rSjP40 suppresses hepatic stellate cell activation by promoting microRNA‐155 expression and inhibiting STAT5 and FOXO3a expression

Abstract Activation of hepatic stellate cells (HSCs) is the central event of the evolution of hepatic fibrosis. Schistosomiasis is one of the pathogenic factors which could induce hepatic fibrosis. Previous studies have shown that recombinant Schistosoma japonicum egg antigen P40 (rSjP40) can inhibit the activation and proliferation of HSCs. MicroRNA‐155 is one of the multifunctional noncoding RNA, which is involved in a series of important biological processes including cell development, proliferation, differentiation and apoptosis. Here, we try to observe the role of microRNA‐155 in rSjP40‐inhibited HSC activation and explore its potential mechanisms. We found that microRNA‐155 was raised in rSjP40‐treated HSCs, and further studies have shown that rSjP40 enhanced microRNA‐155 expression by inhibiting STAT5 transcription. Up‐regulated microRNA‐155 can down‐regulate the expression of FOXO3a and then participate in rSjP40‐inhibited expression of α‐smooth muscle actin (α‐SMA) and collagen I. Furthermore, we observed microRNA‐155 inhibitor could partially restore the down‐regulation of FOXO3a, α‐SMA and collagen I expression in LX‐2 cells induced by rSjP40. Therefore, our research provides further insight into the mechanism by which rSjP40 could inhibit HSC activation via miR‐155.

of SEA, [8][9][10] our laboratory also focused on the effect of the recombinant SjP40 (rSjP40) on HSC activation. We found that rSjP40 could inhibit α-smooth muscle actin (α-SMA) and collagen I expression in transforming growth factor-β1 (TGF-β1)-treated LX-2 cells. 11 Further study also confirmed that the expression of the p27 promoter was enhanced in HSCs through an E2F1-dependent mechanism. 12 Interestingly, rSjP40 could also promote HSC senescence and cell cycle arrest through the STAT3/p53/p21 pathway. 13 MicroRNAs (miRNAs) are a class of noncoding small RNAs that are prevalent in animals and plants. 14 miRNAs can regulate gene expression at the post-transcriptional level by degrading mRNA or inhibiting mRNA translation. miRNAs are involved in a series of important biological processes, such as cell proliferation, differentiation and apoptosis and play an important role in the physiological and pathological process. 15,16 More and more studies have shown that miRNAs play an indispensable role in the progression of hepatic fibrosis. For example, miR-145 inhibits the activation and proliferation of HSCs through regulating the Wnt/β-catenin signalling pathway. 17 miR-30 exerts its inhibitory effect by inhibiting the TGF-β signalling pathway, 18 and miR-15b and miR-16 are reported to be required for the induction of HSC apoptosis by targeting Bcl-2 through the caspase signalling. 19 As a multifunctional miRNA, miR-155 has been shown to involve in the regulation of various biological processes including infection, atherosclerosis, oncogenesis, inflammation and immunity. [20][21][22][23] Recently, miR-155 has been reported to inhibit the activation of HSCs through the ERK1 pathway in activated HSCs and simultaneously inhibit the progression of epithelialmesenchymal transition (EMT) in cells. 24 However, whether miR-155 plays a role in the inhibition of HSC activation by rSjP40 has not been reported yet. Therefore, in this study, we sought to explore the role of miR-155 in the inhibition of HSC activation by rSjP40 and to explore its underlying molecular mechanisms.

