Downregulation of B7-H4 suppresses tumor progression of hepatocellular carcinoma

B7-H4, as a member of the B7 superfamily, was overexpressed in various types of cancers. However, the effects of B7-H4 on the aggressiveness of HCC and the underlying mechanisms have not yet been fully explored. For this purpose, B7-H4 expression was detected by Flow cytometry and Western blotting, it was highly expressed in several HCC cell lines but not in normal LO2 cell line. Knockdown B7-H4 expression induced HCC cells apoptosis by flow cytometry and colony formation assays and increased several apoptosis-related proteins, including survivin, cleaved caspase-3, cleaved caspase-7, and Bax, while the pro-growth protein survivin was reduced. Then the proliferation and cell cycle were suppressed after treated by siB7-H4. Moreover, the level of B7-H4 was significantly correlated with cell migration. In vivo, intra-tumor injection of siRNA targeting B7-H4 can significantly inhibited the growth of HepG2 cells in nude mice. Finally, regions of interest were manually traced on T1WI, T2WI, DWI and ADC of MR images. ADC values were increased in HCC xenografts after B7-H4 siRNA treatment. These data indicated that downregulation of B7-H4 suppressed the proliferation and migration and promoted apoptosis in vitro and in vivo. Blocking the B7-H4 channel might be a potential therapeutic strategy for HCC.


B7-H4 high expression in
Efficient siRNA-mediated B7-H4 gene silencing in HCC cell lines. Real-time PCR and Western blot analysis were employed to evaluate interference efficiency. Data showed that B7-H4 mRNA expression in Huh-7 and HepG2 cells was reduced in the B7-H4 siRNA group compared with that in the non-target siRNA groups (p < 0.01, Fig. 2A). There was no significant difference in the expression of B7-H4 mRNA between the control and non-target siRNA groups (p > 0.05). The same trends were observed by Western blot analysis. The expression of B7-H4 in Huh-7 and HepG2 cells declined in the B7-H4 siRNA group compared with that in the control and non-target siRNA groups (Fig. 2B). In addition, B7-H4 expression was reduced by a more significant level in the B7-H4 siRNA-2 group than in the B7-H4 siRNA-1 group. Consequently, B7-H4 siRNA-2 was used to perform all subsequent investigations.

B7-H4 knockdown increased the apoptotic of HCC cells.
The apoptosis of Huh-7 and HepG2 cells was detected after 72 h in non-transfected cells and in cells transfected with siRNA, including B7-H4 siRNA-2, and non-target siRNA. The level of apoptosis in Huh-7 and HepG2 cells was higher in the B7-H4 siRNA-2 group than in the non-target groups, as measured using annexin V/PI (p < 0.01, Fig. 3A), There were no significant differences between the control and non-target siRNA group (p > 0.05). The down-regulation of B7-H4 by siRNA to B7-H4 was confirmed with Western blot (Fig. 3B). In addition, colony formation assays revealed that Huh-7 and HepG2 cells formed fewer colonies in the B7-H4 siRNA-2 group than in non-target groups (p < 0.01, Fig. 3C,D). There were no significant differences between the control and non-target siRNA group (p > 0.05). To further investigate the mechanisms of HCC apoptosis caused by B7-H4 siRNA-2 transfection, apoptosis-related proteins were analyzed by Western blot. The results showed that the levels of the pro-apoptotic proteins Bax, cleaved caspase-3 and cleaved caspase-7 were increased, but the levels of the anti-apoptotic protein survivin in the B7-H4 siRNA-2 group were decreased compared with those in the control and non-target groups (Fig. 3E).

B7-H4 knockdown suppressed the proliferation of HCC cells.
