PLK1 regulates hepatic stellate cell activation and liver fibrosis through Wnt/β‐catenin signalling pathway

Abstract As an outcome of chronic liver disease, liver fibrosis involves the activation of hepatic stellate cells (HSCs) caused by a variety of chronic liver injuries. It is important to explore approaches to inhibit the activation and proliferation of HSCs for the treatment of liver fibrosis. PLK1 is overexpressed in many human tumour cells and has become a popular drug target in tumour therapy. Therefore, further study of the function of PLK1 in the cell cycle is valid. In the present study, we found that PLK1 expression was elevated in primary HSCs isolated from CCl4‐induced liver fibrosis mice and LX‐2 cells stimulated with TGF‐β1. Knockdown of PLK1 inhibited α‐SMA and Col1α1 expression and reduced the activation of HSCs in CCl4‐induced liver fibrosis mice and LX‐2 cells stimulated with TGF‐β1. We further showed that inhibiting the expression of PLK1 reduced the proliferation of HSCs and promoted HSCs apoptosis in vivo and in vitro. Furthermore, we found that the Wnt/β‐catenin signalling pathway may be essential for PLK1‐mediated HSCs activation. Together, blocking PLK1 effectively suppressed liver fibrosis by inhibiting HSC activation, which may provide a new treatment strategy for liver fibrosis.

important for normal liver development. 6 Studies have confirmed that this pathway is closely related to the formation of HSCs and liver fibrosis. 7,8 However, the mechanisms involved in the activation of HSCs by the Wnt/β-catenin pathway to participate in liver fibrosis remain unclear. PLK1 (polo-like kinase 1) belongs to the polo-like kinase family and regulates cell mitosis, cytokinesis and DNA damage response. 9,10 Importantly, previous studies have identified that PLK1 is highly expressed in a variety of cancers including HCC, pancreatic carcinoma and renal cell carcinoma, and it is associated with decreased survival in cancer patients. 11,12 Several studies have shown that PLK1 is involved in the invasion and metastasis of cancer. 13,14 Furthermore, blocking the expression of PLK1 can effectively inhibit the proliferation of tumour cells and induce their apoptosis. 15 In addition, PLK1 has been demonstrated as a marker associated with the cell cycle in acute idiopathic pulmonary fibrosis (IPF) and is a therapeutic target. 16 Considering the above findings, we speculated whether PLK1 can regulate the proliferation and apoptosis of HSCs to promote the resolution of liver fibrosis. Interestingly, a previous study identified that PLK1 phosphorylation of axin2 facilitated the GSK3-dependent phosphorylation of β-catenin by enhancing binding between GSK3 and β-catenin, offering a novel PLK1-Wnt/β-catenin signalling axis to treat prostate cancer. 17,18 In our study, we investigated whether PLK1 regulates HSCs via the Wnt/β-catenin signalling pathway to influence the development of liver fibrosis.
To the best of our knowledge, this is the first study to demonstrate the essential role of PLK1 in the pathogenesis of liver fibrosis and identify the potential mechanisms involved. We found that reduced PLK1 expression effectively prevented HSC activation. In addition, we revealed that inhibition of PLK1 promoted HSC apoptosis and reduced liver fibrosis via the Wnt/β-catenin signalling pathway in vivo and in vitro.

| Mouse model of liver fibrosis
All experiments were conducted as required of the Ethics Committee and Animal Experimental Committee at Anhui Medical University.
Male C57BL/6J mice (8 weeks of age) were purchased from the Animal Experiment Center of Anhui Medical University. C57BL/6J mice were intraperitoneally injected twice a week with a 10% solution of CCl 4 in olive oil or olive oil alone (vehicle control) at a dose of 0.001 mL/g for 6 weeks. Mice were killed 3 days after the last injection.

| Adeno-associated virus (AAV) infection
Purified adeno-associated viral vector serotype 8 (AAV8) encoding PLK1 was generated by Hanheng Biotechnology (Shanghai, China). C57BL/6J mice received a single tail vein injection of AAV8 encoding PLK1 at a concentration of 1 × 10 12 vg/mL. The transfection efficiency was measured by Western blotting and real-time PCR analysis.

