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Introduction

Hepatocellular carcinoma (HCC) is one of the most common malignancies, and is the second leading cause of cancer-related death worldwide (1). HCC accounts for 91% of primary liver cancer cases and presents poor prognosis with an overall 5-year survival rate of <5% (2). Like other malignant tumors, the metastasis and recurrence of HCC has become one of the major obstacles to the therapeutic treatment of HCC.

Although blood vessels are considered as a main route of HCC metastasis, vasculogenic mimicry (VM) has been certified to be a potential bypass for metastasis. VM is a microvascular channel which occurs de novo without the presence of endothelial cells (3). In VM, tumor cells arrange in lines to form vessel-like structure effectively mimicking a true vascular endothelium, which provide tumors with blood perfusion and promote tumor metastasis (4,5). VM has been reported in HCC (6) and other types of tumors including ovarian cancer (7), melanoma (8), osteosarcoma (9) and prostatic cancer (10).

VM is associated with poor patient prognosis and is considered as a loophole for antiangiogenesis therapy. Currently, antiangiogenic therapy mainly targets endothelium-dependent angiogenesis. Several antiangiogenic agents such as endostatin (ES) have been proved to be effective in inhibiting endothelium-dependent angiogenesis in both mice and humans (11,12). However, in the clinical trials, they failed to decrease tumor metastasis, and instead, tended to increase the risk of occurrence (13). Antiangiogenic therapy can lead to hypoxia, which contributes to the formation of VM, thus, facilitating tumor metastasis. Therefore, an effective treatment strategy should target not only endothelium-dependent vessels but also endothelium-independent vessels such as VM (14,15). Recently, the effects of caspases, including caspase-3, −6 and −7, were found to play a key role in apoptosis and may also play an important role in tumorigenesis, particularly in VM (16,17). However, the mechanism by which VM formation is promoted remains elusive.

Overexpression of migration-inducing gene 7 (MIG7) is found in highly aggressive tumors with VM rather than non-aggressive malignant cells without VM (18–20) suggesting an important role in VM formation and cancer aggression. Recently, it was reported that MIG7 was found to be related to the formation of VM in gastric cancer, and to play a complementary role in growth factors and COX-2/PGE2-related cancer invasion and metastasis (20,21). In the present study, we investigated the association of MIG7 expression with VM formation in HCC and its effects on the potential for HCC invasion and metastasis.

Materials and methods

Patient samples and cell lines

Forty matched pairs of HCC paraffin-embedded specimens from 40 patients (male, n=28; female, n=12; mean age, 54) and 10 normal liver paraffin-embedded specimens were purchased from Shaanxi Chaoying Clinical Pathology Institute (Shaanxi, China). The use of the specimens in the study was approved by the Ethics Committee of the Affiliated Hospital of the Logistics University of Chinese People's Armed Police Forces. Human HCC cell lines (MHCC-97H, MHCC-97L, Huh-7) and human normal hepatocyte L-02 cells were purchased from the Liver Cancer Institute of Fudan University (Shanghai, China). Dulbeccos modified Eagles medium (DMEM) with 10% fetal bovine serum (FBS) was used to culture all 4 types of cells at 37°C and 5% CO2.

Primary antibodies and reagents

Rabbit anti-human MIG7 (cat. no. ab83494) was purchased from Abcam (Cambridge, MA, USA). Mouse anti-human CD34 monoclonal antibody (cat. no. sc-19621), mouse anti-human β-actin monoclonal antibody (cat. no. 130065), goat anti-rabbit IgG (cat. no. sc-2004), goat anti-mouse IgG (cat. no. sc-2005) and laminin rabbit anti-human polyclonal antibody (cat. no. sc-5582) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fluorescence marked (FITC) goat anti-mouse IgG (cat. no. 555988) and fluorescence marked (TRITC) goat anti-mouse IgG (cat. no. 610055) were purchased from BD Biosciences (San Jose, CA, USA). High Fidelity PrimeScript™ RT-PCR kit and PrimeSTAR® HS DNA Polymerase were purchased from Takara Bio Group (Dalian, China). Matrigel and cell culture plates were purchased from Corning Inc. (Corning NY, USA). ES was purchased from Calbiochem (San Diego, CA, USA) and 0.25% tryptase was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Immunostaining analysis