| Cell culture and treatment
LX-2 was a well-characterized human HSCs' line obtained from Nantong Third people's Hospital, and LX-2 cells were cultured in Dulbecco's modified Eagle's Medium (DMEM, Gibco, Thermo Fisher, Waltham, MA, USA) supplemented with 10% of foetal bovine serum (FBS), in a humidified atmosphere containing 5% CO 2 at 37°C. LX-2 cells were seeded into 6-well culture plates and treated with rSjP40 at the concentration of 20 μg/mL. rSjP40 was obtained as previously described. 11 2.2 | Bioinformatics analysis of miR-155 promoter and construction of plasmids containing miR-155 promoter sequence and dual-luciferase reporter assay
Anti-STAT5 antibody was purchased from Cell Signaling Technology (USA) and was used to precipitate the DNA-protein complex. Normal IgG provided in SimpleChIP Kit was used as the negative control.
Purified DNA obtained from SimpleChIP Kit was then used as the template, and PCR was conducted using primers in Table 1, which ZHU ET AL.
| 5487 were designed based on the different STAT5-binding sites in the miR-155 promoter.
Membranes were incubated with specific primary antibodies at 4°C overnight and then incubated with an appropriate second antibody at room temperature. A chemiluminescence (ECL) kit (Merck, Darmstadt, Germany) was used to detect target proteins.
Protein bands were normalized to GAPDH, and protein expression was quantified by Image Lab of Bio-Rad (Berkeley, California, USA).

| Quantitative real-time PCR
The miRNAs were extracted using RNAiso for Small RNA (TAKARA, Kyoto, Japan) and transcribed into cDNA as previously described. 25 cDNA products were worked as the template for All samples were run in triplicate, and the relative expression levels were determined by normalization to U6.

| Statistical analysis
The statistical significance of differences was determined by SPSS software using the methods of independent Student's t test or one-way ANOVA (least significant difference, LSD). All data were harvested from at least three independent experiments and presented as the mean ± SEM. P < 0.05 was considered statistically significant.

| The expression of miR-155 in LX-2 cells is enhanced by rSjP40
To confirm the expression of miR-155 in inhibition of LX-2 activation by rSjP40, RT-qPCR was performed firstly and the results showed that miR-155 expression was up-regulated after LX-2 cells were treated with rSjP40 (20 μg/mL) ( Figure 1). This result is identical to that reported by Dai et al, 24 who reported that the expression of miR-155 was inhibited in TGF-β1-treated primary HSCs and activated HSC-T6 cell line. Hence, we speculated that rSjP40 can inhibit HSC activation by promoting miR-155 expression in LX-2 cells.

Primer
Sequence (5′→3′) Purpose F I G U R E 1 miR-155 expression is up-regulated in LX-2 cells treated with rSjP40. The expression levels of miR-155 in LX-2 cells which were treated with rSjP40 at the concentration of 20 μg/mL for 24 or 48 h were detected by RT-qPCR. *P < 0.05, compared to each untreated group. The data are presented as the mean ± SEM of at least three independent experiments 3.2 | FOXO3a is the potential target gene of miR-

155
Studies have shown that FOXO3a is targeted by miR-155 in renal cell carcinoma 26 and enterocyte. 27 By software prediction, we also found that FOXO3a is a potential target for miR-155 (Figure 2A)

| miR-155 is involved in inhibiting the activation of LX-2 cells by rSjP40 via FOXO3a
Our previous studies show that SEA induces the senescence of activated HSCs through the FOXO3a/SKP2/p27 pathway. 28 As SjP40 is the main antigen of SEA, we further explore the role of miR-155 and FOXO3a in the inhibition process of LX-2 cell activation by rSjP40.
Western blot results showed that FOXO3a protein expression was significantly inhibited in rSjP40-treated LX-2 cells ( Figure 3A), and FOXO3a protein expression was up-regulated in miR-155 inhibitor transfected group, compared with that in NC group ( Figure 2B and 3B). Furthermore, partial increased of FOXO3a protein expression was observed in the groups cotreated with rSjP40 and miR-155 inhibitor, compared to that in the group cotreated with rSjP40 and inhibitor NC ( Figure 3B). These results suggest that miR-155 inhibitors may partially restore rSjP40-induced down-regulation of FOXO3a expression.
As biological markers of hepatic fibrosis, 29 the expression levels of α-SMA and collagen I were detected by Western blot. Compared with the control group, the protein expression of α-SMA and collagen I was significantly down-regulated in rSjP40-treated group (*P < 0.05, Figure 3C). Compared with the data from the group cotreated with rSjP40 and inhibitor NC, the protein expression of α-SMA and collagen I was partially increased in rSjP40 and miR-155 inhibitor cotreated groups ( & P < 0.05, Figure 3C). Hence, these results in Figure 3C demonstrate that miR-155 inhibitor can also partially restore rSjP40-induced down-regulation of α-SMA and collagen I expression. However, although the expression of α-SMA and collagen I was up-regulated slightly in LX-2 cells transfected with miR-155 inhibitor alone, there was no significant statistical difference between miR-155 inhibitor + rSjP40-group and NC inhibitor + rSjP40-group ( NS P > 0.05, Figure 3C). These data above demonstrate that the miR-155 may involve in the inhibition process of HSC activation induced by rSjP40 in LX-2 cells and prevent progression of hepatic fibrosis.