To determine whether the silencing of B7-H4 by siRNA affected the proliferation of human HCC cells, the proliferation of Huh-7 and HepG2 cells were evaluated using a cell proliferation assay. The growth of Huh-7 cells was significantly inhibited in the B7-H4 siRNA group at 3 d (p < 0.01), 4 d (p < 0.01), and 5 d (p < 0.05) time-points in comparison with that in the non-target siRNA groups (Fig. 4A). Similarly the growth of HepG2 cells was reduced in the B7-H4 siRNA group at 3 d (p < 0.01), 4 d (p < 0.05), and 5 d (p < 0.05) time-points (Fig. 4B). The growth of both cell lines were no significant differences between the control and non-target siRNA group (p > 0.05). Furthermore, the cell cycle was determined at 72 h following B7-H4 siRNA-2 transfection by flow cytometry. Most Huh-7 and HepG2 cells were arrested in the G0/G1 phase in the B7-H4 siRNA-2 group compared to the control and non-target siRNA group (Fig. 4C,D). In parallel, cell lysates from control, non-target, or B7-H4 siRNA-transfected cells were harvested and subjected to Western blot analysis of B7-H4 expression (Fig. 4E).
B7-H4 knockdown suppressed the migration of HCC cell lines. The wound healing capacity of Huh-7 and HepG2 cells at 72 h in the B7-H4 siRNA-2 group were reduced compared with that in the non-target groups (p < 0.01, Fig. 5A,B), but there were no significant differences between the control and non-target siRNA group (p > 0.05). Western blot was used to examine the migration-related proteins E-cadherin and vimentin. The E-cadherin expression level of Huh-7 and HepG2 cells were all increased in the B7-H4 siRNA-2 group whereas vimentin were decreased comparied with non-target siRNA group, but there were no significant differences between the control and non-target siRNA group (Fig. 5C). B7-H4 knockdown suppressed human HCC growth in vivo. Tumors volumes of three groups were measured twice a week and drew the tumor growth curves. The tumor growth curves indicated that there was a significant growth inhibition in B7-H4 siRNA2 group (P < 0.05), but no differences of blank control group and non-target siRNA group (P > 0.05) after treated with multi-point injection of siRNA for 4 weeks (Fig. 6A). The tumor volume of three groups were measured at 6 weeks, the result showed that the B7-H4 siRNA-2 group were 1211 ± 193.1 mm 3 , 1730 ± 98.57 mm 3 in the non-target siRNA group and 1700 ± 58.86 mm 3 in the blank control group, while the corresponding weight were 777.7 ± 114.0 mg, 1186 ± 133.5 mg and 1181 ± 95.73 mg respectively. The tumor volume and weight of the B7-H4 siRNA-2 group were obviously reduced compared with that in non-target siRNA group (p < 0.05, p < 0.05), but there was no difference between non-target siRNA group and blank control group (Fig. 6B,C). B7-H4 expression was detected in xenografts of each group by flow cytometry, that the level of B7-H4 expression was lower in the B7-H4 siRNA-2 group than that in the non-target groups (p < 0.01, Fig. 6D a ), but no differences between non-target siRNA group and blank control group (P > 0.05). The down-regulation of B7-H4 by siRNA to B7-H4 was further confirmed with western blot and IHC (Fig. 6D b,c ).
The results of HE stain demonstrated a decrease in cell density and an increase in degree of necrosis in tumors of B7-H4 siRNA group (Fig. 6E).
MRI features of HCC xenografts. T1WI, T2WI, DWI could provide additional information about HCC xenograft tumors, such as position, size, shape, abnormal regions and intensity of tumors in vivo. All tumors and adjacent organ of three groups were detected clearly on T1WI and T2WI images, observed isointensity on T1WI images, hyperintensity and more hyperintensity in necrotic areas on T2WI; In addition, the B7-H4 siRNA-2 group exhibited a major decrease of tumor volume on Coronal T2WI plus fat repression compared with non-target siRNA group and blank control group. DWI images showed hyper-intensity and more hyperintensity in solid lesions of tumor tissues. For ADC maps, the solid of tumor lesions of three groups were clearly exhibited low signal intensity (Fig. 7A). Furthermore, DWI related parameters, ADC values were measured on www.nature.com/scientificreports www.nature.com/scientificreports/ ADC maps, Data showed that the ADC values were decreased in the B7-H4 siRNA group compared with that in the non-target siRNA groups (p < 0.05, Fig. 7B), There was no significant difference between the control and non-target siRNA groups (P > 0.05). The tumor volume of the B7-H4 siRNA-2 group acquired on coronal T2WI in vivo were obviously reduced compared with that in non-target siRNA group (p < 0.05, Fig. 7C), but there was no difference between non-target siRNA group and blank control group (P > 0.05). ADC values of three groups were all negatively correlated with their corresponding tumor volume (Fig. 7D).

