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Effect of Xylosyltransferase-I Silencing on Implanting Growth of Salivary Pleomorphic Adenoma

Background Salivary pleomorphic adenoma is one of the most common salivary gland tumors. It has a relatively high tendency to recur and a high risk of malignant transformation. The present study aimed to study the effect of XT-I gene silencing on the implanting growth of salivary pleomorphic adenoma. Material/Methods Primary cultures of SPA cells and fibroblasts from the same patient were assessed. The adenovirus vector Ad-shRNA-XT-I was constructed and transfected into SPA cells. The expression of XT-I gene and XT-I protein was detected by real-time PCR and Western blot. The contents of proteoglycans were detected. The SPA cells transfected with Ad-shRNA-XT-I (group SPA-XT-I) and Ad-shRNA-HK (group SPA-HK), as well as without transfection (group SPA), were implanted into ADM scaffold with fibroblasts and then transferred into 18 BALB/C-nu nude mice for 3 months. Results Primary cultures showed SPA cells were positive for human CK and S-100 protein and the fibroblasts were positive for human vimentin. The expressions of XT-I gene and protein were decreased by 51% and 51.31%, respectively. The content of proteoglycans was reduced by 48.45%. The results of the implanting growth in vitro and in vivo of nude mice indicated that no tumors grew in the SPA-XT-I group, whereas SPA grew in groups SPA-HK and SPA positive for human α-SMA, S-100 protein, and calponin. Conclusions XT-I gene silencing effectively inhibited the implanting growth of SPA.


Background
Salivary pleomorphic adenoma (SPA) accounts for 69% of salivary gland tumors. It is also called mixed tumor due to its pathologic characteristics: epithelial and modified myoepithelial elements intermingle most commonly with tissue of mucoid, myxoid, or chondroid appearance. It has a relatively high tendency to recur and has a high risk of malignant transformation [1][2][3].
Previous research by the Wang research group have indicated that after the biosynthesis of proteoglycans was suppressed, the malignant biological behaviors of SACC tumorigenesis, neurotropic growth, and lung metastasis were clearly inhibited [12][13][14][15].
However, the effect of proteoglycans on the implanting growth of SPA has been unclear. In the present study, the xylosyltransferase-I (XT-I) gene was silenced by RNAi and then resulted in decreased contents of proteoglycans. The implanting growth of SPA was evaluated in vitro and in vivo in nude mice.

Material and Methods
Cell culture Salivary pleomorphic adenoma (SPA) was obtained from a 35-year-old male patient who presented with a 15-year history of a parotid lump in the clinic of Department of Oral & Maxillofacial Surgery, Hospital of Stomatology, Hebei Medical University. This study was approved by the Ethics Committee of the Hospital of Stomatology, Hebei Medical University. The tumor was diagnosed and confirmed as a pleomorphic adenoma by 2 clinical pathologists. A part of the tumor tissue was isolated and primitively cultured in RPMI-1640 medium supplemented with 20% fetal bovine serum (Gibco, Invitrogen, USA), penicillin (100 U/ml), and streptomycin (100 U/ml) at 37°C in humidified air containing 5% CO 2 . SPA cells were identified by Giemsa stain and immunocytochemistry against human CK8 and S-100 protein (Zhongshan, China).
Ad-shRNA-XT-I was constructed and transfected The sequence of shRNA-WJ4 targeted to XT-I mRNA was: 5'-AAC AGG CAG CCC ATC AAA CCT. The sequence of shRNA-WJ4 is: 5'-GAT CCG CAG GCA GCC CAT CAA ACC TTT CAA GAC GAG GTT TGA TGG GCT GCC TGT TTT TTG TCG ACA-3'; 3'-GCG TCC GTC GGG TAG TTT GGA AAG TTC TGC TCC AAA CTA CCC GAC GGA CAA AAA ACA GCT GTT CGA-5' [12,13]. The Ad-shRNA-HK (GAC TTC ATA AGG CGC ATG C) was not isogenous with any human gene. Ad-shRNA-XT-I and Ad-shRNA-HK were constructed by Wuhan Cell Marker and Machine Company, China. The third generation of SPA cells (2×10 5 cells/100 ml) was cultured and transfected with Ad-shRNA-XT-I (group SPA-XT-I) and Ad-shRNA-HK (group SPA-HK) (100 multiplicity of infection, MOI) at 37°C for 48 h. SPA cells without transfection were used as the control (group SPA). The cell growth curves of the 3 groups were described from the 1 st day to the 7 th day after the 3 groups of cells were transfected for 48 h.

