The Effect Of Biological Behavior In Salivary Pleomorphic Adenoma By Xylosyltransferase-â…¡ Gene Silenced

Objective:Salivary pleomorphic adenoma(SPA) is the most common benign tumour in salivary gland. It is also called mixed tumor because it includes neoplastic myoepithelial cells, neoplastic glandular epithelial cells, double-deck glandular structures, chondroid areas and myxoid areas. As a benign tumor, SPA has the malignant biological behavior of recurrence, perineural invasion and metastasis. It is difficult to avoid recurrence, perineural invasion and metastasis for SPA although SPA was completely resected. In the clinic, SPA has the potential of malignant transformation. For these reasons, molecular study of pleomorphic adenoma became a research hotspot.Proteoglycans(PGs) as the part of extracellular matrix, have a close relationship with the formation and development of tumor. Xylosyltransferase-I(XT-I) and Xylosyltransferase-II(XT-II) are the key enzythesis that initiates glycosaminoglycans(GAGs) chain which by transferring the first sugar residue to the PG core protein. Wang Jie team turned to the study of biological behavior between SPA and PGs nearly one decade. And the team reported that PGs secreated by neoplastic myoepithelial cells of salivary adenoid cystic carcinoma(SACC) has a close relationship with invasion and metastasis of SACC.SPA cells were cultured by adhesive culture methods. The adenovirus vector named Ad-sh RNA-XT-II and Ad-sh RNA-HK was constructed and transducted into SPA cells to silence the XT-II gene by RNA interference technology. The SPA cells which transducted with Ad-sh RNA-XT-II and Ad-sh RNA-HK was named Group SPA-XT-II and Group SPA-HK. The SPA cells without any transducted were named Group SPA. XT-II genes and the XT-II proteins were detected by Real-time RCR and Weastern blot respectively. The content of PGs was detected by silenced XT-II gene. The biological behaviour of SPA was observed by silenced XT-II gene.This study was sponsored by the National Science Foundation. The methors of cultivation of the SPA cells in vitro was established by Wang Jie team. The adenovirus vector Ad-sh RNA-XT-II was transducted into SPA cells by RNA interference technology. XT-II gene and XT-II protein were detected by Real-time PCR and western blot. The synthesis of PGs was detected by silenced XT-II gene. The invasion and metastasis of SPA was observed by decresed the synthesis of PGs. And this team has secured patented technology of constructing tissue-engineered scaffolds and establish animal model of pleomorphic adenoma in 2012. The implanted biological behavior of SPA was observed by silenced XT-II gene.Methods:1 The effect of PGs secreted by neoplastic myoepithelial cells on SPA by XT-II gene silencedThe slivary pleomorphic adenoma cells were cultured and identified by Giemsa and immunocytochemimal stain against calponin, S-100 protein, and α-SMA. The adenovirus vector which was named Ad-sh RNA-XT-II(Group SPA-XT-II) and Ad-sh RNA-HK(Group SPA-HK) was constructed and transducted into SPA cells to silence the XT-II gene by RNA interference technology. The contents of proteoglycans of SPA were detected after the cells were transducted for 48 hours.2 The effect of biological of invasion and metastasis on SPA by XT-II gene silencedThe slivary pleomorphic adenoma cells were cultured and identified by immunocytochemimal stain against calponin, S-100 protein, and α-SMA. The adenovirus vector named Ad-sh RNA-XT-II(Group SPA-XT-II) and Ad-sh RNA-HK(Group SPA-HK) was transducted into SPA cells to silence the XT-II gene by RNA interference technology. The invasion and metastasis biological behaviour of SPA were observed by matrigel invasion assay and mound healing assay after XT-II gene silenced.3 The effect of implanted biological behaviour of SPA by XT-II gene silencedThe SPA cells and fibroblasts from tumor capsule were cultured by adhesive culture methods. Fibroblasts were planted into acellular dermal matrix(ADM) which was soaked by completely cell culture medium containing 5% hydrocortisone. Tissue engineering fibroblasts scaffold were observed by HE staining and scanning electron microscope. The SPA cells and the SPA cells which were transducted with Ad-sh RNA-XT-II and Ad-sh RNA-HK were implaned into tissue engineering fibroblasts scaffold and named Group SPA-XT-II, Group SPA-HK and Group SPA and which were observed by HE staining, immunohistochemistry and scanning electron microscope. The cells in three groups and tissue engineering fibroblasts scaffold were implanted into BALB/C-nu mice. 18 nude mice were randomised into three groups. The transplanted complexus were observed by histology and immunohistochemistry after three months.Results:1 The effect of PGs secreted by neoplastic myoepithelial cells on SPA by XT-II gene silenced1.1 The sample contained neoplastic cells, double-layer glandular structures, and chondroid areas and it was