SATB2 induces stem-like properties and promotes epithelial-mesenchymal transition in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is one of the most prevailing malignancies, and the molecular mechanisms underlying HCC tumorigenesis remain to be further clarified. The aim of our present study was to determine the biological functions and clinical significance of SATB2 in HCC. Quantitative RT-PCR was performed to detect SATB2 mRNA expression in HCC tissues and cells. Cell proliferation, migration and invasion were investigated by MTT assay and transwell assay. Western blotting was performed to evaluate the protein expression levels. Tumor xenografts were established to explore the effects of SATB2 on HCC tumor growth in vivo. Our results indicated that SATB2 was highly expressed in HCC tissues and cell lines, and its high expression was closely correlated with aggressive clinical phenotypes and poor prognosis of HCC patients. Through gain- and loss-of function experiments, we found that SATB2 significantly promoted HCC cell proliferation, migration, and invasion in vitro and HCC tumorigenicity in vivo. Furthermore, SATB2 was identified as an important regulator of stem-like properties and epithelial to mesenchymal transition (EMT) in HCC cells. In summary, our results suggested that SATB2 is a crucial upregulator of HCC, and SATB2 might be a potential prognostic marker and a therapeutic target for HCC.

Introduction

Hepatocellular carcinoma (HCC), accounting for about 90% of primary liver cancers, is the sixth most prevalent cancer and the third most frequent cause of cancer-associated death [1]. China alone accounts for nearly 55% of all global HCC cases [2]. Many HCC patients do not have any symptoms until an advanced stage [3]. Despite of recent advances in surgical resection and medical treatment, the overall survival for HCC patients remains dismal largely due to the high rates of recurrence and metastasis [4]. Therefore, it is imperative to investigate the mechanisms underlying HCC metastasis and develop effective individual therapeutic strategies for HCC patients.

SATB2 (special AT-rich binding protein-2), encoded on chromosome 2q32-33, is an evolutionarily conserved transcription factor, which regulates the expression of diverse sets of genes by binding to the nuclear matrix attachment regions [5,6]. Recent studies have indicated that SATB2 is associated with cancer progression. The expression of SATB2 was upregulated in HCC, and SATB2 rescued the miR-211-mediated inhibition of HCC cell invasion and proliferation [7]. However, the specific mechanisms by which SATB2 regulate the malignant progression of HCC are still largely unknown.

In our study, we detected the mRNA and protein expression levels of SATB2 in HCC tissues and cell lines, and explored the relationship of SATB2 with clinical characteristics and prognosis of HCC patients. Moreover, we further investigated the effects as well as the potential molecular mechanism of SATB2 involved in proliferation, migration and invasion of HCC cells in vitro. Our findings indicated the potential value of SATB2 in the clinical diagnosis and treatment of HCC.

Materials and methods

Human tissue samples

HCC tissue samples and their adjacent non-cancerous tissues (2 cm from the edge of the cancerous region) were obtained from 103 patients diagnosed with HCC who had undergone a routine hepatic resection at Sichuan Provincial People’s Hospital (Chengdu, China). The clinicopathological data of these patients are shown in Table 1. Patients who had preoperative radiotherapy or chemotherapy were excluded. Tissue samples were immediately snap-frozen in liquid nitrogen and stored at -80°C till use. This study was approved by the Ethics Committee of Sichuan Provincial People’s Hospital, and all patients provided written informed consent before enrollment in the study.

Table 1.

Correlation between the clinicopathologic characteristics and SATB2 expression in HCC tissues

Characteristics	Total number (n=103)	SATB2 expression		P value
		Low (n=59)	High (n=44)
Age (years)				0.182
≤50	38	25	13
>50	65	34	31
Gender				0.371
Male	70	38	32
Female	33	21	12
HBsAg status				0.847
Positive	81	46	35
Negative	22	13	9
Serum AFP level (ng/ml)				0.251
≥400	76	41	35
<400	27	18	9
Liver cirrhosis				0.357
Yes	65	35	30
No	38	24	14
Tumor size (cm)				0.049
<5	56	37	19
≥5	47	22	25
Number of tumor nodules				0.082
1	83	51	32
≥2	20	8	12
TNM stage				0.017
I+II	65	43	22
III+IV	38	16	22
Edmondson-Steiner grading				0.215
I+II	61	38	23
III+IV	42	21	21
Vascular invasion				0.023
Yes	43	19	24
No	60	40	20

Cell culture and transfection

Five HCC cell lines (SMMC-7721, Huh-7, HepG2, HCCLM3 and Hep3B) and one normal liver cell line L02, obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China), were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Biowest, Loire, France) in a humidified incubator with 5% CO₂ at 37°C.