To detect XT-I and proteoglycans
Total RNA was extracted from the cells by using TRIzol reagent (Invitrogen, USA) following the manufacturer's instructions at 48 h after transfection. RNA concentration was obtained by spectrophotometric readings at 260 and 280 nm, and quality was evaluated by gel electrophoresis.
The expression of XT-I gene was detected using the 7500 Realtime PCR system (Applied Biosystems, USA). The mRNA level of each group was normalized with that of GAPDH. The total reaction volume was 20 μl, including 1 μl of the cDNA sample. The expression of XT-I gene was calculated by using the Quantification-Comparative C T (DDC T ) method.
XT-I protein concentration of the 3 groups was performed by Western blot and determined by using a bicinchoninic acid protein assay kit. We fractionated 50 μg of total protein in each group by electrophoresis through 10% polyacrylamide gels and transferred them to polyvinylidene difluoride membranes. The membranes were blocked in 5% nonfat dried milk at 37°C for 1 h, followed by incubation with goat anti-human XT-I polyclonal antibody (1: 500, Santa Cruz Biotechnology, USA) at 4°C overnight, and then incubated them with HRP-conjugated anti-goat immunoglobulin G (1: 1000, Zhongshan, China) at 37°C for 1 h. GAPDH was used as loading control. Treatment with the chemiluminescence substrate followed by exposure to X-ray film was used to examine the immunolabeled bands. The data were evaluated using GeneTool from Syngene software.

ANIMAL STUDY
After the cells of the 3 groups were transfected for 48 h, the content of total GAGs, which were the major components of proteoglycans, were measured and quantitated using the Blyscan™ Sulfated Glycosaminoglycan Assay kit (Biocolor, UK).
Software SPSS 13.0 was used for statistical analysis. All data are expressed as mean ± standard. P<0.05 was considered statistically significant.
The implanting growth of SPA Acellular dermal matrix (ADM) scaffold with size of 5×5×2 mm 3 was soaked in complete cell culture medium containing 5% hydrocortisone. Fibroblasts of subcutaneous connective tissue from the same patient were planted into an ADM scaffold, continue to culture for 2 days, and then observed by HE staining.
The cells of the 3 groups were implanted into the scaffold with fibroblasts and cultured for 7 days, after which they were observed by HE staining.
Eighteen female BALB/C-nu mice, 3~4 weeks old, were divided into 3 groups with 6 mice in each group. The nude mice were intraperitoneally injected by ketamine (40 mg/kg). The scaffolds with fibroblasts and SPA cells implanted in the 3 groups were subcutaneously transferred into the nude mice and were grown in vivo for 3 months. The implanting growth of SPA was observed by histology and immunohistochemistry against human a-SMA, S-100 protein, and calponin.

Cell culture
Salivary pleomorphic adenoma (SPA) from the patient showed microscopically by epithelial elements included duct and myoepithelial cells intermingled with myxoid appearances ( Figure 1A, 1B). After 9 days, SPA cells primitively migrated from the tissue ( Figure 1C). The subcultured cells showed polygonal, round, or spindle shape, with some myxoid regions  ( Figure 1D). The modified or neoplastic myoepithelial cells had hyperplasia and secreted proteoglycans, which displayed red granules by Giemsa staining ( Figure 1E). Neoplastic glandular cells were few and scattered with mucous vacuoles ( Figure 1F) and positive for human CK8 ( Figure 1G). Neoplastic myoepithelial cells were positive for human S-100 protein by immunocytochemistry ( Figure 1H). After 14 days, fibroblasts of subcutaneous connective tissue from the same patient were primitively migrated from the tissue (Figure 2A). The subcultured cells showed spindle shape with vortex pattern ( Figure 2B) and were positive for human vimentin ( Figure 2C, 2D).

