The Effect Of Secretion Inhibited Of Proteoglycans On The Growth Of Salivary Adenoid Cystic Carcinoma

ObjectivesProteoglycans are an important glycosylated macromolecules, which represent one of the largest and most complex molecular structures in human body. Proteoglycans are the major components of the extracellular matrices and consist of core protein and glocosaminoglycan (GAGs) chains which attached to the core protein. Proteoglycans are increasingly implicated as important regulators in many biological processes, such as extracellular matrix (ECM) deposition, cell membrane signal transfer, cell differentiation and morphogenesis, cell adhesion and migration, normal and tumor cell growth, and viral infection. More and more reseachers have paid attention to the study of proteoglycans function in the tumorigenesis.Xylosyltransferase (XT-I) catalyzes transfer of xylose from UDP-xylose to selected serine residues in the proteoglycans core protein, which is the initial and rate-limited step in the proteoglycans biosynthesis of human body. The role of XT-I is the key to the biosynthesis of proteoglycans. It had been determined that xylosyltransferase-I activity is a clinical biochemical marker of an altered proteoglycans biosynthesis in 2006. The effect of XT-I on human health has become a new reseach focus in recent two years.Salivary adenoid cystic carcinoma (SACC) is one of most common malignancy in salivary gland tumors, accounting for approximately 10% of salivary gland tumors and 30% of human malignancy. Recurrence or early metastasis are its biological properties. SACC occurred in all ages with a peak incidence in 40-60 years old. Lung is the most common distant organ of SACC metastasis. SACC is composed of two kinds of cells: neoplastic duct epithelial cells and neoplastic myoepithelial cells (NMCs) which are chief proliferative cells in SACC. The neoplastic myoepithelial cells have secreted proteoglycans full of the cribriform or tubular and glandular structures. It is demonstrated that proteoglycans provide the essential microenvironment for the growth and metastasis of SACC. A great deal of studies have approved that there is a close relationship between ECM and the biological feature of SACC. However, there is rare study of the proteoglycans effect on the carcinogenesis and biological behavior of SACC.RNA interference is the most powerful gene blocking technology up to the date. The expression of a special gene can be efficiently and rapidly silenced by RNAi. RNAi can inhibit the transcription product and down regulate the protein expression of the target gene. A number of experimental studies have made it clear that abnormal proliferation and metastasis of tumor cells can be inhibited efficiently by RNAi. All these results provide a good basis for salivary gland tumor gene reseach by using RNAi technology.In this study, the salivary adenoid cystie carcinoma cell line with high tendency of lung metastasis (ACC-M) was selected as the reseach model. Human XT-I gene was knockdown by shRNA (short hairpin RNA) expression system in order to inhibit the biosynthesis of proteoglycans in ACC-M cells. The effect of down-regulated proteoglycans on the growth and biological activities were observed, the relationship between proteoglycans and the carcinogenesis of SACC was discussed. This reseach will provide a new idea for the useage of RNAi in salivary gland tumor treatment.Methods1. Preparation and identification of anti-human polyclonal antibody against xylosyltransferase-IThe epitope of XT-I gene was predicated by the DNAssist software . The DNA fragment of this epitope region was synthesized by PCR and cloned into the pGEX-4T-2 vector. The recombinant plasmid was transferred into E.coli ER2566 and the fusion protein GST-XT was induced and isolated. The purified fusion protein was used to immunize New Zealand rabbits. The antibody titer was determined by ELISA. Purified polyclonal antibody was obtained through affinity chromatography column and the specificity of the