For MIG7 protein detection, formalin-fixed and paraffin-embedded sections were deparaffinized and hydrated in a graded ethanol series. After antigen retrieval, sections were treated with 10% goat serum for 20 min and incubated with rabbit anti-human MIG7 polyclonal antibody (1:100) at 4°C overnight, and in turn biotinylated antibody and streptavidin-peroxidase at 37°C for 30 min. Signaling was detected with DAB substrate for 5 min. For VM formation detection, conventional treated sections were incubated with rabbit anti-human laminin polyclonal antibody (1:200) at 37°C for 30 min, and then treated with DAB for 5 min. The sections were then dehydrated and mounted with Permount and viewed by bright-field microscopy. The results, statistically analyzed according to the positive staining rate of the malignant cells and staining intensity, were assessed by the independent film reading of two professional physicians in a blinded manner. The three levels of staining intensity were: negative (−, 0); weakly positive (+, 1); intensely positive (++, 2).

3D cell culture

3D culture was employed to detect VM formation in vitro. Six-wells of culture plates were coated with Matrigel (50 µl/well). The cells were maintained in DMEM supplemented with 10% FBS and trypsinized, and then, suspended at 1×106/ml in complete medium. Finally, the cells were seeded on gels and incubated at 37°C in 5% CO2/9% air. The tube-like connections were observed under an inverted microscope. The number of tube-like connections per field (×200 magnification) was counted (22). Five random fields were analyzed in each sample.

Semi-quantitative PCR analysis

The semi-quantitative PCR was employed to detect MIG7 mRNA in different cell groups and the β-actin RNA was used as the internal standard control. According to the instructions, TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to harvest the total RNA. Subsequently, according to the manufacturers protocol, the High Fidelity PrimeScript™ RT-PCR kit (Takara Bio) was employed to transform to cDNA with 1 µg of total RNA. Then, the resulting cDNA was used for semi-quantitative PCR with SYBR-Green reagents (Fermentas, Waltham, MA, USA). The sequences of the primers were as follows: MIG7 mRNA forward: 5-TCT CAG GCA GTC AGT GGG-3 and MIG7 mRNA reverse: 5-GTT GGA TGG GAT GTC TCG-3; β-actin mRNA forward: 5-ATC GTG CGT GAC ATT AAG GAG AAG-3 and β-actin mRNA reverse: 5-AGG AAG GAA GGC TGG AAG AGT G-3. The cycling parameters were 94°C for 2 min, then 40 cycles of 94°C for 10 sec and 60°C for 30 sec, followed by a melting curve analysis. Dissociation curve analysis was applied to confirm the specificity of the PCR amplification. Relative expression levels were determined using the housekeeping gene β-actin for normalization.

Western blot assay

All cells (MHCC-97H, MHCC-97L, Huh-7 and L-02) were washed with phosphate-buffered saline (PBS) and the lysates were prepared using modified radioimmunoprecipitation assay buffer at 4°C for 15 min. The proteins were resolved by 0.1% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose filter membranes. Subsequently, blots were blocked by TBST and incubated overnight with primary antibodies (MIG7 1:200, β-actin 1:500) at 4°C. Then, the blots were washed in TBS containing 0.1% Tween-20 and labeled with goat anti-rabbit IgG-HRP (1:1,000) and goat anti-mouse IgG-HRP (1:1,000). MIG7/β-actin ratio was used for relative expression of proteins.