The full-length SATB2 cDNA was amplified and cloned into the pcDNA3.1 (+) vector (Invitrogen, Carlsbad, CA, USA). The oligonucleotides to inhibit SATB2 expression were synthesized and inserted to the shRNA expression vector pGPH1/Neo (GenePharma, Shanghai, China). HepG2 cells were transfected with pcDNA3.1-SATB2 for overexpressing SATB2, and SMMC-7721 cells were transfected with sh-SATB2 for suppression of SATB2 using Lipofectamine 3000 (Invitrogen) following the manufacturer’s protocol.

Quantitative RT-PCR

Total RNA was extracted from using TRIzol reagent (Invitrogen). 1 μg of total RNA was used for complementary DNA synthesis using the Primer Script RT reagent Kit (Takara, Dalian, China). Quantitative RT-PCR was conducted using SYBR Green Real-time PCR Master Mix (TOYOBO, Shanghai, China) on the LightCycler 480 II System (Roche, Basel, Switzerland). Comparative quantification was determined using the 2^-ΔΔCt method [8]. GAPDH was used as an internal control. The following primers were used: SATB2 forward: 5’-GGAGAACGACAGCGAGGAA-3’, reverse: 5’-CCGATGTATTGCTTTGCCTAGT-3’; GAPDH forward: 5’-ACCGCAATTGGGAGTCAGGATT-3’, reverse: 5’-CCAATGTGTCCAGGAGGATT-3’.

Western blotting

Total protein was isolated using RIPA lysis buffer (Beyotime, Shanghai, China) with PMSF (Roche, Basel, Switzerland). Equal amounts of total protein were separated by SDS-PAGE and then transferred onto PVDF membrane (Millipore, Bedford, MA, USA). The membrane was blocked in 5% nonfat dry milk in TBST for 2 h and then incubated with appropriate primary antibodies in 4°C for one night. Next the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The proteins of interest were detected using Image-Pro Plus software 6.0 (Media Cybernetics Inc., Rockville, MD, USA), and GAPDH was used as an internal control.

MTT assay

The proliferation of HCC cells was measured using 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Briefly, approximately 2,000 cells were seeded in a 96-well culture plate incubated for 24, 48, 72 and 96 h. At various time points, 0.5 mg/ml MTT solution was added to each well, and then the plate was incubated at 37°C for 4 h. Next, the supernatants were removed and 100 μL of DMSO was added to solubilize the formed crystals. Absorbance levels were measured at the wavelength of 490 nm.

Transwell assay

HCC cell migration and invasion were evaluated using Matrigel-uncoated and -coated transwell inserts (8 μm pore size; BD Biosciences, Erembodegem, Belgium). Briefly, 2 × 10⁴ cells suspended in 200 μL serum-free medium were into the upper chamber, and the lower chamber was filled with 500 µl medium with 10% FBS. After 48 h of incubation at 37°C, the cells that had traversed the membrane were fixed with using 4% paraformaldehyde and stained with 0.1% crystal violet dye. The images were obtained and cells were counted using a light microscope.

Tumorsphere formation assay

About 1 × 10³ single HCC cells were plated onto 6-well ultralow attachment plates (Corning Inc., Corning, NY, USA) in DMEM/F12 supplemented with 2% B27 (Life Technologies, Invitrogen, USA), 20 ng/ml human EGF, 10 ng/ml human bFGF and 5 μg/ml insulin. After 14 days at 37°C, plates were observed for tumorsphere formation.

Tumor xenografts

The 6-week-old BALB/c-nu mice, purchased from Shanghai Laboratory Animals Center (Shanghai, China), were randomized into two groups (n=8/group). SMMC-7721 cells (1 × 10⁶ cells in 200 μl PBS) transfected with sh-SATB2 or sh-NC were inoculated subcutaneously into the right flank of the nude mice. Tumor volume was measured by an external caliper and calculated using the formula: Volume =0.5 × width² × length. 28 days after inoculation, the mice were killed and the tumors were excised and weighted. All experimental procedures were carried out with the approval of the Animal Care and Use Committee of Sichuan Provincial People’s Hospital.

Statistical analysis

Statistical analyses were performed by using GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA, USA) and SPSS 20.0 software (IBM, Chicago, IL, USA). Data are expressed as the mean ± standard deviation (SD) from at least three independent experiments, and inter-group differences were assessed for significance using two-tailed unpaired Student’s t-test. Chi-square test was applied to analyze the correlation of SATB2 expression and clinicopathological features of HCC patients. Association between SATB2 expression and overall survival was estimated using Kaplan-Meier method and evaluated using the log-rank test. A P value <0.05 was considered to indicate statistical significance.