| Human samples
Ten normal and fourteen human liver fibrosis samples were received from the First Affiliated Hospital of Anhui Medical University (Anhui, China). All specimens were obtained after receiving informed consent from each patient, and the experiment was performed in accordance with the Declaration of Helsinki and with approval by the Ethics Committee of Anhui Medical University. Part of each sample was fixed in 4% paraformaldehyde and embedded in paraffin, whereas the rest was stored at −80°C.

| Isolation of primary HSCs
HSCs were isolated as previously described 19,20 with some modifications. Briefly, mouse primary HSCs were isolated employing a two-step collagenase (Sigma)-pronase (Sigma) perfusion of mouse livers, followed by OptiPrep density gradient centrifugation (Axis Shield, Norway). Finally, the expression level of α-SMA (an HSC marker) was detected by Western blotting and real-time PCR.

| Serum biochemical value analysis
Serum was collected from whole blood samples by centrifugation at 1000 g for 30 minutes at room temperature. Serum alanine aminotransferase and aspartate aminotransferase (ALT and AST) were measured using an ALT and AST Assay Kit (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions.

| Real-time PCR analysis
Total RNA was extracted from primary HSCs or LX-2 cells using the TRIzol reagent (Invitrogen, USA) following the manufacturer's protocols. Quantitative detection was performed using a Spectrophotometer NanoDrop 2000 (Thermo Scientific, USA), and cDNA was synthesized using a PrimeScript™RT Master Mix (Takara, Japan). Real-time PCR samples were run on the CFX96 Real-Time PCR Detection System (Bio-Rad, USA) for 10 minutes at 95°C, followed by 40 cycles at 95°C for 15 seconds and at 60°C for 1 minute.
GAPDH expression was used as an internal control. The sequences of the primers for real-time PCR are listed in Table 1.
LX-2 cells were treated with an empty plasmid as a negative control.
The transfection efficiency was measured by Western blotting and real-time PCR analysis.

| Cell apoptosis analysis
The apoptosis of LX-2 cells was analysed by flow cytometry using the Annexin V-FITC/PI Apoptosis Kit (Best Bio, China). Cells were centrifuged and suspended in 400 µL Annexin binding solution.
Then, 5 µL Annexin V-FITC staining solution was added to the suspension and incubated for 15 minutes followed by the addition of PI and incubated for 5 minutes in the dark at 4°C before flow cytometry analysis (Beckman Coulter, USA).

| Cell cycle analysis
The

| Statistical analysis
Data collected from this study were analysed using one-way analysis of variance (ANOVA), followed by Newman-Keuls post hoc test

| PLK1 was highly expressed in human liver fibrosis tissues
We analysed PLK1 levels in human healthy control and liver fibrosis tissue. The pathological results showed that the liver tissues were structurally disordered and the fibrosis tissues were significantly hyperplastic, as shown by H&E staining in patients with liver fibrosis ( Figure 1A). The fibrotic parts were indicated by Sirius red staining ( Figure 1A), and activated HSCs were identified by α-SMA-positive areas ( Figure 1B). Immunohistochemical analyses revealed low expression of PLK1 in healthy livers, whereas much higher expression of PLK1 was evident in the perivascular region of the portal vein and lesion boundary in patients with liver fibrosis ( Figure 1B). Protein expression of PLK1, α-SMA and Col1α1 was elevated in human liver fibrosis compared with healthy livers by Western blotting ( Figure 1C). Similarly, the mRNA levels of PLK1 and fibrogenic genes (α-SMA, Col1α1 and TIMP-1) were higher in liver fibrosis than in healthy livers, as shown by real-time PCR ( Figure 1D). These results indicate that the expression of PLK1 is elevated in liver fibrosis, and that up-regulated PLK1 expression is associated with liver fibrosis.