MIG7 shRNA constructs

Two target sequences and one negative control (Neo) sequence were synthesized based on the sequence of human MIG7 (GenBank no. DQ080207.2) and cloned into pSIREN vector to make pSIREN-M1 (MIG7 shRNA-1), pSIREN-M2 (MIG7 shRNA-2) shRNA constructs or MIG7 Neo construct pSIREN-MN (MIG7 shRNA-N) construct. The target sequences were as follows: Oligo #1: 5-GAT CCA AAG TTT CAT TCT TCG ACT TCA AGA GAG TCG AAG AAA TGA AAC TTT TTT TTT G-3 and 3-GTT TCA AAG TAA GAA GCT GAA GTT CTC TCA GCT TCT TTA CTT TGA AAA AAA AAC TTA A-5; Oligo #2: 5-GGA TCC CAC AGC TTG AGT GGA ATA CTT CAA GAG AGT ATT CCA CTC AAG CTG TGT TTT TTG-3 and 3-GGT GTC GAA CTC ACC TTA TGA AGT TCT CTC ATA AGG TGA GTT CGA CAC AAA AAA CTT AAG-5.

Transfection

MHCC-97H cells were cultured in DMEM high glucose medium supplemented with 10% FBS (from Gibco), 80 U/ml penicillin, and 100 µg/ml streptomycin at 37°C under 5% CO2. MHCC-97H cells were transfected with MIG7 shRNA constructs or control MIG7 Neo construct using Lipofectamine 2000 (Invitrogen) following the manufacturers instructions. The stable knockdown cells were selected by 3 µg/ml puromycin for 2 weeks. The stable cells in which MIG7 was efficiently knocked down were named as MHCC-97H-1 cells derived from MHCC-97H+pSIREN-M1 and MHCC-97H-2 cells derived from MHCC-97H+pSIREN-M2, and the stable control cell line was named as MHCC-97H Neo. The clones were characterized by semi-quantitative PCR and western blot analysis to assess the expression of MIG7 mRNA and MIG7 protein, respectively.

3D culture after transfection

Matrigel (50 µl/hole) was added to 6-well plates and then the cells were incubated at 37°C for 20 min. The MHCC-97H cells (1×106/ml), from the 6 groups, were maintained in DMEM supplemented with 10% FBS, and were then seeded onto the gels and incubated at 37°C in 5% CO2/95% air. The numbers of tube-like connections were measured in a high-power field (Leica DM ILM inverted microscope; Leica Microsystems GmbH, Wetzlar, Germany).

Transwell invasion assay

Transwell assays were used to evaluate the invasion of the cells in six groups. A total of 4×104 MHCC-97H cells were seeded in the upper chamber (24-well plates, 8-µm pores) coated with Matrigel (10 µg/hole) extracellular matrix (ECM) gel and media containing 10% FBS was placed in the lower chamber. The chambers were incubated at 37°C in 5% CO2 for 48 h. Invasive cells at the bottom of the membrane were stained with 0.1% crystal violet and were counted under a microscopic. Five random fields were analyzed in each chamber.

Transwell migration assay

A total of 1×104 MHCC-97H cells were seeded in the upper chamber (24-well plates, 8-µm pores) coated with Matrigel (10 µg/hole) extracellular matrix (ECM) gel and media containing 20% FBS was placed in the lower chamber. The chambers were incubated at 37°C in 5% CO2 for 24 h. Cells that migrated to the bottom of the insert were stained with 0.1% crystal violet and were counted under a microscopic (Leica DM ILM inverted microscope). Five random fields were analyzed in each chamber.

Cellular adhesion assay

MHCC-97H cells (1 ml cell suspension, 1×106/ml) were placed in an EP tube (1.5 ml) and incubated at 37°C in 5% CO2 for 12 h. The number of individual cells were counted at 2, 6 and 12 h, respectively. The lower proportion of remining single cells, the stronger the cell-to-cell adhesion.

Statistical methods

SPSS ver20.0 (IBM SPSS, Armonk, NY, USA) was used for statistical analyses. Rank-sum test was used for ranked data. P<0.05 was considered statistically significant.

