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HOXD9 promotes epithelial–mesenchymal transition and cancer metastasis by ZEB1 regulation in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a common malignant tumor that severely threatens human health. The poor prognosis of HCC is mainly attributed to intrahepatic and extrahepatic metastases. HOXD9 proteins belong to a superfamily that regulates the development and control of many cellular processes, including proliferation, apoptosis, cell shape, and cell migration. HOXD9 can also function as an oncogene in several cancer cells. However, its biological function in human HCC requires further investigation. In this study, HOXD9 exhibited high expression in invasive HCC cells. HOXD9 overexpression can significantly enhance HCC cell migration, invasion, and metastasis, whereas silencing HOXD9 inhibits these processes. HOXD9 also promotes the epithelial–mesenchymal transition (EMT) of HCC cells. Microarray analysis suggests that ZEB1 can function as a downstream factor of HOXD9. HOXD9 can interact with the promoter region of ZEB1 and promotes ZEB1 expression. ZEB1 knockdown inhibits HOXD9-induced migration and invasion, as well as EMT in HCC cells. This study helps elucidates the oncogenic functions of HOXD9 in HCC. Electronic supplementary material The online version of this article (doi:10.1186/s13046-015-0245-3) contains supplementary material, which is available to authorized users.


Background
Hepatocellular carcinoma (HCC) is the sixth most prevalent cancer and the third leading cause of cancerrelated mortality worldwide [1]. The poor prognosis of this disease is attributed to tumor metastasis, frequent intrahepatic spread, and extrahepatic metastasis during the initial diagnosis [1].
Homeobox (HOX) genes encode a highly conserved family of transcription factors that significantly influence many cellular processes, including proliferation, apoptosis, cell shape, and cell migration. HOX genes contain a conserved 183 bp sequence and encode nuclear proteins called homeoproteins. HOX proteins provide an axial pattern during embryonic development [2][3][4], and HOX genes can also control normal cellular proliferation and differentiation in different tissues [5][6][7]. In neoplasms and cancer cells, HOX proteins participate in proliferation and oncogenic transformation [7]. HOX gene expression has been altered in many cancer tissues, such as liver carcinoma, neuroblastoma, ovarian carcinoma, cervical carcinoma, prostate carcinoma, breast carcinoma, and leukemia [8]. The HOX family has four HOX gene clusters: HOXA, HOXB, HOXC, and HOXD. These clusters are located on different chromosomes in tandem. HOXD9 is one of the HOXD genes located closest to the 3′ end of the chromosome. This gene participates in the development and patterning of the forelimb and axial skeleton [8]. Studies have demonstrated the relation of HOXD9 to epigenetic control in development and diseases [9]. The DNA methylation of the HOXD9 gene promoter affects transcript and protein expression and is significantly higher in melanoma brain metastasis than that in the early stages [10]. HOXD9 is important for embryonic segmentation and limb bud patterning during development; however, the biological function in human HCC remains poorly understood.
Epithelial-mesenchymal transition (EMT) refers to specific morphological and phenotypic alterations in epithelial cells during embryonic development [11]. In the EMT process, the polarity and adhesion of cells surrounding the epithelial cells and matrix is reduced and becomes similar in morphology to fibroblasts; thus, this process enhances migratory ability [12]. The occurrence of EMT is accompanied by different expression-specific epithelial and mesenchymal molecular markers [13]. In EMT, epithelial cell markers such as E-cadherin, αcatenin, β-catenin, and γ-catenin are downregulated, whereas the expression levels of mesenchymal tissue markers such as N-cadherin, α-smooth muscle actin, vimentin, and fibronectin protein are upregulated [12]. EMT is closely associated with tumor progression and resistance to chemotherapy [14,15]. This finding has drawn the attention of academics, clinicians, and pharmaceutical researchers to EMT.
Primary liver cancer is a common malignant tumor and severely threatens human health. The poor prognosis of this type of cancer is mainly attributed to intrahepatic and extrahepatic metastases [16]. A study has shown that EMT increases the invasive ability of cancer cells, thus inducing distant metastasis [17,18]. Exploring the molecular mechanism of EMT can greatly contribute to the understanding of tumor progression and metastasis.
This study demonstrates that HOXD9 overexpression can significantly enhance the migration, invasion, and metastasis of HCC cells. HOXD9 also promotes the EMT of HCC cells. Microarray analysis indicates that HOXD9 performs its functions by regulating ZEB1. ZEB1 knockdown inhibits HOXD9-induced migration and invasion and the EMT of HCC cells. The results of this study can elucidate the oncogenic functions of HOXD9 in liver cancers.