Discussion
HCC is a common malignant tumor with high morbidity and mortality, particularly in China 17 . Compared with other cancers, risk factors for HCC are relatively well known; however, the mechanisms of hepatocarcinogenesis are not well understood at present 18 . Some studies have suggested that B7-H4 is involved in the developmental mechanisms of some types of cancer including pancreatic and breast cancers 11,19 . Previous studies have reported that sB7-H4 is expressed at high levels in the serum and is associated with an advanced clinical tumor stage www.nature.com/scientificreports www.nature.com/scientificreports/ in patients with HCC 13,14 . In addition, the expression of B7-H4 has been detected in cancerous liver tissues 15 . However, to date, this expression remains to be quantified. The mechanism underlying B7-H4 involvement in HCC development and progression also remains to be elucidated. In the present study, we discovered that B7-H4 was highly expressed in Hep3B, HepG2, and Huh-7 cells but was undetectable in normal human hepatocyte cells (LO2). These results strongly suggest that high levels of B7-H4 expression are involved in the molecular physiology of HCC and that this may represent an important mechanism in the development and progression of HCC.
A previous report has shown that B7-H4 plays a role in cell apoptosis in pancreatic and ovarian cancers 11,20 . In the present study, flow cytometry was used to determine whether B7-H4 influenced the apoptosis of human HCC cells. The results showed that the silencing of B7-H4 led to the induction of cell apoptosis. To further verify the anti-apoptotic effect of B7-H4, we quantified colony formation and found lesser number of colonies in the B7-H4 siRNA group than in the control and non-target groups. These results demonstrated that B7-H4 exerts an anti-apoptotic effect on HCC cells, and consequently promotes HCC development. Previous studies have shown that survivin is expressed at high levels in HCC cells, which could inhibit cancer cell apoptosis 21,22 . However, whether B7-H4 exerts an apoptotic effect on HCC cells by disrupting the expression of survivin remains to be www.nature.com/scientificreports www.nature.com/scientificreports/ elucidated. In the present study, we found that survivin was significantly downregulated in HCC cells following B7-H4 siRNA transfection. This result suggests that B7-H4 interfered with the expression of survivin and thereby inhibited HCC cell apoptosis. In addition, survivin exerts its strong anti-apoptotic effects by altering the caspase-3-caspase-7 and Bax pathways 23,24 ; thus, it plays an important anti-apoptotic role. Our current results also showed that the levels of cleaved caspase-3, cleaved caspase-7, and Bax were significantly increased in HCC cells transfected with B7-H4 siRNA. Collectively, these results suggested that B7-H4 alters the expression of survivin, which can subsequently inhibit the caspase-3-caspase-7 and Bax pathways; eventually, apoptosis in HCC cells is blocked.
Some studies have reported that B7-H4 plays a role in cell proliferation in pancreatic and ovarian cancers 11,16 . Shiraki et al. further reported that survivin promotes the aberrant proliferation and cell cycle progression of HCC cells 21,25 . In the present study, we found that the downregulation of B7-H4 could significantly suppress the growth www.nature.com/scientificreports www.nature.com/scientificreports/ of HCC cells by arresting the cell cycle at the G0/G1 phase. Therefore, these data suggested that B7-H4 plays an important role in promoting HCC proliferation by regulating the function of survivin.
An earlier study demonstrated that B7-H4 promotes the invasion and migration of colorectal cancer cells 10 . However, whether B7-H4 is linked to HCC cell migration is an issue that requires further exploration. In the present study, the effect of silencing B7-H4 on HCC cell migration was determined by a cell wound scratch assay, the results of which showing that the silencing of B7-H4 could inhibit HCC cell migration. These results suggested that B7-H4 plays an important role in HCC cell migration, affecting the metastasis of HCC and resulting in HCC progression. Evidence in support of the fact that B7-H4 influences migration-related proteins including E-cadherin and vimentin and then enhances the migratory and invasive potential of pancreatic cancer has been increasing 11 . In the current study, we also evaluated the expression of E-cadherin and vimentin to investigate the potential role of B7-H4 in HCC cell migration. Our results revealed that the expression of E-cadherin increased when B7-H4 expression was blocked, whereas vimentin levels decreased. These data demonstrated that B7-H4 could induce the progression of HCC by reducing the adhesion of HCC cells and by subsequently increasing their potential migration.
