METTL14 Promotes Oral Squamous Cell Carcinoma Progression by Regulating the mRNA and m6A Levels of CALD1.

Oral squamous cell carcinoma (OSCC) still threatens people's daily life. METTL14 is a newly discovered methyltransferase that catalyzes m6A methylation. Hence, this research was carried out to investigate the action mechanism of METTL14 in OSCC. The SCC-4 and UM2 cells, and tumorigenicity assay were utilized to investigate METTL14 roles in vitro and in vivo. Bioinformatic analysis was carried out with the UCSC, TCGA database and The Human Protein Atlas. The gene expression at mRNA and protein levels were measured by qRT-PCR and Western blot. In addition, cell growth and metastasis was analyzed by colony formation and transwell assays. MeRIP assay was performed to test the m6A levels of CALD1. The METTL14 and CALD1 levels were prominently expressed in OSCC cells. METTL14 silencing depleted the cell growth and metastasis. Furthermore, METTL14 silencing depleted the tumor growth in vivo. Additionally, the mRNA and m6A levels of CALD1 were depleted after METTL14 silencing. Overexpressed CALD1 neutralized the si-METTL14 effects in OSCC cells. In conclusion, METTL14 participated in the OSCC progression through modulating the mRNA and m6A levels of CALD1.

Effect of tumor-stromal fibroblasts on the biological behavior of salivary gland pleomorphic adenoma cells in vitro.

OBJECTIVES: This study aims to investigate the effects of tumor-stromal fibroblasts (TSFs) on the proliferation, invasion, and migration of salivary gland pleomorphic adenoma (SPA) cells in vitro. METHODS: Salivary gland pleomorphic adenoma cells (SPACs), TSFs, and peri-tumorous normal fibroblasts (NFs) were obtained by tissue primary culture and identified by immunocytochemical staining. The conditioned medium was obtained from TSF and NF in logarithmic phase. SPACs were cultured by conditioned medium and treated by TSF (group TSF-SPAC) and NF (group NF-SPAC). SPACs were used as the control group. The proliferation, invasion, and migration of the three groups of cells were detected by MTT, transwell, and scratch assays, respectively. The expression of vascular endothelial growth factor (VEGF) in the three groups was tested by enzyme linked immunosorbent assay (ELISA). RESULTS: Immunocytochemical staining showed positive vimentin expression in NF and TSF. Results also indicated the weak positive expression of α-smooth muscle actin (SMA) and fibroblast activation protein (FAP) in TSFs and the negative expression of α-SMA and FAP in NFs. MTT assay showed that cell proliferation in the TSF-SPAC group was significantly different from that in the NF-SPAC and SPAC groups (P<0.05). Cell proliferation was not different between the NF-SPAC and SPAC groups (P>0.05). Transwell and scratch assays showed no difference in cell invasion and migration among the groups (P>0.05). ELISA showed that no significant difference in VEGF expression among the three groups (P>0.05). CONCLUSIONS: TSFs may be involved in SPA biological behavior by promoting the proliferation of SPACs but has no effect on the invasion and migration of SPACs in vitro. Hence, TSF may be a new therapeutic target in SPA treatment.

A proteomics study to explore differential proteins associated with the pathogenesis of ameloblastoma.

BACKGROUND: Reports on the proteomic studies of ameloblastoma and other common odontogenic lesions are limited. We thus explored the differential proteins among ameloblastoma, odontogenic keratocyst, dentigerous cyst, and normal gingival tissue using proteomics and identified hub proteins involved in the local aggressiveness and recurrence of ameloblastoma. METHODS: Samples were obtained from 14 patients with ameloblastoma, 6 with odontogenic keratocyst, 9 with a dentigerous cyst, and 5 with normal gingival tissue. Proteins were then extracted, purified, quantified, and analysed using Easy-nLC chromatography and mass spectrometry. Further functional annotation and enrichment analyses were performed using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes on the target protein collection. Protein clustering and protein-protein interaction network analyses were used to screen the hub proteins. Proteins with significant interactions were screened according to their degree index. These results were verified by immunohistochemical staining. Proteins meeting the screening criteria of expression difference ploidy >1.2-fold (upregulation and downregulation) and p < 0.05 were considered differential proteins. RESULTS: In ameloblastoma, 808 differential proteins were upregulated and 505 were downregulated compared with those in odontogenic keratocyst; 309 were upregulated and 453 were downregulated compared with those in dentigerous cyst; and 2210 were upregulated and 829 were downregulated compared with those in normal gingival tissue. The three groups of differential proteins were associated with cellular exosomes, antigen binding, complement activation, human papillomavirus infection, focal adhesion, cell adhesion molecules, and metabolic pathways. CONCLUSION: CDH3 is associated with the local aggressiveness and recurrence of ameloblastoma and is a potential therapeutic target.