Patients and specimens
One hundred two tumor tissues which, were used for qRT-PCR and immunohistochemical analysis, were randomly collected from HCC patients who underwent curative resection with informed consent between 2007 and 2010 at the First Affiliated Hospital of Dalian Medical University. All tissues were collected immediately upon resection of the tumors in the operation theater, transported in liquid nitrogen, and then stored at −80°C. Tumor staging was based on the 6th edition of the tumornode-metastasis (TNM) classification of the International Union Against Cancer. The clinicopathologic characteristics of the hepatocellular carcinoma tissues are summarized in Additional file 1: Table S1. Follow-up data were summarized at the end of May 2015, with a median observation time of 65.3 months. Study protocols were approved by the First Affiliated Hospital of Dalian Medical University, and written informed consent was obtained from patients based on the Declaration of Helsinki.

Histological and immunohistochemical analysis
The normal human liver tissues, human tumor tissues, and lungs dissected from mice were fixed in 4 % paraformaldehyde in phosphate-buffered saline (PBS) overnight and subsequently embedded in paraffin wax. Sections cut at a thickness of 4 μm were stained with hematoxylin and eosin for histological analysis. Immunohistochemical analysis was performed for different markers in these arrays as described previously [19]. The proportion of stained cells (lower, <30 % staining; higher, ≥30 % staining) was semiquantitatively determined following published protocols [20].

Establishment of stable expression and knockdown cell lines
Retroviral construct containing human pBabe-HOXD9 cDNA and pSuper.retro.puro with shRNA against human HOXD9 and siZEB1s were prepared as described previously [20]. The generation of retrovirus supernatants and transfection of hepatocellular carcinoma cells were conducted as described previously [21]. The expression of HOXD9 and ZEB1 was confirmed by qRT-PCR and Western blotting analysis.

Cell invasion and motility assay
Invasion of cells was measured in Matrigel (BD, Franklin Lakes, NJ, USA) -coated Transwell inserts (6.5 mm, Costar, Manassas, VA, USA) containing polycarbonate filters with 8-μm pores as detailed previously [22]. The inserts were coated with 50 μl of 1 mg/ml Matrigel matrix according to the manufacturer's recommendations. 2 × 10 5 cells in 200 μl of serum-free medium were plated in the upper chamber, whereas 600 μl of medium with 10 % fatal bovine serum were added to lower well. After 24 h incubation, cells that migrated to the lower surface of the membrane were fixed and stained. For each membrane, five random fields were counted at × 10 magnification. Motility assays were similar to Matrigel invasion assay except that the Transwell insert was not coated with Matrigel.

Confocal immunofluorescence microscopy
Cell lines were plated on culture slides (Costar, Manassas, VA, USA). After 24 h, the cells were rinsed with PBS and fixed with 4 % paraformaldehyde, and cell membrane was permeabilized using 0.5 % Triton X-100. These cells were then blocked for 30 min in 10 % BSA and then incubated with primary antibodies overnight at 4°C. After three washes in PBS, the slides were incubated for 1 h in the dark with FITC-conjugated secondary antibodies (Invitrogen, Grand Island, NY, USA). After three further washes, the slides were stained with DAPI for 5 min to visualize the nuclei, and examined using a Carl Zeiss confocal imaging system (LSM 780) (Carl Zeiss, Jena, Germany).