diagnosed with SPA.1.2 The primary culture of human SPA tissue, polygons cells migrated from the tissue after two weeks. Subcultured cells were observed in a polygon, with round and oval nucleus.1.3 Giemsa staining of the second generation of SPA cells showed that neoplastic myoepithelial cells(NMCs) grew around neoplastic glandular epithelium cells(NGECs). Giemsa staining of the fourth generation of SPA cells showed that NMCs were polygon shape and secreted PGs which stained red granules, whereas NGECs were almost vanished in the fourth generation cells. Immuncytochemistry showed calponin, S-100 protein and α-SMA were positive in the NMCs.1.4 1% agarose gel result and the sequencing results showed that Ad-sh RNA-XT-II was constructed successfully. Forty-eight hours after the transduction of Ad-sh RNA-XT-II and ad-sh RNA-HK into the fourth generation of SPA cells, the transduction rate of green fluorescent protein(GFP) in both groups was approximently 100%. There is no expression of GFP in SPA cells without any transduction.1.5 Real-time PCR showed that XT-II gene was silenced by 42.72% in Group SPA-XT-II. There was no XT-II gene silenced in Group SPA-HK. Compared with Group SPA-HK and SPA, Group SPA-XT-II was siginificantly different(P<0.05).1.6 Gene Tool from Syngene software was performed in this study. The results showed that the relative expression of XT-II protein was 0.44±0.09, 0.86±0.16 and 0.77±0.11 in Group SPA-XT-II, Group SPA-HK and Group SPA respectively. The XT-II protein was decreased by 34% in Group SPA-XT-II.1.7 The content of PGs(the total GAGs) in three groups were tested according to the standard curve. The PGs inhibitory rate in Group SPA-XT-II was 41.15%(47.78±3.16 μg) compared with Group SPA(81.18±2.80 μg). The PGs were not inhibited significantly in Group SPA-HK(77.58±2.01 μg). One-way ANOVA showed that Group SPA-XT-II was significantly different compared with Group SPA-HK and Group SPA(P<0.05).2 The effect of biological behaviour of invasion and metastasis on SPA by XT-II gene silenced2.1 SPA cells primary cultured, identification and transduction were same as Section One.2.2 Matrigel invasion assay showed that the number of cells through the membrane were 14.40±2.50, 39.90±4.61 and 40.80±4.63 in Group SPA-XT-II, Group SPA-HK and Group SPA by microscope at 400 magnification respectively. 64% inhibition of cells invasion was recorded in Group SPA-XT-II.2.3 The distance of cells moved to the scratched wound region in Group SPA-XT-II, Group SPA-HK and Group SPA was 374.40±194.05 pixel,1164.50±276.58 pixel and 873.50±229.27 pixel after four days cultured. One-way ANOVA showed that Group SPA-XT-II was significantly different compared with Group SPA-HK and Group SPA(P<0.05). There was no different between Group SPA-HK and Group SPA(P>0.05).3 The effect of implanted biological behaviour on tissue-engineered SPA by XT-II gene silenced3.1 SPA cells primary cultured was same as Section One.3.2 The fibroblasts were spindle-shaped, circinate and radial. And fibroblasts were positive against vimentin by immunofluorescence chemical staining and immunocytochemical staining.3.3 The transduction of SPA cells was same as Section One.3.4 HE stainning and SEM showed that fibroblasts were successfully implanted into ADM.3.5 The cells in Group SPA-XT-II, Group SPA-HK and Group SPA were implanted into the tissue engineering fibroblasts scaffold. HE, immunohistochemistry and SEM showed that fibroblasts were more than tumor cells which were distributed in the fibroblasts. The tumor cells were pyknotic in Group SPA-XT-II.3.6 SPA cells in three groups implanted into tissue engineering fibroblasts scaffold in nude mice3.6.1 SPA cells in three groups implanted and tissue engineering fibroblasts scaffold grew in subcutaneous of nude mice for three months. The capillaries grew on the surface of ADM through macroscopy. The tumors were not seen with unaided eye in each group.3.6.2 Histological observation3.6.2.1 The adipocyte, tumor cells and blood vessels were observed in Group SPA-XT-II, but there were no tumor formation by HE staining. The adipocyte, tumor cells, blood vessels, double-deck glandular structures and neoplastic myoepithelial cells were observed in Group SPA-HK and Group SPA. There was no different between Group SPA-HK and Group SPA.3.6.2.2 The compound contain SPA cells in three groups and tissue engineering fibroblasts scaffold from nude mice were positive against S-100 protein, CK8&18 and α-SMA, but negative against calponin in Group SPA-XT-II, Group SPA-HK and Group SPA by immunohistochemistry stain.Conclusions:1 The adenovirus vector of xylosyltransferase II was successfully constructed.2 The biosynthesis and secretion of PGs in SPA cells could be inhibited by XT-II gene silenced.3 The invasion and metastasis of SPA cells could be inhibited by decreasing the synthesis of PGs.4 The growth of pleomorphic adenoma was inhibited by decreasing the synthesis of PGs.