purified antibody was characterized by Western blot.2. Construction of eukaryotic expression vector of short hairpin RNA targeting human xylosyltransferase-I geneShort chain oligonucleotides were designed according to the mRNA sequence of XT-I provided by Genbank . The DNA segments were gained through annealing after chemosynthesis and cloned into pGgenesil-1 vector . The recombinant XT-I shRNA expression vectors were identified by digestion and sequencing analysis. Meanwhile, a sequence was not isogenous with any human genes selected as negative control.3. The detection of the silince efficiency of shRNA expression vectors and the selection of stable cells in which the biosynthesis of proteoglycans was down-regulatedThe constructed XT-I expression vectors were transfected into ACC-M by LipofectameneTM 2000. The expression of green fluorescent protein (GFP) was detected by inverted fluorescent microscope and the ratio of transfection were detected. G418 (500μg/ml) was added into the transfected cells in order to collect stable cells.Real-Time PCR was used to detect the expression of mRNA level of XT-I and the protein expression of XT-I was detected by Western blot in the stable cells. The proteoglycans contents of the cells were tested by biocolor measure method.4. The effect of down-regulated proteoglycans on the proliferation of ACC-M cell by silencing the XT-I geneACC-M-WJ3 cell (the postive group) was chosen to be detected in this part and ACC-M-HK (the negative control ),ACC-M (the blank control )were detected too.The growth curve and MTT assay were made and clones fomation assay was performed to detect the state of cell proliferation in vitro. The cell cycle and apoptosis were analyzed by flow cytometer.Fifteen four-week-old famale BALB/C nude mice were divided into three groups of 5 mice each randomly (weight: 16-17g), and the nude mice transplant model of the cells were established by subcutaneous injection of ACC-M-WJ3 cells,ACC-M cells and ACC-M-HK cells, respectively, with 0.2ml cell cell suspensions containing 2×106 cells on the left anterior back. The proliferation of the xenograft was measured every 3 days for 4 weeks. The mice were killed in the 28th day of injection. The xenograft were measured, weighed, then fixed in formaldehyde, embedded and HE staining for histological examination.5. The effect of down-regulated proteoglycans on the potential of metastasis of ACC-M by silencing the XT-I geneIn this study, three groups were divided, they were ACC-M-WJ4 cell (the postive group), ACC-M-HK cell (the negative control) and ACC-M cell (the blank control). The cell adhesion ability of the three groups was determined by adhesion assay. Matrigel invasion assay and wound healing assay were performed to detect the ability of cell invasion and metastasis in vitro. Adhesion assay: The three kinds of cells mentioned above were planted in 96-well plates coated with Matrigel and BSA, repectively. After being incubated in 5% CO2 and 37℃for 1h, the absorption value (A value, wavelength was 490nm )of cells in each well was detected by MTT method and the adhesion ratio of different cells was determined.Wound healing assay: The three kinds of cells were planted in 6-well plates and were scraped with a sterile pipette tip to create a wound, thereafter washed by PBS twice. The distance of the wound was recorded. After being incubated in 5% CO2 and 37℃for 24h in DMEM supplemented with 10g/L BSA and 1% FBS, the cells were incubated in 5% CO2 and 37℃for another 12h in complete DMEM supplemented with 10% FBS. The cells migrating into the wound were recorded and the closure hours of the wound was recorded too.Matrigel invision assay: A total of 200μl cell suspensions containg 2×104 cell was plated into the upper millicell chamber coated with matrigel. DMEM supplemented with 10g/L BSA and 1% FBS was added into the upper millicell chamber with micropore PVPF membrane too, 400μl DMEM with chemotactic factor were added into the 12-well plates in which the millipore chamber were placed. After