| Up-regulation of PLK1 expression in HSCs is associated with CCl 4 -induced liver fibrosis
To evaluate whether PLK1 expression is correlated with liver fibrosis, we developed a CCl 4 -induced mouse liver fibrosis model for histopathological studies. Liver injury was observed in liver fibrosis mice by H&E staining ( Figure S1A). The collagen area was indicated by Sirius red staining, and the activated HSCs were highlighted by α-SMA positivity ( Figure S1A). Immunohistochemical analyses demonstrated that PLK1 expression was increased in  Figure S1E). The co-localization positive area quantitative results are shown in Figure S1E.
These studies demonstrate that PLK1 expression is increased in the liver fibrosis mouse model, which is consistent with the results in humans, suggesting that PLK1 may be related to HSCs activation in liver fibrosis.

| Inhibition of PLK1 alleviates CCl 4 -induced liver fibrosis
To define the role of PLK1 in liver fibrogenesis, we investigated the functional effects of PLK1 on liver fibrosis in CCl 4 -induced liver fibrosis mice in vivo. Mice were injected with adeno-associated virus (AAV)-shRNA-PLK1 to knockdown PLK1 expression in the liver, and mice injected with an empty AAV vector were used as controls (Figure 2A). Mice were repetitively treated with CCl 4 for 6 weeks. We detected the knockdown efficiency of AAV-shPLK1 on PLK1 by Western blotting and real-time PCR ( Figure 2B). Inhibition of PLK1 alleviated CCl 4 -induced liver injury and fibrosis compared with AAV-empty-treated mice by H&E and Sirius red staining ( Figure 2C). Consistently, IHC staining results showed that the decrease in PLK1 significantly reduced α-SMA expression ( Figure 2C). The quantification of α-SMA-positive and PLK1-positive areas is shown in Figure 2C. Furthermore, there was a common reduction in the levels of ALT and AST in serum from CCl 4 -induced liver fibrosis mice injected with AAV-shPLK1 compared with the mice that received AAV-empty ( Figure 2D).
Moreover, low expression of PLK1 by AAV-shPLK1 led to a significant decrease in the expression of α-SMA and Col1α1 protein in HSCs from CCl 4 -induced liver fibrosis mice ( Figure 2E). The mRNA levels of α-SMA, Col1α1 and TIMP-1 were notably attenuated in CCl 4 -treated PLK1 knockdown mice compared with AAV-empty mice ( Figure 2F). Collectively, these observations revealed that inhibition of PLK1 alleviates liver fibrosis and the activation of HSCs in vivo.

| Blocking PLK1 attenuates the activation of LX-2 cells stimulated with TGF-β1
To further assess whether PLK1 expression is related to HSCs activation in liver fibrosis, we investigated the functional effects of silencing PLK1 on liver fibrosis in LX-2 cells in vitro. We found that α-SMA and PLK1 expression levels were enhanced

| PLK1 promotes the activation of LX-2 cells stimulated with TGF-β1
Next, LX-2 cells were transfected with GTP-PLK1 plasmid, and the efficiency of PLK1 overexpression was measured by Western blotting ( Figure S2A). PLK1 overexpression significantly increased LX

| Inhibition of PLK1 facilitates apoptosis of HSCs in liver fibrosis
To verify the effect of PLK1 knockdown on the apoptosis of HSCs, we detected the expression of cleaved caspase-3, Bcl-2 and Bax by Western blot analysis. The results showed that the expression of cleaved caspase-3 was increased and that the Bax/Bcl-2 ratio was enhanced in HSCs from CCl 4 -induced liver fibrosis mice injected with AAV-shPLK1 compared with the AAV-empty group ( Figure 5A). Consistently, the expression of cleaved caspase-3 and the Bax/Bcl-2 ratio was increased compared with the Cas9empty group after the transfection of LX-2 cells with Cas9-gRNA-PLK1 ( Figure 5B). In contrast, we identified that the expression of cleaved caspase-3 and the Bax/Bcl-2 ratio were significantly down-regulated in LX-2 cells following GTP-PLK1 overexpression compared with the GTP-empty group ( Figure 5C). In addition, PLK1 silencing increased the percentage of apoptotic LX-2 cells, whereas overexpression of PLK1 reduced the percentage of apoptotic LX-2 cells by flow cytometry (Figure 5D,E). Taken together, our analyses suggest that inhibiting PLK1 promotes the apoptosis of HSCs by mitochondrial-and caspase-dependent pathways in vivo and in vitro, providing evidence that inhibiting PLK1 ameliorates liver fibrosis.