Western blotting
Cells were lysed in lysis buffer and total protein contents were determined by the Bradford method. 30 μg of lysis were separated by reducing SDS-PAGE and probed with specific antibodies. Blots were washed and probed with respective secondary peroxidase-conjugated antibodies, and the bands visualized by chemoluminescence (Amersham Biosciences).

qRT-PCR
Total RNA was extracted using Trizol reagent and cDNA was synthesized using SuperScript II Reverse Transcriptase (Invitrogen). qRT-PCR and data collection were performed with an ABI PRISM 7900HT sequence detection system. The primers used for the amplification of the indicated genes are available upon request.

Gene expression profiling
Total RNA quality and quantity were determined using Agilent 2100 Bioanalyzer and NanoDrop ND-1000. Affymetrix HU U133 plus 2.0 arrays were used according to manufacturer's protocol. The data were initially normalized by robust multiarray average (RMA) normalization algorithms in expression console software (Affymetrix). Significantly altered genes between HOXD9 knockdown and its control cells were considered by scatter plots and the genes up-and down-regulated ≥5-fold. Clustering analysis was done using gene list by Gene Cluster v3.0 software, and heat maps were visualized using Java Tree-View v1.1.4r3 software. Gene set enrichment analysis was carried out using ConceptGen. Gene sets were either obtained from the ConceptGen or from published gene signatures.

Chromatin immunoprecipitation (ChIP)-qPCR
Chromatin Immunoprecipitation kit (Cat. 17-371) was purchased from Millipore and ChIP experiments were carried out essentially as described [22]. Immnuoprecipitated DNA was analyzed on the ABI PRISM 7900HT sequence detection system. The primers used for detection of promoters after ChIP are available upon request.

In vivo tumor metastasis
Nude mice were purchased from the Shanghai Slac Laboratory Animal Co. Ltd and maintained in microisolator cages. All animals were used in accordance with institutional guidelines and the current experiments were approved by the Use Committee for Animal Care. For metastasis assays, cells were resuspended in PBS at a concentration of 1×10 7 cells ml −1 . Cell suspension (0.1 ml) was injected into tail veins of nude mice. All of the mice were killed by CO 2 60 days after inoculation.

Statistical analysis
Data was described as the mean ± s.d.. Association between ZEB1 and HOXD9 expression in HCC tissues was assessed using Spearman's rank correlation test. Comparisons between different groups were undertaken using the Student's two-tailed t-test or ANOVA test. The survival probability was estimated by Kaplan-Meier method, and the comparison of survival curves between groups was done with the log-rank test. The statistical significance of the differences between mean values was determined by P < 0.05. Statistical analysis was done with SPSS/ Win11.0 software (SPSS, Inc., Chicago, Illinois, USA).

HOXD9 is highly expressed in malignant cancers
Compared with normal liver cells, HOXD9 showed a significantly high expression of liver cancer cells, particularly in invasive cancer cells (Fig. 1a, b, and c). In liver cancer cells, the protein level of HOXD9 was thrice higher than that of normal liver cells. The protein level of HOXD9 in invasive cancer cells is approximately 1.5 times higher than in noninvasive cancer cells. The mRNA level of HOXD9 was also determined, and the results were consistent with those described above (Fig. 1c). To further examine the relationship between HOXD9 and distant metastasis, liver cancer tissues were analyzed. HOXD9 expression was significantly correlated with distant metastasis (Additional file 2: Figure S1 and D). Immunohistochemistry reveals that 97 % of tumor tissues were moderately or strongly HOXD9 positive ( Fig. 2a and b). By contrast, 92 % of noncancerous tissues are HOXD9 negative (Fig. 2a and c). The survival curves in Fig. 2d indicates that patients with higher HOXD9 level in liver cancer tissues had shorter longevity than those with lower HOXD9 expression. This finding suggests a negative correlation between HOXD9 and long survival.