OncoPubMiner: a platform for mining oncology publications.

Updated and expert-quality knowledge bases are fundamental to biomedical research. A knowledge base established with human participation and subject to multiple inspections is needed to support clinical decision making, especially in the growing field of precision oncology. The number of original publications in this field has risen dramatically with the advances in technology and the evolution of in-depth research. Consequently, the issue of how to gather and mine these articles accurately and efficiently now requires close consideration. In this study, we present OncoPubMiner (https://oncopubminer.chosenmedinfo.com), a free and powerful system that combines text mining, data structure customisation, publication search with online reading and project-centred and team-based data collection to form a one-stop 'keyword in-knowledge out' oncology publication mining platform. The platform was constructed by integrating all open-access abstracts from PubMed and full-text articles from PubMed Central, and it is updated daily. OncoPubMiner makes obtaining precision oncology knowledge from scientific articles straightforward and will assist researchers in efficiently developing structured knowledge base systems and bring us closer to achieving precision oncology goals.

Comprehensive analysis of DNA methylation for periodontitis.

BACKGROUND: Periodontitis is an infectious disease, and a risk factor for peri-implantitis that could result in the implant loss. DNA methylation has an essential role in the etiology and pathogenesis of inflammatory disease. However, there is lack of study on methylation status of genes in periodontitis. This study sought to explore the gene methylation profiling microarray in periodontitis. METHODS: Through searching in the Gene Expression Omnibus database, a gene methylation profiling data set GSE173081 was identified, which included 12 periodontitis samples and 12 normal samples, respectively. Thereafter, the data of GSE173081 was downloaded and analyzed to determined differentially methylated genes (DMGs), which then were used to perform Gene Ontology analysis and pathway enrichment analyses through online database. In addition, the DMGs were applied to construct the protein-protein interaction (PPI) network information, predict the hub genes in pathology of periodontitis. RESULTS: In total 668 DMGs were sorted and identified from the data set, which included 621 hypo-methylated genes and 47 hyper-methylated genes. Through the function and ontology analysis, these 668 genes are mainly classified into intracellular signaling pathway, cell components, cell-cell interaction, and cellular behaviors. The pathway analysis showed that the hypo-methylated genes were mostly enriched in the pathway of cGMP-PKG signaling pathway; RAF/MAP kinase; PI3K-Akt signaling pathway, while hyper-methylated genes were mostly enriched in the pathway of bacterial invasion of epithelial cells; sphingolipid signaling pathway and DCC mediated attractive signaling. The PPI network contained 630 nodes and 1790 interactions. Moreover, further analysis identified top 10 hub genes (APP; PAX6; LPAR1; WNT3A; BMP2; PI3KR2; GATA4; PLCB1; GATA6; CXCL12) as central nodes that are involved in the immune system and the inflammatory response. CONCLUSIONS: This study provides comprehensive information of methylation status of genes to the revelation of periodontitis pathogenesis that may contribute to future research on periodontitis.

Framework Nucleic Acid-Based Spatial-Confinement Amplifier for miRNA Imaging in Living Cells.

Real-time in situ monitoring of miRNAs in living cells is often appealed to signal amplifiers to tackle their low abundance challenges. However, the poor kinetics of amplifiers and potential interferences from the complex intracellular environment hamper its widespread applications in vivo. Herein, we report a framework nucleic acid (FNA)-based nonenzymatic spatial-confinement amplifier for rapid and reliable intracellular miRNA imaging. The amplifier consists of a localized catalytic hairpin assembly (L-CHA) reactor encapsulated in the inner cavity of an FNA (a 20 bp cube). The L-CHA reactor is certainly confined to the internal frame by integrating two probes (H1 and H2) of the L-CHA within a DNA strand and harnessing it to the opposite angles of the cube. We find that the stability of the amplifier is remarkably improved due to the protection of the FNA. More importantly, the spatial-confinement effect of the FNA can endow the confined L-CHA amplifier with enhanced local concentrations of reagents (5000-fold), thereby accelerating the reaction rate and improving the dynamic performance (up to 14.34-fold). With these advantages, the proposed amplifier can enable accurate and effective monitoring of miRNA expression levels in living cells and poses great potential in medical diagnostics and biomedical research.