Results

SATB2 is highly expressed in human HCC tissues and cell lines

Firstly, the expression levels of SATB2 in HCC tissues and cell lines were measured by quantitative RT-PCR and western blot analysis. The results showed that SATB2 mRNA was expressed at low levels in the adjacent normal liver tissues, while it was significantly upregulated in the HCC tissues (Figure 1A). Then, we detected the expression levels of SATB2 in a panel of HCC cell lines, and found that the expression levels of SATB2 was significantly increased in HCC cells when compared to normal human hepatocyte cell line L02 (Figure 1B, 1C). The expression of SATB2 was most highly in SMMC-7721 cells and was lowest in HepG2 cells. Thus, these two cell lines were chosen for subsequent analysis.

Figure 1.

SATB2 is highly expressed in human HCC tissues and cell lines. A: The relative SATB2 mRNA expression levels in HCC tissues and adjacent non-cancerous tissues were determined by quantitative RT-PCR. B: The relative SATB2 mRNA expression levels in HCC cell lines were determined by quantitative RT-PCR. Data are presented as mean ± SD. *P<0.05 versus L02 cells. C: The relative SATB2 protein expression levels in HCC cell lines were determined by western blotting. D: Kaplan-Meier survival curve analysis showed that HCC patients with high SATB2 expression had significantly shorter overall survival.

Clinical significance of SATB2 expression in HCC patients

In addition, to understand the clinical significance of SATB2 upregulation in HCC, HCC patients were divided into two groups according to the SATB2 expression in HCC tissues, including the high-expression group (n=44) and the low-expression group (n=59). Then we analyzed the relationship between SATB2 expression and clinicopathological characteristics of 103 HCC patients. As shown in Table 1, increased SATB2 expression was significantly associated with tumor size (P=0.049), TNM stage (P=0.017) and vascular invasion (P=0.023). Additionally, the overall survival was higher in HCC patients with lower SATB2 expression than in those with higher SATB2 expression (P=0.015; Figure 1D).

SATB2 promotes HCC cell proliferation, migration and invasion in vitro

Next, we explored the regulatory functions of SATB2 in HCC cells in vitro. We knocked down SATB2 in SMMC-7721 cells and overexpressed SATB2 in HepG2 cells. As expected, the expression of SATB2 was significantly modulated by pcDNA3.1-SATB2 and sh-SATB2 in HepG2 and SMMC-7721 cells (Data not shown). MTT assay revealed that cell proliferation was significantly promoted by overexpression of SATB2 in HepG2 cells; conversely, proliferation of SMMC-7721 cells was suppressed after knockdown of SATB2 (Figure 2A). Additionally, transwell assay demonstrated that HepG2 cells with SATB2 overexpression displayed more invasive and migratory properties, while knockdown of SATB2 significantly reduced the number of SMMC-7721 cells crossing the membrane (Figure 2B).

Figure 2.

SATB2 promotes HCC cell proliferation, migration and invasion in vitro. A: MTT assay was performed to detect the proliferation of HCC cells after transfection with pcDNA3.1-SATB2 or sh-SATB2. B: Transwell assay was performed to detect the migratory and invasive capacities of HCC cells after transfection with pcDNA3.1-SATB2 or sh-SATB2. Data are presented as mean ± SD. *P<0.05 versus pcDNA3.1-NC group or sh-NC group.

Downregulation of SATB2 inhibits tumorigenesis of HCC cells in vivo

The effects of SATB2 on tumorigenesis of HCC cells were further determined in vivo. SMMC-7721 cells transfected with sh-SATB2 or sh-NC were injected into the flanks of nude mice. As shown in Figure 3A, 3B, downregulation of SATB2 markedly inhibited the growth of HCC xenografts in nude mice. Moreover, the average tumor weight was also remarkably decreased in the sh-SATB2 group compared with that of sh-NC group (Figure 3C).

Figure 3.

Downregulation of SATB2 inhibits tumorigenesis of HCC cells in vivo. A: Tumor volume was measured and calculated every three days. B: Representative pictures of the tumors. C: Tumor weight was measured after four weeks of implantation. Data are presented as mean ± SD. *P<0.05 versus sh-NC group.