Results

Positive association of MIG7 expression with VM formation in HCC tissues

MIG7 expression and VM formation in 40 matched pairs of HCC specimens from 40 patients were detected by immunostaining. The results (Table I) showed that the expression of MIG7 (Fig. 1A) was negative in 20% of the cases (8/40), weakly positive in 45% of the cases (18/40) and strongly positive in 35% of the cases (14/40). Meanwhile, VM formation (Fig. 1B) was negative in 15% of the cases (6/40), weakly positive in 50% of the cases (20/40) and strongly positive in 35% of the cases (14/40). The results showed that there was a positive correlation between MIG7 expression and VM formation (rs=0.554; P<0.001). There was no MIG7 expression and VM formation in normal liver tissues.

Figure 1. Immunohistochemical analysis of MIG7 expression and vasculogenic mimicry (VM) formation detection in HCC specimens. (A) The expression of MIG7 was detected by immunostaining. (B) VM formation was detected by immunostaining. The results showed that there was a positive correlation between MIG7 expression and VM formation. Magnification, ×200.

Table I. Expression of MIG7 protein and vasculogenic mimicry (VM) formation in HCC tissues.

Table I.

Expression of MIG7 protein and vasculogenic mimicry (VM) formation in HCC tissues.

	MIG7			VM
	−, 0	+, 1	++, 2	−, 0	+, 1	++, 2
HCC specimens, n/total (%)	8/40 (20)	18/40 (45)	14/40 (35)	6/40 (15)	20/40 (50)	14/40 (35)

[i] r_s=0.554, P<0.001.

Positive correlation between VM formation and the metastatic potential of the HCC cell lines in 3D culture

After 6 h of incubation, the difference in proliferation among the 4 types of cells (MHCC-97H, MHCC-97L, Huh-7 and L-02) was observed, and then it became more notable after 12 and 18 h. We found that (Fig. 2) the density of VM formation in the MHCC-97H group (x¯=21.2) was higher than that of the MHCC-97L group (x¯=6.8) (P=0.000) and Huh-7 group (x¯=1.6) (P=0.000). VM formation in the different HCC cell lines varied, and it was coincident with the metastatic potential of the HCC cell lines. Moreover, there was no VM formation observed in the normal liver L-02 cells after 6, 12 and 18 h. These results showed that there was a positive correlation between VM formation and the metastatic potential of the HCC cell lines.

Figure 2. Vasculogenic mimicry (VM) formation was detected by 3D culture. After 18 h of culture of the 4 types of HCC cells (MHCC-97H, MHCC-97L, Huh-7) and a human normal hepatocyte cell line L-02, the results indicated that the density of VM in the MHCC-97H group was higher than that of the MHCC-97L group and Huh-7 group. There was no VM formation in the normal liver cell L-02 group. Scale bars, 10 µm. *P<0.01, (P=0.000), compared with MHCC-97L, Huh-7 and L-02 groups; #P<0.01, (P=0.000), compared with Huh-7.

Positive correlation between MIG7 expression and the metastatic potential of the HCC cell lines

The expression of MIG7 was detected in the different cell groups. The results (Fig. 3A) showed that the HCC cell lines with different metastatic potential had diverse expression of MIG7 mRNA. Among them, the level of MIG7 mRNA expression in MHCC-97H cells was higher than that of MHCC-97L (P=0.000) and Huh-7 (P=0.000), and it was negative in L-02 cells. Western blot analysis was used to detect the MIG7 protein in the 4 types of HCC cell lines. The results (Fig. 3B) indicated that the expression of MIG7 protein in MHCC-97H cells was higher than that in the MHCC-97L and Huh-7 cells. No MIG7 protein was detected in the normal liver L-02 cells.

Figure 3. MIG7 mRNA and protein were detected by semi-quantitative PCR and western blot assay, respectively. (A) MIG7 mRNA was detected with semi-quantitative PCR. The results showed that the expression of MIG7 mRNA in MHCC-97H cells was higher than that in the MHCC-97L, Huh-7 and L-02 cells. *P<0.01, (P=0.000), compared with MHCC-97L, Huh-7 and L-02 groups; #P<0.01, (P=0.000), compared with Huh-7 and L-02 groups. (B) MIG7 protein was detected by western blot assay. The results indicated that the expression of MIG7 protein in MHCC-97H cells was higher than that in the MHCC-97L and Huh-7 cells.