| rSjP40 induces miR-155 expression by promoting its promoter expression in HSCs through a STAT5-dependent mechanism
To explore the mechanism by which rSjP40 promotes miR-155 expression, we constructed a plasmid containing the miR-155 promoter sequence. Compared with the pGL3-basic group, the luciferase activity of the miR-155 promoter group was higher than that of the pGL3-basic group ( Figure 4A). This result confirmed that the  Figure 4D). To verify that STAT5 binds directly to miR-155 promoter, primers (Table 1)  The data are presented as the mean ± SEM of at least three independent experiments. *P < 0.05, compared to untreated group. # P < 0.05, compared to NC inhibitor + rSjP40− group. $ P < 0.05, compared to miR-155 inhibitor + rSjP40− group. & P < 0.05, compared to NC inhibitor + rSjP40 + group. NS represents P > 0.05 and shows that there is no significant statistical difference between the compared two groups by SEA or its component, whether miRNAs can be involved in is an interesting issue to attract us.
Recently, many miRNAs affecting HSC activation have been identified, suggesting that miRNAs play a potential role in the pathogenesis of hepatic fibrosis. [40][41][42] For instance, miR-122, the most abundant miRNA in liver, was down-regulated significantly during hepatic fibrosis. 43,44 It has been demonstrated that miR-29 is negatively correlated with ECM deposition in HSCs and involves an interaction with the PDGF-mediated signalling pathway. 45 Lakner et al 46 showed that high expression of miR-19b inhibits the TGF-β signalling pathway in activated HSCs by decreasing TGF-β receptor II (TGFβRII) expression. In our preliminary studies, we also confirmed some asso- transcription factors BRCA1, p53, p63 and STAT3 can also inhibit miR-155 promoter activity in various cells. [56][57][58] In our study, we also found that transcription factor STAT5 regulates miR-155 transcription by binding to the position of −989 bp to −977 bp F I G U R E 4 rSjP40-mediated enhancement of miR-155 promoter activity is related to STAT5 in LX-2 cells. A, Luciferase activities of pGL3-basic and pGL3-promoter miR-155 in LX-2 cells were determined by dual-luciferase reporter assay. *P < 0.05, compared to pGL3-basic group. B, The effect of rSjP40 on the luciferase activities of pGL3-basic or pGL3-promoter miR-155 in LX-2 cells was determined by dual-luciferase reporter assay. *P < 0.05, compared to each untreated group. C, STAT5 and P-STAT5 protein expression levels in LX-2 cells treated with rSjP40 at the concentration of 20 μg/mL were evaluated by Western blot. *P < 0.05, compared to each untreated group. D, Diagram of STAT5 binding sites in the miR-155 promoter was shown. E, ChIP analysis was performed to confirm the binding of STAT5 to the miR-155 promoter. M, DL2300 from SMOBIO. Lane 1 represents anti-Histone H3 group for positive control group. Lanes 5, 9, 13 and 17 represent anti-STAT5 group for target group. Lanes 2, 6, 10, 14 and 18 represent normal IgG group. Lanes 3, 7, 11, 15 and 19 represent input group. Lanes 4, 8, 12, 16 and 20 represent water group using ddH 2 O as the template for PCR. Primer RPL30 was used for the positive control group in promoter of miR-155 ( Figure 4E) and rSjP40 could inhibit the expression levels of P-STAT5 and STAT5.
In conclusion, our results indicate that rSjP40 enhances the expression of miR-155 by inhibiting the expression of STAT5, thereby inhibiting the expression of FOXO3a and inhibiting the activation of HSCs.