Interleukin‐34 mediated by hepatitis B virus X protein via CCAAT/enhancer‐binding protein α contributes to the proliferation and migration of hepatoma cells

Abstract Objectives Interleukin‐34 (IL‐34) is associated with hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC). However, the role and associated mechanisms of IL‐34 in HBV‐related HCC remain unclear. In this study, the expression, biological function and associated mechanisms of IL‐34 in HBV‐related HCC cells were investigated. Methods IL‐34 expression induced by HBV and HBV X (HBX) gene was measured in hepatoma cells. The role of CCAAT/enhancer‐binding protein α (CEBP/α) in HBX‐induced IL‐34 expression was examined. The signal pathways involved in the expression of CEBP/α and IL‐34 induced by HBX were assessed. The role of IL‐34 in the proliferation and migration of HCC cells, and related mechanisms were explored. Results Dependent on HBX, HBV increased IL‐34 expression in hepatoma cells, and HBX upregulated and interacted with CEBP/α to enhance the activity of IL‐34 promoters. CEBP/α mediated by HBX was associated with the activation of PI3‐K and NF‐κB pathways to promote IL‐34 expression. Via CSF1‐R and CD138, IL‐34 promoted the proliferation and migration of hepatoma cells, and contributed to the activation of ERK and STAT3 pathways and the upregulation of Bcl‐xl and c‐Myc mediated by HBX. Conclusion We demonstrate that IL‐34 contributes to HBX‐mediated functional abnormality of HCC cells and provides a novel insight into the molecular mechanism of carcinogenesis mediated by HBX.


Hepatitis B virus (HBV) is one of the most vital aetiological factors
for the occurrence and progression of hepatocellular carcinoma (HCC). 1,2 However, the molecular mechanisms of hepatocarcinogenesis mediated by the virus are not well clarified. HBV genome contains four open reading frames (ORF): S, P, C and X. S ORF has HBS, preS1 and preS2 genes that encode three viral envelope proteins. P ORF encodes viral polymerase (HBP). The C ORF contains C and precore genes that responsible for the expression of viral core protein (HBC) and HBe protein. X is the smallest ORF that encodes HBV X protein (HBX). Among viral proteins encoded by HBV genome, HBX is considered as a cancer cofactor and modulates tumorigenesis via the regulation of expression and activity of multiple host factors. 1,[3][4][5][6] Especially, current studies indicate that HBX is capable of regulating various cytokines, including IL-6, 7 IL-12 8 and TGF-β, 9 to mediate the proliferation, apoptosis and migration of HBV-related HCC. Further exploring the role and related mechanisms associated with the cytokines mediated by HBX will help us identify new therapeutic targets to improve the outcomes of HCC patients with HBV infection.
Interleukin-34 (IL-34) is a newly identified cytokine from a comprehensive human protein library. 10 Binding to three receptors, including colony-stimulating factor 1 receptor (CSF1-R), CD138 and PTP-ζ, 11 IL-34 could regulate the differentiation and function of various target cells. Until now, collective evidence has demonstrated that IL-34 is involved in the development of viral infection, autoimmune diseases and cancers. 12,13 Importantly, recent studies show that IL-34 is involved in the HBV infection and associated with liver fibrosis. 14,15 Besides, the report from Zhou S et al shows that increased IL-34 is related to the poor survival and tumour recurrence in HCC patients, and modulates the invasion and metastasis of HCC cells via macrophages. 16 However, whether IL-34 contributes to the development of HBV-infected HCC is still unclear.
In this study, we investigated the expression, biological function and associated mechanisms of IL-34 in HBV-related hepatoma cells. We found that, in HBV associated HCC cells, via a transcription factor CCAAT/enhancer-binding protein α (CEBP/α), HBX contributed to the increase of IL-34. In addition, IL-34 mediated by HBX contributes to the proliferation and migration of HCC.
These results could improve our understanding on the underlying mechanism of hepatocarcinogenesis mediated by HBX during HBV infection.

| MATERIAL S AND ME THODS
The source and culture of HepG2, Huh7 and HepG2.2.15 cells were described previously. 17,18 See the Supplementary Information for details regarding reagents, plasmids and clinical samples, and other materials and methods used in the study.