Decreased VM formation in MHCC-97H cells with MIG7 knockdown

After 24 h from transfection with the constructs for knockdown, the green fluorescent protein, expressed by the constructed plasmid, was observed under a fluorescence inverted microscope and the transfection efficiency was 10–15% (Fig. 4B). The transfected cells were selected by puromycin and green fluorescent protein labeling the positive cells increased gradually. Then, the cells were cultivated and selected by limited dilution twice. Finally, the green fluorescent protein-labeled positive cells accounted for >90% (Fig. 4C). The knockdown efficiency of MIG7 shRNA was detected by semi-quantitative PCR (Fig. 5A) and western blot analysis (Fig. 5B). The results showed that MIG7 mRNA and protein was deceased significantly in both the MIG7 shRNA-1 and MIG7 shRNA-2 groups, especially in the MIG7 shRNA-1 group. The inhibitory efficiency of shRNA on MIG7 expression in the MIG7 shRNA-1 group was ~70% compared with MIG7 shRNA-N group. 3D culture was utilized to detect the inhibitory effect of MIG7 shRNA on VM formation. The results (Fig. 6) showed that VM formation was significantly decreased in the MIG7 shRNA-1 (x¯=1.8) and MIG7 shRNA-2 groups (x¯=3.2) compared with the control group (x¯=26.4; P=0.001, 0.001). MIG7 shRNA-N group (x¯=25.8), empty vector group (x¯=23) and ES group (x¯=25.6) did not have a significant difference compared with the control group (P=0.842, P=0.284 and P=0.819, respectively). The results indicated that MIG7 plays an important role in VM formation.

Figure 4. Transfection. (A) After 24 h from transfection with the constructs for knockdown, the green fluorescent protein was observed under a fluorescence inverted microscope and the transfection efficiency was 10–15% (original magnification, ×400). (B) The transfected cells were selected by puromycin, and green fluorescent protein-labeled positive cells increased gradually. (C) Finally, the green fluorescent protein-labeled positive cells accounted for >90% (original magnification, ×400).

Figure 5. After transfection, MIG7 mRNA, MIG7 protein and vasculogenic mimicry (VM) formation were detected by semi-quantitative PCR, western blot assay and 3D culture, respectively. (A) Expression of MIG7 mRNA was detected with semi-quantitative PCR. *P<0.01, compared with MIG7 shRNA-2 (P=0.005), MIG7 shRNA-N (P=0.000), vector (P=0.000), ES (P=0.000) and control groups (P=0.000); #P<0.01, compared with MIG7 shRNA-N (P=0.005), vector (P=0.005), ES (P=0.007) and control groups (P=0.002). (B) The expression of MIG7 protein was detected by western blot assay. The results showed that MIG7 protein was deceased significantly in both MIG7 shRNA-1 and MIG7 shRNA-2 groups, especially in the MIG7 shRNA-1 group.

Figure 6. After transfection, the results of 3D culture showed that the quantity of VM in the MIG7 shRNA-1 and MIG7 shRNA-2 groups was significantly lower than that in the MIG7 shRNA-N, ES, empty vector, and the control group (P=0.000). MIG7 shRNA-N group, ES group and empty vector group did not have significant difference compared with the MHCC-97H cell group (P>0.05). *P<0.01, compared with MIG7 shRNA-N, vector, ES and control groups; #P<0.01, compared with MIG7 shRNA-N, vector, ES and control groups.

Suppressed invasive properties and increased cellular adhesion in MHCC-97H cells with MIG7 knockdown