| PLK1 promotes the proliferation of HSCs in liver fibrosis by regulating the Wnt/β-catenin signalling pathway
Previous studies have demonstrated that the Wnt/β-catenin signalling pathway is involved in HSCs proliferation. The present study questioned whether PKL1 activates the Wnt/β-catenin signalling pathway to regulate HSCs proliferation. We found that  Figure 6E). Additionally, we used a TGF-β1 inhibitor to block TGF-β1 signalling. As shown in Figure S3A,B, the expression of PLK1 was significantly decreased in LX-2 cells treated with TGF-β1 inhibitor. These results indicate that PLK1 promoted the proliferation of HSCs by regulating the Wnt/β-catenin signalling pathway in liver fibrosis in vivo and in vitro (Figure 7).

| D ISCUSS I ON
Liver fibrosis is an important global concern, and there is currently no approved treatment. 22 It is a reversible pathological process in which an imbalance between the synthesis and degradation of the ECM leads to abnormal deposition of fibrous connective tissue in the liver. 23 The activation, proliferation and transformation of HSCs caused by liver injury are important factors in the process of liver fibrosis. 24 However, the molecular mechanisms by which HSCs are activated to induce fibrosis have not been fully elucidated. 25 In our study, we found that increased PLK1 expression was associated with liver fibrosis and generated reliable data to support the pro-fibrosis effect of PLK1. We demonstrated that PLK1 was increased in human liver fibrosis samples and primary HSCs from CCl 4 -induced fibrosis mice using Western blot and real-time PCR, whereas immunofluorescence and IHC assays further showed PLK1 was up-regulated in the cytoplasm. In addition, we demonstrated that the up-regulation of PLK1 in HSCs induced the expression of α-SMA, Col1α1 and TIMP-1 and promoted the activation of HSCs. We also found that the induction of PLK1 expression in HSCs further promoted the proliferation and activation of HSCs by increasing the expression of specific transcription factors associated with the Wnt/β-catenin signalling pathway. In CCl 4 -induced liver fibrosis mice, we found that reduced expression of PLK1 by AAV-shPLK1 significantly decreased liver fibrosis. Our results suggest that PLK1 may be a potential therapeutic target for liver fibrosis.
PLK1 is a highly conserved serine/threonine kinase. 26 Recent studies have revealed that PLK1 has a broad regulatory role in cell mitosis and plays a critical role in cell proliferation. 27,28 It has been confirmed that PLK1 is overexpressed in many different tumour types and thus has a critical effect on tumour development. 12,29 We found that inhibition of PLK1 expression could reduce the ac- We conclude that PLK1 is an important molecule for HSCs activation in liver fibrosis. We identified low expression of PLK1 in liver fibrosis. Down-regulated PLK1 in HSCs further alleviated the expression of proteins associated with HSCs proliferation (c-Myc and Cyclin D1) and enhanced the expression of apoptosis-related proteins (cleaved caspase-3 and Bax/Bcl-2). The current study demonstrates that inhibition of PLK1 expression prevents HSCs activation and proliferation to reduce liver fibrosis through the Wnt/β-catenin signalling pathway. Our present study suggests that PLK1 is an attractive novel therapeutic target for liver fibrosis.

ACK N OWLED G EM ENTS
This work was supported by the National Science Foundation of China (Nos. 81770609).

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
YC and XC conceived and designed the study. YC, XC and SZ performed the experiments. FTB collected and coordinated the data.
YRJ provided the mouse. XSD and YC wrote the manuscript. XMM, CH and JL reviewed and modified the manuscript. All authors read and approved the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are included within the article.