| HBX is responsible for IL-34 expression in HBV-related HCC cells
To investigate whether HBV could promote IL-34 expression in HCC cells, the expression level of IL-34 was measured in HepG2 and HepG2.215 cells (HepG2 cells with HBV genome). Compared with HepG2 cells, the expression of IL-34 was increased in HepG2.215 cells ( Figure 1A). Next, HBV and control plasmids were transfected into HepG2 and Huh7 cells, and we found that HBV could increase IL-34 expression in both two types of hepatoma cells ( Figure 1A). We evaluated serum IL-34 levels in chronic hepatitis B (CHB) patients, HBV-related HCC patients and HBV-negative HCC patients. The results showed that the levels of serum IL-34 were significantly higher in HBV-related HCC patients than those in CHB and HBV-negative HCC patients ( Figure 1B). IL-34 protein expression in HBV-negative HCC, HBV-negative adjacent tissues, HBV-positive tumour tissues and HBV-positive adjacent tissues were examined. Compared with HBV-negative adjacent tissues, HBV-positive adjacent tissues and HBV-negative HCC, the expression of IL-34 was elevated in HBV-related tumour tissues ( Figure 1C).
We next investigated the role of different HBV genes on IL-34 expression, and only HBX was found to significantly increase IL-34 expression in HCC cells ( Figure 1D). In addition, we transfected hepatoma cells with HBV plasmids or HBV plasmids with HBX mutation to further detect the effect of HBX on IL-34 expression in HCC cells with HBV infection. When HBX was deleted in the HBV genome, the expression of IL-34 mediated by the virus was significantly inhibited in HCC cells ( Figure 1E). We transfected HepG2.215 cells with HBX shRNA vectors and found that the knockdown of HBX could attenuate IL-34 expression in HepG2.215 cells ( Figure 1F). We also explore the expression of IL-34 in hepatoma cells stably transfected with HBX or control plasmids. Compared with control cells, IL-34 expression was elevated in HBX stable expressing cells ( Figure 1G). Taken together, these results suggest that, dependent on HBX, HBV could facilitate IL-34 expression in HCC cells.

| HBX activates IL-34 promoter through CEBP/α in hepatoma cells
To elucidate the mechanisms related to HBX-mediated expres- The transcription factor binding sites in IL-34 promoter regions (−745/−491) were predicted using tfsearch and aliBaBa2.0 software. 17 The binding site of CEBP/α was found to locate in the promoter region (−745/−491) of IL-34. CEBP/α is associated with the dysregulation of different genes mediated by HBX. 19,20 We assumed that HBX might activate the promoter of IL-34 via CEBP/α. As expected, we found that the promoter activity of PGL3-P(−745/+155) was inhibited when CEBP/α binding site sequences (−602/−596) were mutated in HCC cells ( Figure 2B Previous studies show that HBX can interact with CEBP/α to regulate the expression of target genes. [19][20][21][22] However, whether the interaction of HBX with CEBP/α mediates the activity of IL-34 promoter is unclear. We measured the interaction of HBX with CEBP/α in HCC cells. The results showed that HBX could bind with CEBP/α in HCC cells using coimmunoprecipitation analysis ( Figure 2G). In addition, the colocalization of HBX and CEBP/α was observed in the cytoplasm and nucleus of HBX-positive cells via immunofluorescence assay ( Figure 2H), indicating that HBX interacted with CEBP/α in these locations. Furthermore, we observed that IL-34 promoter fragment containing CEBP/α binding site could be examined in the anti-HBX immunoprecipitated candidates using ChIP assay, whereas the interaction of IL-34 promoter with HBX was significantly decreased when these cells were transfected with CEBP/α shRNA plasmids ( Figure 2I). Taken together, these results suggest that HBX could bind to the promoter region of IL-34 via the interaction with CEBP/α in hepatoma cells.