To evaluate the invasive properties of the MHCC-97H cells with MIG7 knockdown, we performed Transwell invasion assay (Fig. 7A) and Transwell migration assay (Fig. 7B). The result of the Transwell invasion assay (48 h) showed that the number of cells that invaded to the lower portion of the chamber in the MIG7 shRNA-1 (x¯=112) and MIG7 shRNA-2 groups (x¯=146) was significantly lower than that of in MIG7 shRNA-N group (x¯=360; P=0.000, 0.000), empty vector group (x¯=358; P=0.000, 0.000), ES group (x¯=365.2; P=0.000, 0.000) and MHCC-97H cell group (x¯=367; P=0.000, 0.000). The results also indicated that there were no significant differences between the MIG7 shRNA-N group, empty vector group, ES group and MHCC-97H cell group (P=0.549). The Transwell migration assay showed that the cell migration in the MIG7 shRNA-1 (x¯=51.25) and MIG7 shRNA-2 groups (x¯=74.25) was significantly lower than that of the MIG7 shRNA-N (x¯=231; P=0.000, 0.000), empty vector (x¯=229.25; P=0.000, 0.000), ES (x¯=225.25; P=0.000, 0.000) and MHCC-97H cell group (x¯=229.75; P=0.000, 0.000) after 24 h from transfection. We also assessed the cellular adherent ability using cellular adhesion assay. The results (Fig. 7C) showed that after 6 h of incubation, the number of single cells in the MIG7 shRNA-1 (x¯=51.2) and MIG7 shRNA-2 groups (x¯=65.2) was significantly lower than that of in MIG7 shRNA-N group (x¯=88.2; P=0.000, 0.000), empty vector group (x¯=89.8; P=0.000, 0.000), ES group (x¯=90.6; P=0.000, 0.000) and MHCC-97H cell group (x¯=91.4; P=0.000, 0.000). Moreover, the number of single cells in the MIG7 shRNA-1 group was significantly lower than that of the MIG7 shRNA-2 group (P=0.000); there were no significant differences among the MIG7 shRNA-N group, empty vector group, ES group and MHCC-97H cell group (P=0.431). These results suggest that MIG7 plays an important role in regulating HCC cellular adhesion and invasive properties.

Figure 7. Transwell invasion, migration and cellular adhesion assay. (A) The Transwell invasion assay showed that the MIG7 shRNA inhibited cell invasive ability. *P<0.01, compared with MIG7 shRNA-2 (P=0.008), MIG7 shRNA-N (P=0.000), vector (P=0.000), ES (P=0.000) and control groups (P=0.000); **P<0.01, compared with MIG7 shRNA-N (P=0.000), vector (P=0.000), ES (P=0.000) and control groups (P=0.000). There was no significant difference between MIG7 shRNA-N, vector, ES and control groups (P>0.05). (B) The Transwell migration assay showed that the cell migration ability was suppressed by MIG7 shRNA. *P>0.05, compared with MIG7 shRNA-2, MIG7 shRNA-N, vector, ES and control groups; **P<0.05, compared with MIG7 shRNA-N (P=0.031), vector (P=0.000), ES (P=0.000) and control groups (P=0.000); ∆P>0.05, compared with MIG7 shRNA-N, vector, ES and control groups; ∆∆P<0.01, compared with MIG7 shRNA-N (P=0.000), vector (P=0.000), ES (P=0.000) and control groups (P=0.000). There was no significantly difference between MIG7 shRNA-N, vector, ES and control groups (P>0.05). (C) The results of cellular adhesion assay showed that after 6 h from incubation, MIG7 shRNA increased cellular adhesion. *P>0.05, compared with MIG7 shRNA-2, MIG7 shRNA-N, vector, ES and control groups; **P<0.01, compared with MIG7 shRNA-2 (P=0.003), MIG7 shRNA-N (P=0.000), vector (P=0.000), ES (P=0.000) and control groups (P=0.000); ∆P<0.01, compared with MIG7 shRNA-N, vector, ES and control groups. There was no significantly difference between MIG7 shRNA-N, vector, ES and control groups (P>0.05).

Discussion

The metastasis of HCC is one of the major reasons responsible for the failure of HCC treatment and the death of patients. Therefore, more attention should be focused on the mechanism of HCC metastasis. In the present study, we found a positive correlation between MIG7 and VM in both clinical specimens and in vitro experiments. MIG7 knockdown in 3D cultured MHCC-97H cells reduced the VM formation and weakened the invasive properties accompanied by enhanced cellular adhesion. Thus, this study provides evidence for a causal association of MIG7 with VM formation in HCC, which suggests that MIG7 could be a potential treatment target for cancer invasion and metastasis.