In situ growth of ZIF-8 on gold nanoparticles/magnetic carbon nanotubes for the electrochemical detection of bisphenol A.

We herein report a facile and scalable strategy for the fabrication of a metal-organic framework (MOF) based composite by in situ growing ZIF-8 on gold nanoparticle (AuNP) loaded magnetic carbon nanotubes (mCNTs). AuNPs were firstly loaded on PEI (polyethylenimine) modified mCNTs by electrostatic forces, and then AuNPs/mCNTs were encapsulated into the ZIF-8 frame through in situ self-assembling of zinc ions and 2-methylimidazole. The morphology, spectroscopy and structural properties of the AuNP/mCNT@ZIF-8 nanocomposites were systematically characterized. The conductivity-strain tests revealed that the in situ insertion of AuNPs/mCNTs in ZIF-8 could not only shorten the electron transfer distance between active sites and mCNTs, but also increase the dispersion of mCNTs, which would benefit the electron and mass transfer. Besides, by adopting the AuNP/mCNT@ZIF-8 nanocomposite-modified glassy carbon electrode (GCE) as the working electrode, a novel electrochemical sensor was successfully developed for the detection of bisphenol A (BPA). A linear range of BPA detection from 1 µM to 100 µM with a limit of detection of 690 nM was favorably obtained. Moreover, the developed sensor exhibited satisfactory reproducibility and superior stability with excellent anti-interference ability, and was successfully applied in the detection of BPA in real samples.

NT-3 Promotes Oligodendrocyte Proliferation and Nerve Function Recovery After Spinal Cord Injury by Inhibiting Autophagy Pathway.

BACKGROUND: Spinal cord injury (SCI) is a serious medical problem, leading to lifelong disability and increasing the health burden worldwide. Traditional treatments have limited effects on neuronal function recovery. Previous studies showed that neurotrophin-3 (NT-3) promoted oligodendrocyte survival and improved neuronal functional recovery after SCI. However, the mechanism by which NT-3 promotes oligodendrocyte survival after SCI remains unclear, which limits its application. MATERIALS AND METHODS: A total of 75 female Sprague-Dawley rats were randomly divided into three groups: the NS group, NT-3 group, and NT-3 + rapamycin group. After successful modeling, the spinal cord specimens were taken at the corresponding time points. Western blot was used to detect autophagy-related proteins and Olig1 protein expression and combined with pathology, immunohistochemistry, flow cytometry, and other methods to detect the proliferation of oligodendrocytes after NT-3 application. RESULTS: NT-3 was found to significantly promote the recovery of motor function by Basso-Beattie-Bresnahan scores analysis in the rat SCI model. Furthermore, intraspinal administration of NT-3 could downregulate the expression of Beclin-1 in oligodendrocytes, indicating that NT-3 could inhibit excessive autophagy of oligodendrocytes after SCI. The effects of NT-3 on oligodendrocyte survival could be blocked by an autophagy activator rapamycin. CONCLUSIONS: This study found that NT-3 could promote the recovery of motor function after SCI in rats. The underlying reason may be that NT-3 inhibits the expression of autophagy proteins in oligodendrocytes and promotes oligodendrocyte proliferation. This study provided evidence for the future clinical application of NT-3 in SCI patients.

Cytocompatibility and biologic characteristics of synthetic scaffold materials of rabbit acellular vascular matrix combining with human-like collagen I.

Scaffold material provides a three-dimensional growing environment for seed cells in the research field of tissue engineering. In the present study, rabbit arterial blood vessel cells were chemically removed with trypsin and Triton X-100 to prepare rabbit acellular vascular matrix scaffold material. Observation by He&Masson staining revealed that no cellular components or nuclei existed in the vascular intima and media after decellularization. Human-like collagen I was combined with acellular vascular matrix by freeze-drying to prepare an acellular vascular matrix-0.25% human-like collagen I scaffold to compensate for the extracellular matrix loss during the decellularization process. We next performed a series of experiments to test the water absorbing quality, biomechanics, pressure resistance, cytotoxicity, and ultra-micro structure of the acellular vascular matrix composite material and natural rabbit artery and found that the acellular vascular matrix-0.25% human-like collagen I material behaved similarly to natural rabbit artery. In conclusion, the acellular vascular matrix-0.25% human-like collagen I composite material provides a new approach and lays the foundation for novel scaffold material research into tissue engineering of blood vessels.