Our above results demonstrate that B7-H4 plays an important role in HCC cells proliferation, invasion, and cell apoptosis. Furthermore, a strong anti-tumor effect of chemically modified B7-H4 siRNA in vivo was observed, as tumor growth in nude mice with xenograft was significantly suppressed when the expression of B7-H4 was down-regulated by intra-tumor injection of B7-H4 siRNA. Our results were consistent with the findings of Zhu et al., injected with B7-H4 siRNA inhibits tumor growth in nude mice in pancreatic cancer 26 . In addition, silencing of B7-H4 which stably transfected SMMC7721 cells with B7-H4 shRNA could inhibits tumorigenicity in HCC 27 . These results indicate that the aberrant expression of B7-H4 have many important roles in the development and progression of hepatocellular carcinoma. To further investigate the response of the HCC xenografts after B7-H4 siRNA treatment, we collected tumor xenografts for pathologic and histological analysis by means of HE. Our study showed that the tumor cell density was obviously decreased, the necrosis area was enlarged, the extracellular space was widened and cell swelling was aggravated after B7-H4 siRNA therapy by the analysis of HE staine. These results indicate that the aberrant expression of B7-H4 have many important roles in the development and progression of hepatocellular carcinoma in vivo.
In clinic, MRI was widely used to diagnose the several tumors included HCC 28 . MRI can dynamic track and judge the tumor development in living animal. In present study the advanced 7.0T MRI was firstly used to explore the influence of B7-H4 siRNA on progression of HCC. The T1WI, T2WI and DWI of this study were obtained on a 7.0-T MRI scanner in HCC nude mice model. The xenograft tumors of three groups were all clearly exhibited on T1WI. Moreover, we acquired the volume of tumors of the three groups on coronal T2WI in vivo, the tumor volume of B7-H4 siRNA-2 group were obviously reduced compared with that in non-target siRNA group and blank www.nature.com/scientificreports www.nature.com/scientificreports/ group, the result was consent with vernier caliper in vitro. In addition, DWI is a powerful sequence of quantitative assessment of the water diffusion behavior to evaluate therapeutic efficiency of targeted therapy in HCC 29 . ADC of DWI related parameter, which is a quantitative biomarker of water diffusion in the microscopic environment, can be used to detect benign and malignant lesions, assess tumor invasive potential and motility, and even predict early treatment responses [29][30][31] . Some studies have reported that ADC values significantly increased after days of drug treatment in triple-negative breast-cancer (TNBC) and hepatocellular carcinoma xenograft nude mice model 32,33 . However, the effect B7-H4 siRNA on HCC progression was analyzed with ADC value of MRI was still not reported. In our study, the ADC values were increased in the HCC tumor xenografts of B7-H4 siRNA mice compared with that non-target siRNA and blank control mice. Previous studies also showed that ADC values were positive correlated with the extent of tumor cell apoptosis and negative with tumor cell proliferation in nude xenograft model 32 . So the increase of ADC values in B7-H4 siRNA group indicated that tumor cell apoptosis was up-regulated, while tumor cell proliferation was down-regulated. Also, ADC values of three groups were all negatively correlated with their corresponding tumor volume in this study. These results declared that silence of B7-H4 can suppresses HCC tumor progression, obviously B7-H4 plays an important role in development of HCC.

conclusion
In summary, siRNA targeting B7-H4 can inhibit the growth of HCC in vitro and in vivo. The results of the study illuminate that the increase expression of B7-H4 on HCC enhance their proliferation and migration but inhibit their apoptosis. So, blocking the B7-H4 expression might be used as a potential target molecule for HCC therapy.