Previously, VM has been described in HCC and was associated with advanced tumor grade, invasion, metastasis and poor patient prognosis (23,24). More studies investigated the relevant mechanisms and signaling pathways of VM formation, such as hypoxia inducible factor 1-α (HIF-1α), (25) matrix metalloproteinases (MMPs), phosphoinositide 3-kinase (PI3K), laminin 5 (Ln-5) γ2 chain (26,27), focal adhesion kinase (FAK) (28) vascular endothelial-cadherin (VE-cadherin) (29) and epithelial cell kinase (EphA2) (30). However, the exact and detailed mechanisms underlying VM remain unclear. In the present study, we found that MIG7 and VM were highly expressed in HCC tissues and that MIG7 has a significant positive correlation with VM formation. We also found that MIG7 expression in different HCC cell lines was coincident with VM formation, invasion and metastasis. The result was consistent with previously reported studies which showed a positive correlation of MIG7 with VM in human lung cancer and gastric carcinoma (20,31).

Based on the analyses above, we proposed that MIG7 may induce the invasion and metastasis of HCC by regulating VM formation. Furthermore, the metastasis of HCC may be inhibited if the expression of MIG7 is downregulated. Downregulation of MIG7 may be an effective and safe therapeutic strategy. Based on the data above, the overexpression of MIG7 generally leads to a poor prognosis and there is no MIG7 expression and VM formation in normal liver tissues. Therefore, MIG7 can be considered as a potential safe and specific molecular target for HCC gene therapy. The invasion and metastasis of HCC could be suppressed if we transport MIG7 antagonist into HCC cells in order to inhibit VM formation. This therapeutic strategy, targeting MIG7, for HCC has great application potential. Moreover, MIG7-targeting therapy combined with various treatment methods at present, especially endothelial-targeting angiogenesis inhibitors (such as ES), may show better efficacy than any single use of angiogenesis inhibitors alone.

Subsequently, in order to verify the hypothesis, RNAi technique was employed to construct the recombinant retrovirus MIG7 shRNA expression vector plasmid. Additionally, because of the high expression of MIG7 in the MHCC-97H cell line, MHCC-97H cells were chosen as effector cells to explore the role of MIG7 in VM formation and HCC metastatic regulation. After the transfection of MIG7 shRNA into MHCC-97H cells, we found that MIG7 mRNA and protein were significantly decreased according to semi-quantitative PCR and western blot assay. Moreover, VM formation, invasion, migration of MHCC-97H cells were inhibited significantly by 3D culture, Transwell invasion and migration assay, respectively (P<0.05), while the adhesion capability of MHCC-97H cells was increased significantly (P<0.05). However, there was no significant effect of ES on MIG7 expression and intercellular adhesion, invasion and metastasis.

A recent study reported that intra-tumor heterogeneity does exist in patients with HCC (32). The authors performed genome sequencing on 43 lesions from 10 patients with hepatitis B virus (HBV)-associated HCC and compared the genetic features of different lesions from each patient. They found that the mutations which were shared by all the lesions in each patient varied from 8 to 97%, indicating the indetermination existing in intra-tumor heterogeneity. Thus, the genomic features of HCC in patients might not be characterized by sequence analysis of one single lesion, and this would be a challenge for precision medicine in patients with HCC. Therefore, more specific targets and the key genes that link to them are still needed to be identified in order to facilitate effective and specific precision medicine for tumor treatment.

In closing, MIG7 expression in HCC tissue is correlated positively with VM formation and MIG7 expression in different HCC cell lines is coincident with their VM formation, invasion and metastasis. Moreover, MIG7 shRNA inhibits MIG7 expression, VM formation and HCC invasion and metastasis stably and effectively. There was no significant effect of ES on MIG7 expression, VM formation and intercellular adhesion, invasion and metastasis. However, relevant in vivo experiments are still necessary for investigation.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (no. 81272547) and Tianjin Science and Technology Project (15ZXLCSY00040).

References