HOXD9 promotes migration and invasion of liver cancer cells
The silencing and stable overexpression of HOXD9 in liver cancer cells were retrovirally established by using MHCC97H and Huh7 cell lines, respectively, and designated as MHCC97H-shHOXD9 #1, #2, and #3 and Huh7-HOXD9. The levels of HOXD9 in these cell lines were verified on protein and mRNA levels ( Fig. 3a and  b; Fig. 4a and b). Transwell and Matrigel assays were performed to evaluate the effects of HOXD9 on migration and invasion in liver cells. Silencing HOXD9 can significantly reduce the number of cells migrating through the membrane to the bottom of the aperture. Statistical analysis indicated that only about half of the cells with HOXD9 silencing migrate through the membrane (Fig. 3c). By contrast, the number of cells with high HOXD9 expression was five times higher than the cells with an empty vector (Fig. 4c). Matrigel assay was used to evaluate the invasive potential of liver cancer cells with altered HOXD9 expression. The number of invaded liver cancer cells was significantly reduced by silencing HOXD9,whereas that in the cell line with high HOXD9 expression increased (Figs. 3c and 4c). These results indicate that HOXD9 promotes the migration and invasion of liver cancer cells.
To detect the function of HOXD9 in distant metastasis in vivo, MHCC97H-shHOXD9#3, Huh7-HOXD9, and their corresponding control cells were injected into nude mice by the tail vein. Silencing HOXD9 significantly decreases the number of mice with distant metastasis (Fig. 3d). Furthermore, a low number of metastasis foci in liver ( Fig. 3e and f ) were counted in each mouse injected with liver cancer cells silencing HOXD9. By contrast, HOXD9 expression increases the number of metastatic mice (Fig. 4d) and metastatic foci in liver ( Fig. 4e and f ).

HOXD9 regulates the transition between epithelial and mesenchymal phenotypes in liver cancer cells
The expression levels of EMT protein markers were evaluated to determine the relationship between HOXD9 and EMT. In the HOXD9 silencing cell lines, epithelial cell markers (E-cadherin and α-catenin) were upregulated, whereas mesenchymal cell markers (N-cadherin, fibronection, and vimentin) were downregulated, as determined by Western blot analysis (Fig. 5a) and qRT-PCR (Fig. 5b) assay. Furthermore, HOXD9 overexpression significantly increases the mesenchymal cell markers and decreases the epithelial cell markers (Fig. 5c and d). The same results were obtained by immunofluorescence analysis (Additional file 3: Figure S2a and b). The mRNA of the EMT markers exhibited a trend consistent with the results mentioned above. These results indicate that HOXD9 plays important roles in EMT.