being incubated in 5% CO2 and 37℃for 24h, the millicell chambers were taken out and fixed with methanol for 15min, HE staining. The cells migrating to the lower side of PVPF membrane were counted, and the inhibitory rate was determined.Tweenty four-week-old famale BALB/C nude mice were divided into four groups of 5 mice each randomly (weight: 16-17g). To establish nude mice lung metastasis models, ACC-M-WJ4 cells, ACC-M cells, ACC-M-HK cells of 0.2 ml cell suspensions containg 2×106 cells, and normal saline were injected into the lateral tail vein of each group, respectively.The mice were killed in the 6th week of injection. The nude mice and their fresh lung samples were weighed. The number of metastatic nodes on the surface of lung was observed, then the lung samples were fixed in formaldehyde for histological examination by light microscope to evaluate the metastatic tumors.Statistical analysis:All data were expressed as mean±SD. Data of the re- presentatives were analyzed for statistical significance by using one way ANOVA. P< 0.05 was considered statistically significant.Results1. Preparation and identification of anti-human polyclonal antibody against xylosyltransferase-IThe amino acid 175-205 of XT-I was selected as an antigen epitope. The synthesized DNA fragment of XT-I was successfully inserted into pGEX-4T-2 vector and the protein GST-XT was expressed. The purified fusion protein GST-XT was used as the immunogen to immunize rabbits, and the polyclonal antibody against XT-I protein was obtained. The result of ELISA showed that the antibody titer was 1: 6.4×105. Western blot analysis showed that the antibody had a good specificity.2. Construction of eukaryotic expression vector of short hairpin RNA targeting human xylosyltransferase-I gene. Two eukaryotic expression vectors of short hairpin RNA targeting XT-I gene were constructed and called shRNA-WJ3 and shRNA-WJ4. Successful constructions were identified by digestion and sequencing analysis.3. The plasmids expressing shRNA targeting XT-I gene was transfected into ACC-M cells by Lipofectemane 2000. ACC-M cells transfected with the plasmids expressed GFP successfully. After G418 selection, two stably XT-I silenced sub-clone cell lines named ACC-M-WJ3 and ACC-M-WJ4 were isolated. The stable cell of negative control transfected with shRNA-HK was selected too.ShRNA-WJ3 and shRNA-WJ4 showed powerful RNAi gene silencing of inhibitory. The inhibitory rate of shRNA-WJ3 was 83.7% of mRNA level and 79.60% of protein level, respectively. The inhibition rate of shRNA-WJ4 was 86.81% of mRNA level and 80.1% of protein level, respectively. The contents of proteoglycans was down–regulated by 49.7%- 66.10% per 24h.4. The effect of down-regulated proteoglycans on the proliferation of ACC-M cells by silencing the XT-I gene.By inverted microscope, ACC-M cells transfected with shRNA expression vectors showed apoptosis obviously. There were many apoptotic cells with their cytoplasm shrank in group ACC-M-WJ3 and ACC-M-WJ4. The chromatin condensed into block and dispersed unevenly. There was no change observed in the controls.The growth curve and MTT assay showed: the cell growth and proliferation were inhibited significantly. Clones fomation assay showed that the fomation rate of group ACC-M-WJ3 (18.93±5.43%) was much lower than that of group ACC-M (37.40±2.86%) and group ACC-M-HK (32.87±5.70%) ; P<0.05.The percentage of cell distribution: in group ACC-M-WJ3, S phrase was decreased and G1/G0 phrase was increased compared with group ACC-M-HK and group ACC-M (P<0.05). There was no difference in G2/M phrase among the three groups (p>0.05)。