CXCR7 correlates with the differentiation of hepatocellular carcinoma and suppresses HNF4α expression through the ERK pathway

  • Authors:
    • Tong-Chun Xue
    • Qing-An Jia
    • Yang Bu
    • Rong-Xin Chen
    • Jie‑Feng Cui
    • Zhao-You Tang
    • Sheng-Long Ye
  • View Affiliations

  • Published online on: September 19, 2014     https://doi.org/10.3892/or.2014.3501
  • Pages: 2387-2396
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Abstract

Hepatocellular carcinoma (HCC) is a malignancy with dysregulated differentiation. However, effective differentiation therapy for HCC is lacking. Previous evidence suggests that CXCR7 is associated with the differentiation of embryonic stem cells. Here, we evaluated the potential role of CXCR7 in the differentiation of HCC. In HCC cell lines, the expression of cancer stem cell-related markers was assessed by flow cytometry and confirmed by western blot and immunofluorescence analyses. Dimethyl sulfoxide, oncostatin M and dexamethasone were used to induce the differentiation of HCC. Immunohistochemical assay was performed on a tissue microarray based on 112 HCC cases that received hepatectomy. Ligand activation, inhibition assays and RNA interference were used to analyze the regulation of hepatocyte nuclear factor 4α (HNF4α) by the CXCR7 pathway. Huh7 and HCCLM3 cell lines were screened for differentiation induction based on biomarkers of hepatic cancer stem cells. CXCR7 was found to be closely associated with the differentiation of HCC, and an inverse expression trend between CXCR7 and HNF4α was found upon induced differentiation. Clinically, high CXCR7 expression was negatively correlated with HNF4α expression in patients with relatively well-differentiated HCC. Moreover, high CXCR7 expression was correlated with poor overall survival and accelerated post-resection metastasis in HCC with a low HNF4α level. Mechanistically, CXCR7 signaling inhibited HNF4α through extracellular regulated protein kinase (ERK) activation, which was inhibited by U0126, an inhibitor of MAPK/ERK kinases 1 and 2. Knockdown of CXCR7 further confirmed that CXCR7 signaling can regulate HNF4α expression. Taken together, our findings indicate that CXCR7 participates in the differentiation of HCC by regulating HNF4α. The CXCR7-ERK-HNF4α cascade represents a new target for the differentiation therapy of HCC.

Introduction

Hepatocellular carcinoma (HCC) is a malignancy with dysregulated differentiation (1). Differentiation-related genes in HCC have been enumerated; for example, the well-known biomarker α-fetoprotein (AFP), only expressed during early stages of liver development, is always highly expressed in HCC. Differentiation therapy has been quite successful for various malignancies, particularly acute promyelocytic leukemia (2). However, effective differentiation therapy is lacking for HCC (3,4), partly due to the limited knowledge of dysregulated differentiation in HCC (5).

During normal liver development, key transcription factors, including hepatocyte nuclear factor 4α (HNF4α), HNF1α, HNF1β, CCAAT/enhancer binding protein α (C/EBPα) and C/EBPβ, control differentiation (6). HNF4α plays a critical role in hepatocyte differentiation (7) and controls the expression of more than 40% of hepatocyte genes. Recently, the critical roles of HNF4α in the dysregulated differentiation and carcinogenesis in HCC have been identified (8,9). A nude mouse model of HCC showed that a recombinant adenovirus carrying HNF4α potently promoted the differentiation of HCC into normal hepatocytes and suppressed tumorigenesis (4).

CXCR7 was formerly known as RDC1 or orphan receptor since its ligands were unknown. Knowledge concerning CXCR7 increased after the functional ligands SDF-1α (or CXCL12) and ITAC (or CXCL11) were found (10,11). CXCR7 is a G-protein-coupled seven-transmembrane receptor. However, it also mediates β-arrestin-biased signaling (12). Functionally, CXCR7 has been shown to be involved in cardiac development (13). In addition, evidence suggests links between CXCR7 and tumor proliferation or invasion (14). Moreover, our previous research demonstrated the critical role of CXCR7 in HCC (15,16), which is related to differentiation (unpublished data). Additionally, during the differentiation of embryonic stem cells, the epigenetic suppression of CXCR7 by SUZ12 is lost, and the expression of CXCR7 can be increased 20-fold (17), suggesting a critical role for CXCR7 in stem cell differentiation. Therefore, we directed our attention to the potential role of CXCR7 in the dysregulation of the differentiation in HCC.