HOXD9 can regulate ZEB1 expression
To elucidate the mechanisms in which HOXD9 is engaged in the development and progression of liver cancer cells, microarray assay was performed by using cell line MHCC97H-shHOXD9 #3 and its control cells with an empty vector. Microarray results reveal that a list of genes is significantly differentially expressed after HOXD9 silencing (Fig. 6a). Gene set enrichment analysis indicates that ZEB1 related gene signatures are significantly enriched in HOXD9 knockdown cells (Fig. 6b). This result supports the hypothesis that HOXD9 regulates EMT and that cancer invasion and metastasis may be mediated by ZEB1.
To further verify the relationship between HOXD9 and ZEB1, we evaluated the ZEB1 expression in liver cancer cell lines constructed above by using Western blot analysis and qRT-PCR. In cell lines with HOXD9 silencing, ZEB1 expression decreases significantly compared with the control vector cells (Fig. 7a and  b). HOXD9 overexpression can increase the ZEB expression in protein and mRNA (Fig. 7c and d). We analyze the ZEB1 mRNA expression in the same human HCC tissues to determine any clinical correlation between HOXD9 and ZEB1. A highly positive correlation between the HOXD9 and ZEB1 expression levels is indicated similar to HOXD9 expression (Fig. 7e). This result verifies the results of in vitro and in vivo analyses.
We used a luciferase reporter controlled by the ZEB1 promoter to determine whether the binding of HOXD9 to ZEB1 influences the transcriptional activity of the gene. This reporter was inactivated in MHCC97H-shHOXD9 #3 cells but activated in Huh7-HOXD9 cells, thus reflecting the endogenous activation of ZEB1 expression ( Fig. 8a  and b). Quantitative chromatin immunoprecipitation (qChIP) assays were performed in MHCC97H-shHOXD9, Huh7-HOXD9, and their control cells to detect whether HOXD9 regulates ZEB1 on a transcriptional level. Antibodies against HOXD9 and IgG were used to pull down the chromatin complex, and three pairs of primers (#1, #2, and #3) against the ZEB1 gene promoter region were used to assess the occupancy of the ZEB1 gene promoter (Fig. 8c). Silencing HOXD9 expression was associated with decreased levels at #1 and #3 of the ZEB1 gene Fig. 6 HOXD9 regulates ZEB1 expression. a Clustering of genes differentially expressed after HOXD9 silencing. b Enrichment scores of differential gene expression in the HOXD9-silencing cell line promoter region in MHCC97H-shHOXD9s cells (Fig. 8d  and e). A greater occupancy of the ZEB1 gene promoter regions by HOXD9 was detected in Huh7-HOXD9 cells ( Fig. 8f and g). These results clearly indicate that HOXD9 induces the transcriptional activation of ZEB1 expression by increasing HOXD9 to the ZEB1 gene promoter in HCC cells.

ZEB1 mediates HOXD9-induced migration, invasion, and EMT in HCC cells
To evaluate whether the HOXD9-induced metastatic capacity was mediated by ZEB1, siRNAs were used to silence the ZEB1 gene expression by virally transfecting Huh7-HOXD9 cells with three distinct ZEB1 siRNAs. Three cell lines with ZEB1 silencing were verified by Fig. 7 ZEB1 expression is affected by HOXD9. a and b The ZEB1 expression levels in the HOXD9 silencing cell line were assayed by Western blot analysis (a) and qRT-PCR (b). c and d Detecting ZEB1 expression in HOXD9 overexpression cell line by Western blot analysis (c) and qRT-PCR (d). e ZEB1 expression was positively correlated with HOXD9 expression in liver cancer tissue arrays. P < 0.01 is based on the Student's ttest. Error bars, SD Western blot analysis and qRT-PCR ( Fig. 9a and b). The knockdown of ZEB1 decreased the migration and invasion capacity of Huh7-HOXD9 cells (Fig. 9c). Epithelial markers, E-cadherin, and α-catenin increase, whereas mesenchymal markers, N-cadherin, vimentin, and fibronectin decrease (Fig. 9d). These results indicate the occurrence of EMT. Overall, these results show that ZEB1 mediates HOXD9-induced EMT, migration, and invasion in HCC cells.