The PI of ACC-M-WJ3,ACC-M and ACC-M-HK was:25.1,60.63,52.63, respectively (P<0.01). The apoptosis rate of group ACC-M-WJ3 was (28.44±3.45)%, significantly higher than that of the two others (group ACC-M: 3.21±1.50 %; group ACC-M-HK: 7.34±1.50 % ), P<0.01.In nude mice transplantation experiment, the growth of xenograft in group ACC-M-WJ3 was significant inhibited. The mean volume and weight of the implanted tumor in group ACC-M-WJ3 [(1.78±0.71)cm2 , (2.21±0.70) g ] were significantly lower than those of group ACC-M-HK [(2.83±0.14)cm2, (3.63±0.12) g ] and group ACC-M [ (3.09±0.06) cm2, (4.97±0.54) g ] (P<0.01). The weight inhibitory rate of group ACC-M-WJ3 was 42.39%.There were many regions showed apoptosis in the transplanted tumors of group ACC-M-WJ3. The cell nuclear disappearance could be observed in many regions of ACC-M-WJ3, and the cytoplasm of some apoptotic cell shrank. Apoptotic bodies were formed in some regions of group ACC-M-WJ3. The tumor cells of group ACC-M and group ACC-M-HK showed small, round in mass, no obvious apoptosis was observed.5 The effect of down-regulated proteoglycans on the potential of metas -tasis of ACC-M by silencing the XT-I geneThe adhesion rate of group ACC-WJ4 was (13.43±1.91)%, much lower than that of group ACC-M (23.30±1.45)% and group ACC-M-HK (22.43±2.78)% ; P<0.05. The inhibitory rate was 42.36%. Matrigel invasion assay showed that the cell number of group ACC-M-WJ4 migrating to the lower side of the PVPF membrane was 41.50±8.3; much lower than that of group ACC-M-HK (83.53±10.21) and group ACC-M (90.50±15.28); P<0.01. The inhibitory rate was 54.14 %.Wound healing assay showed that the cell of group ACC-M-WJ4 migrating into the wound was 115.92±6.81, much lower than that of group ACC-M-HK (283.53±9.72) and group ACC-M (289.50±23.02); P<0.01.In the experimental lung metastatic models, the rate of lung metastasis of group ACC-M-WJ4 was 40% (2/5), significantly lower than that of group ACC-M-HK (100%) and group ACC-M (100%), P<0.05. The number of metastatic nodes on the surface of lungs was (49.4±4.81) in group ACC-M, (41.6±8.09) in group ACC-M-HK and (4.8±3.89) in group ACC-M-WJ4, respectively; P<0.01.The lung weight of group ACC-M-WJ4 (0.226±0.83) was significantly lower than that of group ACC-M (0.897±0.21)and group ACC-M-HK (0.786±0.28g), P<0.01.Concluson1. The rabbit polyclonal antibody against human XT-I protein has been prepared successfully with good titer and specificity. It can be used to detect the expression of XT-I protein in human cells.2. The eukaryotic expression vector of short hairpin RNA targeting XT-I gene are effective with high efficiency of XT-I gene silence. The inhibitory rate of shRNA-WJ3 was 83.70% of mRNA level and 79.60% of protein level; the inhibition rate of shRNA-WJ4 was 86.81% of mRNA level and 80.10% of protein level, respectively.3. The silence of XT-I gene effectively resulted in the inhibition of the biosynthsis of proteoglycans in ACC-M cells. The contents of proteoglycans were down–regulated by 49.7%-66.06% per 24h in ACC-M-WJ3 cell and ACC-M-WJ4 cell.4. The reduction of proteoglycans could inhibit the proliferation of ACC-M significantly and induce cell apoptosis in vitro. The inhibitory rate of clones formation was 49.39% and the apoptosis rate was 28.44±3.45%. The percentage of cell distribution was changed too: S phrase was decreased and G1/G0 phrase was increased.5. The nude mice transplantation experiment showed that the growth of xenograft in group ACC-M-WJ3 with down-regulated proteoglycans biosynthsis was significant inhibited. Cell apoptosis could be observed.6. The reduction of proteoglycans could inhibit the ability of cell adhesion, invision and metastatic properties of ACC-M cells. The inhibitory rate of cell adhesion was 42.36%. The inhibitory rate of invision was 54.14 %.7. The reduction of proteoglycans could inhibit the metastatic ability of ACC-M cell significantly, the rate of lung metastasis was decreased to 40%.In conclusion, by using RNAi technology, We showed that inducible knockdown of XT-I gene expression in ACC-M cells resulted in significant inhibition of proteoglycans biosynthesized by ACC-M cells. The reduction of proteoglycans could inhibit the biological activities of ACC-M cell significantly including: cell growth, adhesion, invision and metastasis. This reseach approved that there is a close relationship between proteoglycans and the biological behavior of SACC.