiting the activation and viability of HSCs is considered as an effective strategy for preventing HF development [8]. Nevertheless, the mechanisms underlying the modulation of HSC activation remain unclear.

Emerging studies indicate that microRNAs (miRNAs) play vital roles in HF [9]. For instance, miR-101 inhibited liver fibrosis via modulating TGF-β signaling [10]. miR-219 could regulate the expression of pro‑fibrotic markers via targeting TGF-β receptor 2 [11]. The vital role of miR-15a has been identified in various diseases. Chen et al reported that silencing of miR-15a promoted fibroblast activation and lung fibrosis by upregulating Twist expression [12]. Sun et al indicated that miR-15a inhibited extracellular collagen deposition, fibroblasts viability, migration and differentiation in lung fibroblasts [13]. Another study reported that miR-15a was downregulated in HF [14]. Nevertheless, the mechanism of miR-15a in HF has not been investigated.

SOX9 is considered as a vital modulator in several diseases. Zhang et al [15] showed that SOX9 could exert a pro-fibrotic role in the development of renal tubular cell epithelial-mesenchymal transition via the PI3K/AKT signaling. Scharf et al [16] demonstrated that SOX9 activation in fibroblasts could promote cardiac fibrosis and inflammation. In addition, a previous study revealed that loss of SOX9 could suppress the development of HF [17]. However, the effects of SOX9 regulated by miR-15a in HF remain unknown.

Through using bioinformatics tools, we identified a direct binding site between miR-15a and SOX9. Thus, we hypothesized that miR-15a might play an essential role in HF by regulating SOX9. Our research aimed to explore the function and role of miR-15a during the development of HF, and the results revealed that miR-15a could inhibit LX-2 cell viability and HF pathogenesis by targeting SOX9.

Materials and methods

Bioinformatic analysis

To evaluate the expression of miR-15a in HF, three datasets GSE40744, GSE49012, and GSE63046 from Gene Expression Omnibus (GEO) database were analyzed. The basic information of the datasets from GEO database were presented in Table 1.

Table 1.

Datasets from GEO database

ID	Sample	Platform
GSE40744	CTRL = 19	Affymetrix multispecies miRNA-2
	HCV-CH = 18
GSE49012	CTRL = 12	Thermo scientific dharmacon microRNA human array
	NASH-CH = 9
	PBC-CH = 13
GSE63046	CTRL = 9	Illumina HiSeq 2000
	HBV/HCV-CH = 11

CTRL: Normal control; HCV: Hepatic C virus; CH: Liver fibrosis/cirrhosis; NASH: Nonalcoholic steatohepatitis; PBC: Primary sclerosing cholangitis; HBV: Hepatic B virus.

Cell treatment

The human HSC line LX2, obtained from Bena (Beijing), were maintained in DMEM containing 10% FBS. To construct an in vitro HF cell model, TGF-β1 (5 ng/ml; Sigma-Aldrich) was employed to treat LX-2 cells for 24 h. The expression of collagen I and α-SMA was subsequently detected as indicators of fibrosis.

Cell transfection

Short hairpin RNA (shRNA) targeting SOX9 (shSOX9), the negative control shRNA (shNC), miR-15a mimics and inhibitors and their controls (NC mimics and NC inhibitor) were purchased from GenePharma (Shanghai). Full length of SOX9 cDNA was sub-cloned into a pcDNA3.1 vector to overexpress SOX9. The transfection was performed using Lipofectamine 2000 (Invitrogen).

Colony formation assay

Cells were seeded into 6-well plates and cultured for 2 weeks. Subsequently, colonies were fixed with paraformaldehyde, stained with crystal violet and counted.

RT-qPCR

Total RNA was isolated from the cultured LX-2 cells using a TRIzol® reagent (Takara). Then, RNA was reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara). qPCR was performed on ABI Step One System (Applied Biosystems) using the SYBR Premix Ex Taq II kit (Takara). The primer sequences were as follows: miR-15a forward 5′-GCAGCACATAATGGTTTG-3′ and reverse, 5′- GAACATGTCTGCGTATCTC-3′; SOX9 forward, 5′-AGGAAGCTCGCGGACCAGTAC-3′ and reverse, 5′-GGTGGTCCTTCTTGTGCTGCAC-3′; GAPDH forward, 5′-GTCTCCTCTGACTTCAACAGCG-3′ and reverse, 5′-ACCACCCTGTTGCTGTAGCCAA-3′; and U6 forward, 5′-CTCGCTTCGGCAGCACAT-3′ and reverse, 5′- TTTGCGTGTCATCCTTGCG-3′. U6 and GAPDH were used as internal controls for normalization of miR-15a and SOX9 mRNA expression levels, respectively.