Study of composite vascular scaffold combining with differentiated VSMC- and VEC-like cells in vitro and in vivo.

Although research into the tissue engineering of vessels has proceeded at a tremendous pace, many deficiencies still need to be resolved. A well-adopted constructed vessel requires both functional and structural properties to stimulate the native vessel and resist stress and tension in vivo. In the present study, we developed a novel three-layer composite vascular scaffold consisting of differentiated vascular smooth muscle cell-, vascular endothelial cell-like cells, and a rabbit acellular vascular matrix (ACVM)-0.25% HLC-I scaffold. HE staining, immunohistochemical assays, immunofluorescence assays (IFAs), and scanning electron microscopy were performed to monitor the growth status of cells on the scaffold material in vitro. After the vascular endothelial cell -vascular smooth muscle cell-scaffold was implanted into nude mice for three, six, and nine weeks, samples were harvested from the implanted mice and observed visually or by HE staining and IFAs for cell viability and morphology. Additionally, burst pressure resistance experiments were used to assess the maximal pressure that the engineered vessel could resist. We found that the engineered vascular endothelial cell-vascular smooth muscle cell-scaffold vessel possessed favorable biocompatibility and considerable strength, matching native vessels in vivo and in vitro, and may be significant in the future clinical implantation of tissue-engineered vasculature.

Induced differentiation of human gingival fibroblasts into VSMC-like cells.

Vascular smooth muscle cells (VSMCs) are major component of the vascular wall, and they play an essential role in maintaining the basic physiological function and stable structure of the vascular wall. In the present study, human gingival fibroblasts (HGFs) were cultured and induced into VSMC-like cells in vitro to confirm that HGFs with properties of stem cells have the potential for differentiation. The epithelium isolated from patients was extracted from normal human gingiva consisting of epithelium and connective tissue. HGFs were first identified by morphological examination, as well as specific gene and protein expression, and then induced by 10ng/mL PDGF-BB combined with 2ng/mL of TGF-ß1 for 28 days. After induction, ICS data indicated that induced VSMC-like cells were positive for α-SMA and SM-MHC, and IFA data showed that induced cells were positive for SM22α and Cnn1. RT-PCR results demonstrated that α-SMA and SM-MHC mRNA were specifically expressed, and myofilament-like structures also appeared in induced cells. In conclusion, the data indicated that HGFs could differentiate to VSMC-like cells with typical VSMC morphologic, ultrastructural, and immunological characteristics via induction with PDGF-BB and TGF-ß1.

Human gingival fibroblasts induced and differentiated into vascular endothelial-like cells.

A novel method for repair of vascular disease, mechanical damage, and tissue rebuilding is urgently required. Vascular endothelial cells (VECs) play an essential role in vascular rebuilding and vasotransplantation. In the present study, human gingival fibroblasts (HGFs) were cultured and induced into endothelial-like cells in vitro in order to confirm that HGFs with stem cell properties possessed the potential for differentiation into endothelial-like cells. The epithelium was extracted from normal human gingiva consisting of epithelium and connective tissue, which was isolated from patients. The identification of HGFs and induced endothelial-like cells were confirmed by flow cytometry, reverse transcription polymerase chain reaction (RT-PCR), immunocytochemical stain (ICS), and immunofluorescence stain (ISA). The morphology of human gingival fibroblasts with 8 ng/mL VEGF165 induced for different periods of days were observed by inverted microscope. Before induction, flow cytometry analysis showed that HGFs were positive for vimentin, but negative for CD31. RT-PCR, ICS, and ISA showed vimentin, S100A4, α-SMA, collagen III, and S100A4 were specifically expressed in these fibroblast cells. After induction, ICS showed induced vascular endothelial-like cells were positive for CD34 and CD31; ISA showed cells induced were positive for vWF and E-cadherin; RT-PCR results demonstrated that tie2 was specifically expressed in the cells induced. Flow cytometry analysis of the transformation efficiency from HGFs to endothelial-like cells. In conclusion, we found that HGFs possessed capacity for being induced and differentiated into vessel endothelial-like cells with typical and specific morphological, ultrastructural, and immunological characters of endothelial-like cells by induction with VEGF.