Ad-shRNA-XT-I constructed and transfected
Group SPA-XT-I and group SPA-HK cells were transfected by Ad-shRNA-XT-I and Ad-shRNA-HK and showed the expression of green fluorescent protein (100%) (Figure 3A, 3B). There was no green fluorescent protein expressed in group SPA ( Figure 3C). The cells of the 3 groups displayed a continuous proliferation after transfection. The maximum level of cell proliferation of group SPA-XT-I was lower than those of the other 2 groups ( Figure 3D, P<0.05).

To detect XT-I and proteoglycans
The results of real-time PCR showed that XT-I gene was 51% silenced in group SPA-XT-I ( Figure 4A-4C) and was significantly different from the other 2 groups (P<0.05). Western blot analysis showed that XT-I protein was decreased by 51.31% in group SPA-XT-I, which was significantly different from the other 2 groups ( Figure 4D, 4E, P<0.05 and Table 1). The content of proteoglycans of the 3 groups was tested according to the standard curve. The proteoglycans inhibitory rate of group SPA-XT-I was 48.45%, P<0.05 ( Figure 4F; Tables 2, 3).

The implanting growth of SPA
Regarding the implanting growth of SPA in vitro, fibroblasts were implanted into ADM scaffolds and grew well ( Figure 5A). The tumor cells of the 3 groups were successfully implanted into the scaffold with fibroblasts. The results indicated that no tumors grew in group SPA-XT-I ( Figure 5B), but in group     Table 3. The GAGs contents of 3 groups (μg/10 6 cells) (mean ±SD, n=3).
Significant difference between group shRNA-XT-I and group SPA (P<0.05). Significant difference between group shRNA-XT-I and group shRNA-HK (P<0.05). No significant difference between group shRNA-HK and group SPA (P>0.05).
SPA-HK and group SPA, the tumors grew well and showed rich epithelial elements with some myxoid and chondroid appearances ( Figure 5C, 5D).
Regarding the implanting growth of SPA in vivo of nude mice: after 3 months, 18 healthy nude mice survived. The results showed that no tumors grew in group SPA-XT-I ( Figure 5E), and the tumors that grew in group SPA-HK and group SPA showed the typical characteristics of SPA with nodules or masses with rich epithelial elements, including ducts and mucoid appearances ( Figure 5F, 5G). Neoplastic myoepithelial cells were positive for human a-SMA, S-100 protein, and calponin ( Figure 5H-5J).

Discussion
Pleomorphic adenoma is the most common benign tumor of the major salivary glands, especially of the parotid gland, and it rarely occurs in others sites in the head and neck (e.g., intraosseous, lacrimal gland, chest, or maxillary vestibular sulcus) [16,17]. It has a relatively high tendency to recur, depending on the location and histological subtype of the tumor [18,19].
Xylosyltransferase-I (XT-I) is the initialized enzyme in biosynthesis of glycosaminoglycan chains in proteoglycans; it catalyzes the transfer of xylose from UDP-xylose to serine residues in proteoglycan core proteins [20,21]. Khair et al. found that in human primary chondrocytes, IL-1b regulated XT-I gene expression into an early phase of induction and a late phase of downregulation [22]. XT activity also is as a new biomarker for myofibroblast differentiation and fibrotic development [23]. Research on XT-I has focussed more on human diseases [24][25][26] and less on tumors.
In the mid-1990s, Wang et al. indicated that neoplastic myoepithelial cells have the function secreting proteoglycan, which constructs the typical histological architecture in different salivary gland tumors [4][5][6][7]. However, it is not clear what the role of neoplastic myoepithelial cells secreting proteoglycan is. A few years ago, the Wang research group revealed that the XT-I gene of SACC cells was silenced by RNAi, and the reduction of proteoglycans inhibited cell adhesion, neurotropic invasion, and lung metastasis [12,13]. Furthermore, the effect of XT-I gene silencing on the implanting growth of SPA is not known.
In the present study, the XT-I gene was silenced by RNAi; subsequently, XT-I protein was decreased and the biosynthesis of proteoglycans was suppressed, leading to obvious inhibition of the implanting growth of SPA. We demonstrated that XT-I is the key initialized enzyme in biosynthesis of proteoglycans and that it plays a critical role in the implanting growth of SPA.
For tumorigenesis, it is necessary for tumor cells to modify the surrounding stroma. The cellular components in the surrounding 3778