SATB2 induces stem-like properties in HCC cells

Considering stem-like cells as an important factor for tumor metastasis, we also examined assessed whether SATB2 modulated the stem-like properties in HCC cells. The result showed that overexpression of SATB2 increased the numbers and sizes of spheres formed by HepG2 cells, whereas knockdown of SATB2 reduced the numbers and sizes of spheres made by SMMC-7721 cells (Figure 4A). Stem cell markers, including Nanog, Oct4 and Sox2, were upregulated in SATB2-overexpressing HepG2 cells and downregulated when SATB2 was silenced in SMMC-7721 cells (Figure 4B).

Figure 4.

SATB2 induces stem-like properties in HCC cells. A: Sphere formation assay was performed to detect sphere-forming abilities of HCC cells after transfection with pcDNA3.1-SATB2 or sh-SATB2. B: The protein levels of stemness-related markers were determined in HCC cells after transfection with pcDNA3.1-SATB2 or sh-SATB2 by western bolt analysis. Data are presented as mean ± SD. *P<0.05 versus pcDNA3.1-NC group or sh-NC group.

SATB2 induces epithelial to mesenchymal transition in HCC cells

EMT is of critical importance for migration and invasion of HCC cells. Accordingly we determined to find whether SATB2 could exert effects on EMT of HCC cells. The results demonstrated that overexpression of SATB2 in HepG2 cells led to a remarkable increase in the mesenchymal makers, including N-cadherin and Vimentin, and a significant decrease in the epithelial markers (E-cadherin) (Figure 5A). On the contrary, as shown in Figure 5B, inhibition of SATB2 in SMMC-7721 cells decreased the mesenchymal markers whereas increased epithelial markers.

Figure 5.

SATB2 induces epithelial to mesenchymal transition in HCC cells. A: The protein levels of EMT-related markers were determined in HepG2 cells after transfection with pcDNA3.1-SATB2 by western bolt analysis. B: The protein levels of EMT-related markers were determined in SMMC-7721 cells after transfection with sh-SATB2 by western bolt analysis. Data are presented as mean ± SD. *P<0.05 versus pcDNA3.1-NC group or sh-NC group.

Discussion

Surgery is still the best therapeutic strategy for HCC. Metastasis is a key cause of poor long-term survival after surgical resection in HCC patients [9]. It is widely accepted that cancer metastasis is a complex multistep process that involves a myriad of genetic alterations. SATB2 is universally expressed in a variety of tissues, and serves important roles in multiple biological processes. However, the impact of SATB2 in cancerogenesis is still controversial. Overexpression of SATB2 suppressed cell proliferation and tumor progression ability in laryngeal squamous cell carcinoma [10], gastric cancer [11] and non-small cell lung cancer [12]. But other study also reported that SATB2 was upregulated in breast cancer cell lines [13]. Therefore SATB2 has been shown to serve as either an oncogenic factor or a tumor suppressor, with their functions depending on specific tissue samples. In the present study we found that SATB2 was significantly upregulated in HCC tissues and cell lines, and SATB2 expression was positively associated with unfavorable prognosis of HCC patients. Besides, gain- and loss-of-function experiment validated that SATB2 overexpression promoted the migratory and invasive capacities of HCC cells whereas SATB2 knockdown reduced these metastatic behaviors in vitro.

The dissemination of cancerous cells from the primary tumor must acquire specific characteristics, including migratory and invasive abilities, which allow them to reestablish in a secondary site [14]. Epithelial-to-mesenchymal transition (EMT) plays an important role in regulating metastasis and invasion in malignant tumors, including HCC [15]. During EMT, epithelial cells lose their apical-basal polarity and transition to a mesenchymal phenotype [16]. Downregulation of SATB2 is sufficient to induce EMT and promote migration and invasion in non-small cell lung cancer cells [17]. We found that overexpression of SATB2 significantly up-regulated the expression of N-Cadherin and Vimentin, and down-regulated the expression of E-Cadherin, further promoting the process of EMT in HCC cells. EMT has emerged as an important regulator of cancer cells exhibiting stem cell-like properties [18]. Stemness characteristics in cancer cells are also responsible for cancer relapse and metastasis [19]. SATB2 has been reported to induce cellular transformation and stemness in pancreatic cancer [20]. But SATB2 might serve as a negative regulator of stemness in colorectal cancer cells [21]. Here, we revealed that overexpression of SATB2 promotes stem-like characteristics in HCC cells, as evidenced by an increase in cell spheres and the expression of stem cell markers.

In summary, our data demonstrated that the aberrant high expression of SATB2 in HCC promotes the process of EMT and stemness. In view of our present results, we hypothesize that SATB2 might be an attractive target for prognostic and therapeutic interventions in HCC. Future studies should be performed to determine the precise mechanisms of the aberrant expression of SATB2 in HCC.

Disclosure of conflict of interest

None.