| HBX promotes the expression of CEBP/α via different signal pathways to upregulate IL-34 in hepatoma cells
Next, we explored the mechanisms associated with the increase of CEBP/α mediated by HBX to promote IL-34 expression in HCC cells. Current reports showed that CEBP/α expression is mainly mediated by PI3-K, 23 NF-κB, 24 JNK and p38 pathways in different cells. 25 We examined whether HBX was able to promote CEBP/α expression through these pathways. The results showed that HBX could activate PI3-K, NF-κB, JNK and p38 pathways in HCC cells ( Figure 3). When the cells were treated with the inhibitors of PI3-K, NF-κB, JNK and p38 pathways, the activities of AKT (a F I G U R E 2 The effect of CEBP/α in activating the promoter of IL-34 mediated by HBX in HCC cells. A, The identification of the regulatory region of IL-34 promoter mediated by HBX via luciferase reporter gene assays. B, The mutation information of CEBP/α binding site in core region of the IL-34 promoter in PGL3-P (−745/+155) plasmid. C, The influence of the mutation in the CEBP/α binding site on activation of the core region of the IL-34 promoter in PGL3-P (−745/+155) plasmid. D, The expression of CEBP/α protein in the cytoplasm and nucleus of HBX-positive HCC cells. E, The inhibition of CEBP/α using shRNA plasmids on the expression of IL-34 protein. F, The inhibition of CEBP/α using shRNA plasmids on the activity of the core region of the IL-34 promoter in PGL3-P (−745/+155) plasmid. G, The interaction of HBX with CEBP/α using Co-IP assay. H, The collocation of HBX (green) and CEBP/α (red) in HCC cells. The white arrows show the colocalization of HBX and CEBP/α in HCC cells. I, The role of CEBP/α in interaction between HBX and IL-34 promoter as determined by ChIP assay. Mock: cells transfected with the negative control plasmids. HBX: cells transfected with HBX plasmids. CEBP/α-WT: the CEBP/α site in the wild type, the CEBP/α-MUT: the CEBP/α site with a mutation. HBX-shCON: The HBX-positive cells transfected with control plasmids. HBX-shCEBP/α: The HBX-positive cells transfected with shRNA plasmids against CEBP/α. *P < .05

| IL-34 contributes to the proliferation and migration of hepatoma cells mediated by HBX
Our previous studies indicate that HBX could promote the growth and migration of hepatoma cells. 18 In addition, current researches suggest that IL-34 contribute to the proliferation and migration of breast cancer cells. 26

| IL-34 promotes the activation of signal pathways and the expression of associated proteins in HBX-positive HCC cells via CSF1-R and CD138
Three receptors, including CSF1-R, CD138 and PTP-ζ, were found to bind with IL-34. 12, 13 We detected the mRNA and protein expression of these receptors in HBX and HBV-positive cells. The results indicated that HBX and HBV had no effect on CSF1-R. Both HBX and HBV could upregulate CD138 but downregulate PTP-ζ in HCC cells. In addition, the role of HBV in increasing the expression of CD138 but inhibiting PTP-ζ expression was mainly dependent on HBX ( Figure 6). These results suggest that the effect of IL-34 on HCC cells might be dependent on the interaction with CD138 or CSF1-R in HCC cells.
Previous study showed that CD138 could enhance the activation of CSF1-R signal pathways mediated by IL-34, 29

| IL-34 promotes the proliferation and migration of HBX-positive hepatoma cells via CSF1-R and CD138
We next assessed whether IL-34 promotes the proliferation and mi-

| D ISCUSS I ON
IL-34 is considered to be associated with HBV infection and HCC. [14][15][16] However, the role and associated mechanisms of IL-34 in HBV-related HCC are not well clarified. In this study, we found that HBV contribute to the increase of IL-34 via HBX in HCC cells. Furthermore, the role of HBX on IL-34 expression is mainly dependent on CEBP/α, and HBX could activate different signal pathways for the upregu- binding with IL-34. 29 Besides, PI3-K, ERK and STAT3 signal pathways are reported downstream pathways of CSF1-R. 30,31 In addition, cyclinD1, c-Myc and Bcl-xl are the downstream molecules of CSF1-R. 32 In the study, we detected whether IL-34 could activate these signal pathways and associated downstream molecules in HBX-positive cells. Our results suggested that HBX could induce the activity of ERK and STAT3 pathways and promote the expression of c-Myc and Bcl-xl through IL-34. AKT is a downstream factor of CSF1-R. 30

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.