Here, CXCR7 was identified to be closely associated with the differentiation of HCC, and a close relationship between CXCR7 and HNF4α was found, which was confirmed by the immunohistochemical (IHC) staining of tissue microarrays (TMAs). High CXCR7 levels and low HNF4α levels were correlated with poor survival. Furthermore, ligand activation, inhibition assays, and RNA interference (RNAi) demonstrated that the regulation of HNF4α by CXCR7 was mitogen-activated protein kinase (MAPK)-dependent.

Materials and methods

Reagents and antibodies

Oncostatin M (OSM), SDF-1α and CXCL11 were purchased from Peprotech (Rocky Hill, NJ, USA). U0126 and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Monoclonal antibodies used in the flow cytometric analysis were mouse anti-human monoclonal antibodies (mAbs) CD44-PE, CD133-APC, CD90-PE, CD24-FITC, the IgG-PE isotype, the IgG-APC isotype and the IgG-FITC isotype (all purchased from Miltenyi Biotec, Bergisch Gladbach, Germany). Antibodies used for immunofluorescence, immunoblotting and immunohistochemistry were as follows: mouse anti-human monoclonal CD90 (Abcam, Cambridge, MA, USA), rabbit anti-human polyclonal CD133 (Abnova, Walnut, CA, USA), mouse anti-human monoclonal CD44, rabbit anti-human albumin (ALB), phospho-p44/42 MAPK (Erk1/2), and rabbit anti-human HNF4α mAb (Cell Signaling Technologies, Danvers, MA, USA), rabbit anti-human polyclonal CD24, rabbit anti-human β-actin mAb (Epitomics, Burlingame, CA, USA), rabbit anti-human polyclonal transferrin (TF) (Proteintech Group Inc., Chicago, IL, USA), rabbit anti-human AFP mAb, rabbit anti-human C/EBPα and C/EBPβ mAb, Erk2/p42 MAPK antibody (Epitomics), rabbit anti-human CXCR7 IgG (Novus Biologicals, Littleton, CO, USA), and horseradish peroxidase-conjugated goat anti-rabbit IgG F(ab’)2 antibodies (Jackson ImmunoResearch, West Grove, PA, USA).

Cell lines and culture

Human HCC cell lines with elevated lung metastatic potential (MHCC97L, MHCC97H, and HCCLM3) were established at the Liver Cancer Institute of Fudan University. The human HCC cell lines with low metastatic potential were SMMC-7721 (established at the Second Military Medical University), Huh7 and HepG2 (obtained from the American Type Culture Collection). These cell lines were cultured in high-glucose DMEM (Gibco-BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA).

To induce differentiation, 10 to 40 ng/ml OSM and 1 or 5 μM dexamethasone (Dex) were used. Chemical differentiation inducer DMSO was also used to help establish a model of differentiation induction. For the CXCR7 stimulation assay, 100 ng/ml recombinant human SDF-1α and 200 ng/ml CXCL11 were used. For the inhibition assay, cells were serum-starved for at least 8 h before the MEK1/2 inhibitor U0126 (10 μM) was added.

Flow cytometry

The expression levels of cancer stem cell (CSC)-related markers were determined by flow cytometry. Briefly, tumor cells were grown to 80% confluency. After trypsin digestion, the cells were re-suspended in medium at a concentration of 1×106 cells/ml and incubated with antibodies against CD44, CD133, CD90 and CD24 (diluted 1:11) at 4°C for 15 min. After washing 3 times with PBS, the cells were analyzed using a FACS flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Immunofluorescence

Cell surface expression of CSC-related markers and CXCR7 was also determined by immunofluorescence analysis. Cells were grown on glass coverslips to 40–50% confluency and fixed, permeabilized, blocked, and incubated with primary monoclonal antibodies overnight at 4°C. Slides were washed and incubated with an anti-mouse or an anti-rabbit Cy3-conjugated secondary antibody (Jackson ImmunoResearch). Cells were counterstained with 4–6-diamidino-2-phenylindole to visualize cell nuclei and for inspection by fluorescence microscopy (Olympus).

Western blot analysis

Western blotting was performed according to the wet transfer protocol using the Bio-Rad Transfer Cell System (Bio-Rad, Ontario, Canada). Analyses of protein expression were performed according to the manufacturer’s instructions. Images were examined using Image Lab Software® (Bio-Rad).