Discussion
To our knowledge, this is the first study to show that HOXD9 plays a functional role in liver cancer EMT and distant metastasis. Silencing HOXD9 in liver cancer cells Fig. 8 HOXD9 binding to the promoter of ZEB1 were assayed by ChIP-qPCR. a Effect of silencing HOXD9 expression on the activity of the ZEB1 promoter in MHCC97H and its control cells. b Effect of ectopic HOXD9 expression on the activity of the ZEB1 promoter in Huh7 and its control cells. Data are presented as the ratio of firefly luciferase activity to sea pansy luciferase activity. pGL3 was used as a negative control for background. c Schematic of the ZEB1 promoter region. d and f qChIP was performed in HOXD9 silencing (d) and overexpression (f) cell lines. e and g IgG was used as a negative control. P < 0.01 is based on Student's t-test. Error bars, SD inhibits EMT, migration, invasion, and EMT in vitro and decreases the tumorigenic and metastatic capacities in vivo. By contrast, HOXD9 overexpression reverses these events in otherwise aggressive and invasive HCC breast cells. Microarray data show that HOXD9 influences ZEN1 expression in HCC cells. Silencing ZEB1 in HOXD9 overexpression cell lines obtains results that are similar to those caused by HOXD9 knockdown.
Our results suggest that HOXD9 expression in HCC cells was higher than that in normal cells. HOXD9 expression in glioma cancer stem cells has also been shown to be higher than that in normal astrocytes and NSPCs [8]. In this respect, HOXD9 may function as an oncogene in cells. The expression disorder of genes belonging to the HOX family in cancer tissues has been reported [23,24]. A non-coding RNA residing in the HOXC locus can act in translation to regulate the transcription of the HOXD locus with polycomb-repressive complex 2 [25]. The downregulation of non-coding RNA expression and the changes in the methylation status of the promoters may be related to the mechanism of HOX gene misexpression in cancer cells [8]. HOXB9 has been found to regulate the tumor growth factor in HCC and the metastatic behavior of HCC cells [26]. The mechanism demonstrates the significance of the TGF-β1 pathway in HOXB9-induced EMT in HCC cells [27]. The effect on cell migration, invasion, and metastasis also indicates that HOXD9 functions as an oncogene in cancer cells. Silencing HOXD9 can significantly reduce the migration and invasion of cancer cells in vitro. The process also weakens the metastatic ability of cancers in vivo. HOXD9 overexpression confers the opposite action on the cell. Our study determines a novel function of HOXD9 in HCC cell metastasis by regulating EMT.
EMT is an important cellular process in embryonic development, tissue repair, and disease occurrence. EMT was first introduced in the 1980s as a cellular phenomenon in the primitive streak of chick embryos; this process controls many developmental processes, such as neural crest development and palate and lip fusion [28]. EMT is a developmental programming mechanism. EMT also significantly influences cancer progression and confers certain fundamental abilities to cancer cells that are essential for distant metastasis [29,30]. The precise contribution of EMT to cancer metastasis remains unclear. One argument is that EMT simply reflects genomic instability in cancer cells. Reports have also suggested that EMT-driven cancer metastasis can be induced by generating cancer stem cells that are capable of colonizing other tissues to form secondary tumors [11,31].
The occurrence of EMT is accompanied by the altered expression of molecular markers; thus, EMT can be determined by detecting the expression of these molecular markers [32]. Different expression levels of HODX9 lead to different cell status. HOXD9 overexpression in HCC cells promotes EMT. Our results also indicate that HOXD9 promotes EMT, and silencing HOXD9 leads to MET. This observation suggests that EMT-MET is a fluid process. High HOXD9 expression also increases the number of distant metastasis in vivo. This phenomenon is consistent with the previous theory that EMT is essential for tumor cells to disseminate from adjacent tissues and seed new tumors in distant sites. These results indicate that HOXD9 affect cell migration and invasion via EMT regulation.
Microarray analysis was conducted to investigate the mechanism of HOXD9 in regulating the development of cancer. Zinc finger E-box-binding HOX 1, short for ZEB1, was identified as an effective mediator of these HOXD9-induced phenomena. Silencing ZEB1 in HOXD9 overexpression cell lines resulted in the similar phenomenon caused by HOXD9 knockdown. ZEB1 has been proven to induce EMT by acting repressing Ecadherin, silencing E-cadherin by binding to the proximal E-boxes [33,34]. HOXD9 may be involved in the development of HCC cells through the ZEB1 transcription factor.

Conclusions
In conclusion, HOXD9 upregulation can be correlated with HCC development. The HOXD9-mediated induction of EMT is ZEB1 dependent, and HOXD9 inhibition results in induction of a mesenchymal phenotype. These findings suggest that HOXD9 activation can promote ZEB1 in HCCs and elucidate HOXD9 signaling. These results can also help identify potential targets in HCC prevention and therapy.