TUNEL

The cultured cells were fixed with paraformaldehyde and then permeabilized with Triton-X 100, followed by incubated in TUNEL reaction mixture. Subsequently, the TUNEL-stained cells were counterstained with DAPI, and the TUNEL-positive cells were counted.

Transwell assay

The transfected cells in 200 μl serum-free DMEM were added into the upper chamber of Matrigel-coated membranes. The lower chamber was supplemented with complete DMEM. After 48 h, cells in the bottom chamber were stained and calculated.

Luciferase reporter assay

The wild-type (WT) or mutant-type (MUT) regions of miR-15a in the SOX9 3’-UTR were synthesized by GenePharma (Shanghai) and were then subcloned into a pmirGLO plasmid (Promega). Then, pmirGLO-SOX9-WT or pmirGLO-SOX9-MUT and miR-15a or NC mimics were co-transfected into cells, and the luciferase activities were then detected using a Luciferase Reporter Assay System (Promega).

Experimental animal model

To induce liver fibrosis, male C57BL/6 mice (6-weeks-old, n = 30) received 0.4 ml/kg body weight CCl4 (Sigma-Aldrich) by intraperitoneal injection, which was dissolved in corn oil, twice a week for eight weeks. Control mice (n = 10, Control) were intraperitoneally injected with the same amount of saline. Next, CCl4-treated mice were randomly divided into 3 experiment groups: treatment with 0.4 ml/kg body weight of CCl4 twice a week in parallel with one intraperitoneal injection of PBS (n = 10, CCl4 group), NC mimics (n = 10, CCl4+ NC mimics group), and miR-15 mimics (n = 10, CCl4+ miR-15a group). The liver histological sections were examined by light microscopy after staining with hematoxylin and eosin (H&E). All experimental procedures were approved the ethics Committee of the First People’s Hospital of Kunshan (No.20200315A).

Western blot

Total proteins were isolated from LX-2 cells using a RIPA buffer (Beyotime), separated by 10% SDS-PAGE, and then transferred onto PVDF membranes (EMD Millipore). The membranes were incubated with primary antibodies, including Collagen1 (1:1000), α-SMA (1:1000), SOX9 (1:1000) and GAPDH (1:1000), followed by exposure to secondary antibodies. The proteins were visualized using an ECL kit.

Statistical analysis

The data were analyzed using SPSS software and presented as mean ± standard deviation (SD). All experiments were performed at least 3 times. Differences between groups were determined using one-way ANOVA or Student’s t-test. P < 0.05 was considered statistically significant.

Results

In this study, we first determined the expression of miR-15a in HF in vitro and in vivo. Subsequently, we demonstrated that miR-15a inhibits HSC activation and proliferation via targeting SOX9, which suggests that miR-15a has therapeutic potential for HF.

miR-15a expression is downregulated in HF

Firstly, results from 3 datasets (GSE40744, GSE49012, and GSE63046) indicated that miR-15a was lowly expressed in HF (Figure 1a). Then, an in vitro HF cell model was successfully established, as confirmed by the increased expression of collagen I and α-SMA in TGF-β1-induced LX-2 cells (Figure 1b,c). Furthermore, miR-15a expression was markedly reduced in HF cell model compared to control cells (Figure 1d), suggesting that miR-15a might be associated with the development of HF.