RNA interference

Small interfering RNAs (siRNAs; GenePharma Corp., Shanghai, China) demonstrated to be effective for the knockdown of CXCR7 expression (15) were used. siRNA transfection of the cells was performed using the Lipofectamine 2000 protocol (18). A negative control siRNA, 5′-UUC UCC GAA CGU GUC ACG UTT-3′, was also used. A FAM-labeled negative control siRNA was used to monitor transfection efficiency.

Gene microarray

The Oligo GEArray Human Chemokine and Chemokine Receptors Chip (SuperArray Bioscience, Frederick, MD, USA) was used to compare profiles of the HCCLM3, MHCC97-L, and SMMC-7721 cells according to the manufacturer’s protocol. Total-RNA was extracted, isolated, and purified according to the TRIzol reagent (Invitrogen Corp., Carlsbad, CA, USA) protocol. GEArray® Analyzer Software (SuperArray Bioscience) was used for data analysis. The microarray analysis was performed twice with similar results.

Patients and follow-up

Ethical approval was obtained from the Zhongshan Hospital Research Ethics Committee, and informed consent was obtained from each patient. Data from 112 patients were retrieved from the prospectively designed database. These patients underwent hepatectomy by the same surgical team from January 2000 to May 2004. The hepatectomy for HCC was carried out as described previously (19). All patients were classified as Child-Pugh A, and all tumors were identified as HCC by histological analysis.

Regular follow-up procedures in our clinic include the following: AFP assay and liver ultrasonography every 3 months during the first year and every 6 months thereafter; and magnetic resonance imaging or computed tomography scanning after 1 month and every 6 months thereafter. Chest computed tomography scanning is regularly used to identify lung metastasis. Lung metastasis was confirmed by biopsy through endoscopy or histology after partial pulmonary resection. Until May 2009, lung metastasis was found in 56 patients. Ten patients with resectable lung metastases received partial resection of the lung.

TMA and IHC

Hematoxylin and eosin-stained slides were screened for optimal tumor content and tissue adjacent to the tumor (TAT) with a distance of 2 cm. The TMA was constructed in accordance with standard procedures (20) based on 112 tumor tissues and 46 TATs. Two cores were taken from each formalin-fixed, paraffin-embedded HCC sample by using punch cores that measured 1.0 mm in diameter from the center of the tumor foci and TAT. A two-step method of IHC including heat-induced antigen-retrieval procedure was performed as previously described (20). Detection without the primary antibody was considered as the negative control.

Scoring and categorization of CXCR7 expression

CXCR7 expression was defined by staining intensity and the percentage of positive tumor cells as described previously (21). Two pathologists observed the results independently. We simplified the assay results of CXCR7 into CXCR7Low (weak staining) and CXCR7High (strong staining). Similarly, HNF4α expression was categorized as HNF4αLow or HNF4αHigh, except that HNF4αLow included the negatively stained population due to the generally weak staining.

Statistical analysis

When two groups of cells or tissues were compared, analysis was performed with the Student’s t-test. The Pearson χ2 test was used to compare qualitative variables in the clinical pathology analysis. When expected sample values were <5, Fisher’s exact test was used. Spearman’s rank test was used to detect the correlation between variables. Overall survival (OS), time to progression (TTP), and time to extrahepatic metastasis were observed in the survival analysis. OS was calculated from the date of hepatectomy to the date of death regardless of cause. TTP was calculated from the date of hepatic resection to the date of recurrence. Time to lung metastasis was calculated from the date of hepatectomy to the date of lung metastasis with definite clinical diagnosis. The patients lost in the follow-up and the patients who had not achieved the desired results at the end of this study were recognized as censored cases. The Kaplan-Meier method was used to describe the survival curves, and the log-rank test was used to compare survival distributions between groups. The Breslow test was also used when survival curves indicated greater differences during the early follow-up period. All P-values were obtained using two-tailed tests and the statistical significance was set at 0.05. Statistical analyses were carried out by SPSS 18.0 Software (SPSS Inc., Chicago, IL, USA).

Results

Screening of the HCC cell lines for differentiation induction

Due to the highly heterogenetic character of HCC, we first detected HCC-related stem cell biomarkers in four HCC cell lines (HepG2, SMMC-7721, Huh7 and HCCLM3) with different characteristics and backgrounds. Flow cytometric analysis indicated that Huh7 cells had the highest ratios of CD133 (72.6%) and CD24 (98.8%), which was confirmed by western blot and immunofluorescence analyses (Fig. 1). HCCLM3 cells had the highest CD90-positive ratio (3.2%) and a CD44-positive ratio of >90%. Since these CSC-related markers have been linked to HCC progression and dysregulated differentiation potential (2224), Huh7 and HCCLM3 cells were selected for further analysis.