Materials and Methods
Cell culture. Human HCC lines including HepG2, Hep3B, and HuH-7 and a normal human liver cell line (LO2) were obtained from the Cell Research Institute of the Chinese Academy of Sciences (Shanghai, China); they were cultured in DMEM (Hyclone) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. All cells were incubated at 37 °C in a humidified atmosphere containing 5% CO 2 .
Cell lysis. Cells were harvested and washed twice with PBS, lysed in ice-cold radio immunoprecipitation assay buffer (Beyotime, Shanghai, China) containing a freshly prepared 0.01% protease inhibitor cocktail (Beyotime), and incubated on ice for 40 min. A cell lysis solution was centrifuged at 12,000 g for 20 min at 4 °C, and the supernatant was harvested. Protein concentrations were determined with a BCA protein assay kit (Beyotime, Shanghai, China) using bovine serum albumin as a standard.
Western blot analysis. Cell lysates were denatured for 5-10 min at 99 °C with an SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer separated on 12% SDS-PAGE gels and were transferred onto polyvinylidene fluoride membranes (Solarbio, Beijing, China). After blocking in 5% non-fat milk, membranes were incubated overnight with 1:800 primary antibodies (B7-H4, GAPDH, Bax, E-cadherin, vimentin, survivin, cleaved caspase-3, and cleaved caspase-7) at 4 °C and then with a goat anti-rabbit secondary antibody for 2 h at room temperature. For each membrane, band intensity was analyzed using enhanced chemiluminescence reagents (Thermo Fisher Scientific, Shanghai, China). siRNA oligonucleotides. Two sets of siRNA were prepared, as previously described 11 . Two B7-H4 siRNA oligonucleotides were synthesized by GenePharma (Shanghai, China). Negative control siRNA (non-target siRNA) was also purchased from GenePharma. Oligonucleotide sequences of siRNA are listed in Table 1.
Transfection with siRNA oligonucleotides. Huh-7 (3 × 10 5 /well) and HepG2 (1 × 10 6 /well) cells were seeded in 6-well plates 24 h prior to transfection. When cells reached 70%-80% confluency in each well, they were transfected with 50 nM B7-H4 siRNA or a negative control using 7.5 µL Lipofectamine 2000 (Invitrogen, Shanghai, China) in accordance with the manufacturer's protocol and were cultured in DMEM without FBS for 4-6 h. Then, the cells were cultured in DMEM with 10% FBS. All experiments were performed in triplicate. After 24 h, the transfected and non-transfected cells were collected and evaluated for mRNA levels by quantitative  In vivo tumor growth and treatment. Female Balb/c athymic nude mice aged 4 weeks were obtained from Vital River Laboratories (Beijing, China) and allowed one week of acclimatization to their new surroundings. Then these mice were housed in temperature-controlled rooms with a 12 h alternating light-dark cycle of Specific Pathogen Free animal laboratory. As some previous studies described HepG2 cells could form subcutaneous tumors in nude mice 34,35 . HepG2 cells (1 × 10 7 ) were injected subcutaneously into the dorsal region near the hind leg of the nude mice, when the tumor tumor volumes reaching approximately 100 mm3, 18 mice with equivalently sized tumors were randomized into three groups. Animals were treated with an intratumoral multi-point injection every 3 days with 25 ul PBS (Blank control group) or with complexes of 15 μg siRNA, a set of 2′-o-Me and 5′ cholesterol-modified B7-H4 siRNA-2 (B7-H4 siRNA-2 treatment group) or negative siRNA (Non-target siRNA treatment group) together with 5 ul Lipofectamine 2000 respectively as previous studies described 36,37 . Tumors were measured twice a week and tumor volumes were calculated by using the formula: volume (A × B 2 )/2, where A is the larger and B is the smaller diameter. After 4 weeks, All mice were killed after Magnetic resonance imaging (MRI), and tumors were collected for histological analysis. Serial section of tumor tissues were stained with hematoxylin and eosin (H&E) and immunohistochemistry (IHC) as studies previously described 12 . Briefly, immunostaining analysis was independently performed by two pathologists. Five fields were