Figure 1.

miR-15a expression is downregulated in HF. (a) The miR-15a expression was analyzed in GSE40744, GSE49012, and GSE63046 datasets. (b and c) RT-qPCR and western blot showed the mRNA and protein levels of Collagen I and a-SMA in LX-2 cells treated with TGF-β1. (d) The relative miR-15a expression in LX-2 cells treated with TGF-β1 was measured. *p < 0.05.

miR-15a modulates HSC proliferation to participate in HF pathogenesis

Subsequently, the regulatory role of miR-15a in HF pathogenesis was explored. RT-qPCR demonstrated miR-15a levels were enhanced in TGF-β1-induced LX-2 cells transfected with miR-15a mimic and reduced in miR-15a inhibitor-transfected cells (Figure 2a,b). Additionally, miR-15a upregulation inhibited TGF-β1-treated LX-2 cell viability, which was enhanced by miR-15a knockdown (Figure 2c,d). In addition, miR-15a overexpression restrained LX-2 cell invasion and promoted apoptosis. However, cell transfected with miR-15a inhibitor had the opposite effects (Figure 2e,f). Furthermore, the mRNA and protein levels of collagen I and α-SMA were repressed and upregulated following miR-15a overexpression and silencing, respectively (Figure 2g,h). These data demonstrated that miR-15a modulated HSC viability and apoptosis to regulate HF.

Figure 2.

miR-15a modulates HSC proliferation to participate in HF pathogenesis. (a and b) The transfection efficiencies of miR-15a mimics or inhibitor in TGF-β1-induced LX-2 were confirmed by RT-qPCR. (c and d) CCK-8 and colony formation assays showed that the proliferation of TGF-β1-treated LX-2 cells transfected with miR-15a mimics or inhibitor. (e and f) Transwell and TUNEL assays indicated cell invasion and apoptosis of TGF-β1-treated LX-2 cells. (g and h) RT-qPCR and western blot assays showed the levels of fibrosis markers a-SMA and collagen I in TGF-β1-treated LX-2 cells. *p < 0.05.

SOX9 directly targets miR-15a in LX-2 cells

Through using starBase website, the potential bind sites between SOX9 and miR-15a were predicted (Figure 3a). Moreover, miR-15a overexpression distinctly decreased the activity of SOX9-WT, but the activity of SOX9-MUT was not affected in LX-2 cells (Figure 3b), which further confirmed the binding ability between miR-15a and SOX9. In addition, miR-15a overexpression downregulated SOX9 expression (Figure 3c), while miR-15a silencing promoted SOX9 expression (Figure 3d). In summary, these results revealed that miR-15a could target SOX9 and downregulate its expression.

Figure 3.

SOX9 directly targets miR-15a in LX-2 cells. (a) StarBase website showed the potential binding sites in miR-15a with SOX9. (b) The binding ability was confirmed by luciferase reporter assay. (c and d) RT-qPCR showed the levels of SOX9 in LX-2 cells. *p < 0.05.

miR-15a regulates HSC proliferation and fibrosis by regulating SOX9 expression

Subsequently, the effect of SOX9 in miR-15a-mediated HF progression was investigated. Firstly, SOX9 was significantly upregulated or downregulated in LX-2 cells transfected with pcDNA3.1/SOX9 or shSOX9 (Figure 4a). The inhibitory effects of SOX9 protein expression mediated by miR-15a overexpression were rescued by SOX9 overexpression (Figure 4b). Furthermore, miR-15a mimics attenuated TGF-β1-treated LX-2 cell proliferation, while this suppressive effect was abolished by the upregulation of SOX9. By contrast, miR-15a knockdown promoted cell proliferation, while SOX9 silencing abrogated these effects (Figure 4c,d). Additionally, SOX9 overexpression or knockdown restored the impacts of miR-15a upregulation or downregulation on cell invasion and apoptosis (Figure 4e,f). Western blotting revealed that the collagen I and α-SMA levels were reduced in TGF-β1-induced cells transfected with miR-15a mimics, which were restored by SOX9 overexpression (Figure 4g). Overall, these data showed that miR-15a modulated HSC cell viability and fibrosis via suppressing SOX9 expression.

Figure 4.

miR-15a regulates HSC proliferation and fibrosis through regulating SOX9 expression. (a)RT-qPCR showed the levels of SOX9 in LX-2 cells transfected with pcDNA3.1/SOX9 or shSOX9. (b)Western blot assay showed the levels of SOX9 in LX-2 cells transfected with miR-15a mimics and pcDNA3.1/SOX9 under TGF-β1 treatment. (c-f) CCK-8, colony formation, transwell and TUNEL assays showed cell proliferation, invasion and apoptosis. G) Western blotting was used to measure the protein levels of LX-2 cells under TGF-β1 treatment. *p < 0.05.