HCC cells differentiate into hepatocyte-like cells upon induced differentiation

The differentiation induction model for HCC was established using potential differentiation inducers. DMSO was used as a chemical inducer of differentiation. OSM, which has the ability to maintain the maturation and differentiation capacity of hepatocytes (25), was also used. Both in the Huh7 and in the HCCLM3 cells, DMSO showed a strong ability to induce HCC differentiation into normal hepatocytes, based on hepatocyte differentiation markers or elevated expression of plasma proteins ALB and TF, and the typical morphological features of differentiated hepatocytes (Fig. 2A and B). The typical morphology of differentiation was maintained from day 2 until late stages of the induction process. Meanwhile, results from HCCLM3 cells revealed that OSM had the ability to induce elevated expression of ALB and TF (Fig. 2C). However, no typical morphological features were noted, which indicated the main role of OSM in maintaining the maturation of cells. Dex was unable to induce differentiation when used alone (data not shown). In addition, AFP was also increased early during induction, which was consistent with previously reported results (26). Next, AFP levels decreased gradually accompanied by the elevated ALB and TF, and the occurrence of hepatocyte-like morphology.

CXCR7 is inversely correlated with HNF4α upon induced differentiation

Based on the model of HCC differentiation in vitro, we further observed CXCR7 levels following induced differentiation by 1% DMSO and 40 ng/ml OSM. After DMSO treatment, CXCR7 expression was increased early and was decreased during the late stages of differentiation, which showed an inverse expression trend to HNF4α, which has a critical role in liver-specific gene expression. As shown in Fig. 3A, during early stages of induced differentiation, CXCR7 was elevated and the HNF4α level was decreased in both Huh7 and HCCLM3 cells. Whereas during the late stage, CXCR7 levels were decreased and HNF4α levels were elevated. In addition, CXCR7 levels were elevated while the opposite trend was observed for HNF4α during the early stage of differentiation in response to 40 ng/ml OSM treatment (Fig. 3B).

High expression of CXCR7 is correlated with decreased HNF4α expression

Furthermore, IHC analysis of the TMA was performed to confirm the relationship between CXCR7 and HNF4α. Immunopositivity for CXCR7 was mainly observed at the membrane or in the cytoplasm of the HCC cells, whereas for HNF4α it was expressed mainly in the nucleus. The expression intensity and localization of CXCR7 and HNF4α are shown in Fig. 4A and B, respectively. Immunostaining of normal hepatocytes indicated the specificity and selectivity of anti-HNF4α. Stratum analysis based on cell differentiation (Edmondson grade 1/2; n=57) indicated that CXCR7 was negatively correlated with HNF4α expression (Spearman’s rho=−0.296; P=0.025). Among the tumors with low HNF4α expression (n=48), 62.5% were CXCR7High, whereas only 22.2% of the tumors with high HNF4α expression (n=9) were CXCR7High. Furthermore, immunostaining analyses indicated that the distribution and expression levels of CXCR7 were inversely associated with those of HNF4α on a relatively well-differentiated background (Fig. 4C). In addition, CXCR7HighHNF4αLow tumors tended to be larger (Fisher’s exact test, P=0.002), suggesting a relationship between differentiation and proliferation of HCC (Table I).

Table I

Clinicopathological factors and combined CXCR7 and HNF4α expression.

Table I

Clinicopathological factors and combined CXCR7 and HNF4α expression.

No. of patients

Clinicopathological characteristics CXCR7High/HNF4αLow (n=30) CXCR7Low/HNF4αHigh (n=7)P-value
Age, (years)1.000
 ≤60246
 >6061
Gender0.570
 Male267
 Female40
HBsAg0.571
 Negative60
 Positive247
Cirrhosis0.327
 Absent244
 Present63
AFP, μg/l0.360
 ≤2073
 >20234
Tumor size (cm)0.002
 ≤566
 >5241
No. of tumor nodules0.213
 Single213
 Multiple94
Tumor encapsulation0.217
 Well encapsulated176
 Poorly encapsulated131
Microvascular invasion0.674
 Negative165
 Positive142
Portal lymphatic status1.000
 No287
 Yes20

[i] Fisher’s exact test was used in these comparisons.

High expression of CXCR7 predicts poor prognosis when combined with HNF4α

There was a statistically significant difference in OS between the CXCR7High and CXCR7Low groups on the HNF4αLow background (log-rank test; P=0.032). In particular, there was a strong difference during the early stages of follow-up (Breslow test; P=0.007; Fig. 5A). The median OS of CXCR7High patients was much shorter than that of CXCR7Low patients (36 vs. 81.2 months). Similarly, on the HNF4αLow background, the TTP of the CXCR7High patients was significantly shorter than that for the CXCR7Low patients (log-rank test; P=0.038), particularly during the early stages of follow-up (Breslow test; P=0.008; Fig. 5B). In addition, the time that relapsed before observing lung metastasis was shorter for CXCR7High patients than for CXCR7Low patients (log-rank test; P=0.007) (Fig. 5C). In addition, in patients with high HNF4α expression in tumors, there was no significant difference between CXCR7High patients and CXCR7Low patients (data not shown).

Since there was a greater correlation between CXCR7 and HNF4α in relatively well-differentiated HCC, the prognostic value of CXCR7 was further evaluated in patients with Edmondson grade 1/2 HCC (n=48) (Fig. 5D and E). We found that CXCR7 expression had a strong prognostic value in the time-to-metastasis analysis (log-rank test; P=0.004; Fig. 5F).

Activated CXCR7 suppresses HNF4α through ERK-dependent signaling

Since CXCR7 is an atypical G-protein-coupled receptor that has been reported to mediate extracellular regulated protein kinase (ERK) signaling, we explored the possibility of a CXCR7-ERK-HNF4α pathway. Gene chip analysis indicated that HCCLM3 cells highly expressed CXCR7. CXCR4 and CXCR3, however, were nearly undetectable (Fig. 6). Therefore, we utilized HCCLM3 cells as the CXCR7+CXCR4CXCR3 model to observe CXCR7 signaling stimulated by SDF-1α and CXCL11. Phosphorylation of MAPKs ERK1/2 was decreased at 1, 8 or 16 h, and was accompanied by increased HNF4α expression (Fig. 7A), which indicated suppression of the ERK pathway by HNF4α. Then, following stimulation with SDF-1α or CXCL11, elevated phosphorylation of ERK1/2 was observed (Fig. 7B). Meanwhile, western blotting indicated elevated HNF4α levels after exposure to U0126 (Fig. 7C), which indicated that the use of U0126 abrogated the inhibitory effect of CXCR7 ligands on HNF4α expression. Furthermore, after downregulating CXCR7 in the HCCLM3 cells, HNF4α was increased in all positive siRNA groups (Fig. 7D). However, another hepatic-enriched nuclear factor C/EBPα was not obviously affected. Another cell line, MHCC97H, with the same genetic background as HCCLM3 and also expressing high levels of CXCR7 and extremely low levels of CXCR4 and CXCR3 was used to confirm these results. The results with this cell line were similar to those using the HCCLM3 cell line.

Discussion

As a chemokine receptor, CXCR7 plays roles in crest cell movement in development. Similarly, the role of CXCR7 in invasion and metastasis has been demonstrated in tumors. However, as an atypical G-protein-coupled receptor, other functions of CXCR7 have just begun to be unveiled. Evidence suggests the potential role of CXCR7 during the differentiation of embryonic stem cells (17). In the present study, the close correlation between CXCR7 and differentiation of HCC was found, including its opposite expression pattern with HNF4α upon induced differentiation. Moreover, the relationship between CXCR7 and HNF4α was confirmed by histological analysis of HCC samples after hepatic resection. Furthermore, high CXCR7 expression levels were closely correlated with poor survival and extrahepatic metastasis in tumors with low HNF4α expression. These findings strongly suggest a critical role for CXCR7 in the differentiation of HCC.

Differentiation therapy is a useful therapeutic strategy for malignancy. However, effective differentiation therapy is lacking for HCC. In this study, a new pathway mediated by CXCR7 in de-differentiated HCC was demonstrated, as summarized in Fig. 7E. Although the screened HCC cell lines have distinct genetic backgrounds and different CSC-like biomarkers, the typical differentiation phenotype can be observed in each, indicating that the CXCR7-MAPK-HNF4α cascade is the general pathway in the differentiation of HCC. In addition, adenoviral targeting of HNF4α has achieved success in mouse models of HCC (4). Therefore, the CXCR7-MAPK-HNF4α pathway discovered in this study represents a promising target for differentiation therapy of HCC.