Overexpression of miR-15a attenuates HF progression in vivo

To further investigate the effect of miR-15a on HF in vivo, we first detected miR-15a expression in fibrotic mouse liver tissues. RT-qPCR exhibited a decreased level of miR-15a in CCl4-induced liver fibrosis mouse model, while overexpression of miR-15a reversed this effect (Figure 5a). Western blotting revealed that the protein levels of collagen I and α-SMA were enhanced in CCl4 group, whereas miR-15a augmentation downregulated collagen I and α-SMA expression (Figure 5b). Moreover, H&E showed that miR-15a overexpression partially abrogated HF induced by CCl4 (Figure 5c). These results indicated that the upregulation of miR-15a prevented the progression of HF in CCl4-induced mice.

Figure 5.

The upregulation of miR-15a attenuates HF progression in vivo. (a) RT-qPCR showed miR-15a level in mice treated with CCl4, CCl4+ NC mimics and CCl4+ miR-15a mimics. (b) Western blot assay showed the levels of a-SMA and collagen I in mice treated with CCl4, CCl4+ NC mimics and CCl4+ miR-15a mimics. (c) H&E showed histological images of mouse livers treated with CCl4, CCl4+ NC mimics and CCl4+ miR-15a mimics. *p < 0.05.

Discussion

HF is a major risk factor for cirrhosis and chronic liver failure [18]. The proliferation and activation of HSCs may result in the deposition of excessive ECM, thus leading to liver fibrosis [19]. Emerging evidence has suggested that HF is a dynamic process, while early cirrhosis may be reversible. Currently, no effective treatment is available for liver fibrosis [20]. Liver biopsy is considered as the gold standard to diagnose and stage HF. Nevertheless, its application has several limitations for accurately defining liver diseases and evaluating their progression, including invasiveness, cost and sampling variability [21,22]. Therefore, understanding the cellular mechanism underlying abnormal HSC viability is important to develop novel therapeutic approaches against HF. In the current work, LX-2 cells treated with TGF-β1 were used to establish an in vitro HF model. The successful establishment of the in vitro HF model was confirmed by the increased levels of collagen I and α-SMA [23,24]. Furthermore, functional assays revealed that miR-15a inhibited HSC activation and proliferation via targeting SOX9.

MiRNAs has been reported to modulate several biological processes in the liver and are directly associated with inflammation, cirrhosis, and malignant transformation, thus considered as useful therapeutic biomarkers [25]. Numerous studies have verified that miR-15a exerts an inhibitory effect on fibrotic disease. For example, miR-15a inhibited peritoneal fibrosis caused by peritoneal dialysis via targeting VEGF [26]. Additionally, another study demonstrated that long non-coding RNA pulmonary fibrosis-associated RNA facilitated fibrogenesis in lung fibroblasts by downregulating miR-15a [13]. Herein, we found that miR-15a was lowly expressed in HF, and miR-15a inhibited HSC activation and proliferation. To further elucidate the biological role of miR-15a in HF in vivo, a CCl4-triggered HF model was established. Our results indicated that miR-15a overexpression partially reversed the HF induced by CCl4, as evidenced by decreased collagen I and α-SMA expression, which further demonstrated that miR-15a could alleviate HF progression.

SOX9 is widely expressed in bile duct cells and is involved in bile duct development and liver regeneration. In vitro, SOX9 was expressed during the activation of HSCs by profibrotic signaling-related factors, thus facilitating ECM component production, such as collagen I and osteopontin [17,27]. Our study showed that miR-15a could bind with SOX9 to inhibit SOX9 expression. Functional assays indicated that SOX9 overexpression reversed the suppressive impact of miR-15a upregulation on HSC activation and viability. The aforementioned results indicated that miR-15a attenuated HSC viability and fibrosis via targeting SOX9.

Conclusions

The current research revealed that miR-15a modulated HSC cell viability and HF pathogenesis through repressing SOX9 expression. These results might provide novel targets for developing new drugs against HF.