In this study, the suppression of HNF4α by CXCR7 signaling was ERK-dependent, which was inhibited by a specific MEK inhibitor. Similar to our finding, the MAPK pathway has been found to control HNF4α directly in hepatoma cells (27). Moreover, mucroporin-M1 was found to selectively activate MAPK signaling and lead to the down-regulation of HNF4α expression (28). On the other hand, as an atypical G-protein-coupled receptor, CXCR7 has been shown to mediate β-arrestin-biased signaling. As a polyfunctional adaptor molecule, β-arrestin can mediate multiple downstream pathways (29). Functionally, CXCR7 has been shown to activate MAPK through β-arrestin. Meanwhile, it is well known that the MAPK pathway is one of the main pathways to control differentiation (30). Therefore, β-arrestin may participate in the CXCR7-ERK-HNF4α signaling cascade, which warrants further study.

In addition to our finding that CXCR7 controlled HNF4α expression, HNF4α can also be modified at the post-translational level (31). Additionally, cyclin D1 inhibits the function of HNF4α (32), whereas GSK3β promotes the function of HNF4α (33). In addition, TGFβ has been found to impair the efficiency of HNF4α through GSK3β inactivation (34). Recently, the Wnt/β-catenin pathway was reported to converge with HNF4α-driven transcription in liver zone specification (35). However, unlike these previous reports, our findings clearly suggest a signaling pathway involved in the differentiation of HCC.

The best proof of principle for differentiation therapy has been the treatment of acute promyelocytic leukemia with all-trans-retinoic acid. However, retinoic acid has a very limited role in HCC differentiation, which suggests that HCC has its own differentiation features. The HCC cell lines used in the present study have distinct genetic backgrounds and different CSC-like biomarkers. However, the selected HCC cell lines can be induced successfully to differentiate into hepatocyte-like cells, which suggests the existence of CSC-like cells or progenitor-like cells in HCC tumor cells and the possibility for differentiation therapy. In addition, upregulation of AFP and downregulation of HNF4α were observed during the early stages of induced differentiation, similar to a report concerning the differentiation of hepatocytes from embryonic stem cells (26). These findings suggest that HCC cells reside in different de-differentiated stages. Additionally, BMP4 administration has been found to induce the differentiation of CD133+ hepatic CSCs and block their contributions to HCC. Therefore, differentiation therapy based on hepatic CSCs may be a promising strategy.

In conclusion, our data indicate that the transmembrane receptor CXCR7 is closely associated with the differentiation of HCC. Activated CXCR7 signaling can suppress the key transcriptional factor HNF4α through ERK activation, implicating the CXCR7-ERK-HNF4α cascade as a potential target for HCC differentiation therapy.

Acknowledgements

This study was supported by the State Key Project on Infectious Diseases of China (no. 2012ZX10002-016) and the Youth Backbone Fund from Fudan University (B-233).

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December-2014
Volume 32 Issue 6

Print ISSN: 1021-335X
Online ISSN:1791-2431

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Spandidos Publications style
Xue T, Jia Q, Bu Y, Chen R, Cui JF, Tang Z and Ye S: CXCR7 correlates with the differentiation of hepatocellular carcinoma and suppresses HNF4α expression through the ERK pathway. Oncol Rep 32: 2387-2396, 2014
APA
Xue, T., Jia, Q., Bu, Y., Chen, R., Cui, J., Tang, Z., & Ye, S. (2014). CXCR7 correlates with the differentiation of hepatocellular carcinoma and suppresses HNF4α expression through the ERK pathway. Oncology Reports, 32, 2387-2396. https://doi.org/10.3892/or.2014.3501
MLA
Xue, T., Jia, Q., Bu, Y., Chen, R., Cui, J., Tang, Z., Ye, S."CXCR7 correlates with the differentiation of hepatocellular carcinoma and suppresses HNF4α expression through the ERK pathway". Oncology Reports 32.6 (2014): 2387-2396.
Chicago
Xue, T., Jia, Q., Bu, Y., Chen, R., Cui, J., Tang, Z., Ye, S."CXCR7 correlates with the differentiation of hepatocellular carcinoma and suppresses HNF4α expression through the ERK pathway". Oncology Reports 32, no. 6 (2014): 2387-2396. https://doi.org/10.3892/or.2014.3501