Molecular characterization of the liver-expressed antimicrobial peptide 2 (LEAP-2) in a teleost fish, Plecoglossus altivelis: antimicrobial activity and molecular mechanism.

Liver-expressed antimicrobial peptide 2 (LEAP-2) is widespread in fish and plays an important role in the host's innate immune system. In this study, full-length cDNA for LEAP-2 (PaLEAP-2) gene was cloned and sequenced from ayu, Plecoglossus altivelis. PaLEAP-2 mRNA was detected in a wide range of tissues, with the highest level of transcripts found in the liver. Upon induction by Vibrio anguillarum, its expression significantly increased in the liver, kidney, spleen, gill, and heart, but decreased in the intestine. The PaLEAP-2 mature peptide was chemically synthesized; it exhibited selective antimicrobial activity against various bacteria in vitro. PaLEAP-2 at high concentration reduced the bacterial load and improved the survival rate of V. anguillarum-infected ayu. Moreover, it inhibited the expression of mRNAs for TNF-α and IL-1ß in V. anguillarum-infected ayu, both at high and low concentrations. PaLEAP-2 induced hydrolysis of the pET-22b plasmid DNA and bacterium genomic DNA. These results suggest that PaLEAP-2 plays a role in ayu immune responses against bacterial infection.

The role of GPC5 in lung metastasis of salivary adenoid cystic carcinoma.

OBJECTIVE: Proteoglycans play a crucial role in salivary adenoid cystic carcinoma (SACC) tumorigenesis, neurotropic growth and lung metastasis. In this study, we investigated the role of GPC5 in the lung metastasis of SACC. DESIGN: The expression of heparan sulfate proteoglycans (HSPGs) was detected in SACC-M (high lung-metastatic cell line), SACC-2 (low lung-metastatic cell line) and SACC-83 (low lung-metastatic cell line) cells by relative quantitative Real-Time PCR. The expression of GPC5 was detected by immunofluorescence and Western blot analysis in SACC-M, SACC-2 and SACC-83 cells, and by immunohistochemical analysis in primary tumours from 16 cases of SACC patients with or without lung metastasis. GPC5 expression was silenced in SACC-M cells, cell proliferation and tumour growth was evaluated by MTT assay and nude mice model, respectively. RESULTS: Expression of most HSPGs was decreased in SACC-M cells, but GPC5 expression increased 3.24-fold and 815.69-fold (more than 3-fold) in SACC-M compared with SACC-2 and SACC-83 cells, respectively. Immunohistochemical analysis showed higher expression of GPC5 in SACC with lung metastasis compared to SACC without lung metastasis. The lung metastasis of SACC-M cells in nude mice was obviously decreased after GPC5 silencing (P<0.05), although GPC5 silencing had no significant effect on SACC-M cell proliferation. CONCLUSION: GPC5 may contribute to lung metastasis of SACC.

Molecular characterization and functional analysis of two distinct liver-expressed antimicrobial peptide 2 (LEAP-2) genes in large yellow croaker (Larimichthys crocea).

Liver-expressed antimicrobial peptide 2 (LEAP-2) plays a vital role in the host innate immune system. In the present study, two LEAP-2 genes (LcLEAP-2A and LcLEAP-2C) from large yellow croaker (Larimichthys crocea) were cloned, both of which consist of 3 exons and 2 introns. The LcLEAP-2A transcripts were expressed in a wide range of tissues, with the highest mRNA levels found in the liver and intestine, while LcLEAP-2C transcripts showed obvious lower mRNA levels in all tested tissues compared to LcLEAP-2A. Upon infection by Vibrio alginolyticus, LcLEAP-2A transcripts were significantly up-regulated in liver, trunk kidney, spleen, head kidney, and gill, but down-regulated in intestine. In addition, significant up-regulation of LcLEAP-2C transcripts were also detected in all tissues tested, including intestine. The LcLEAP-2A and LcLEAP-2C mature peptides were chemically synthesized and found to exhibit selective antimicrobial activity in vitro against various species of bacteria. LcLEAP-2C, but not LcLEAP-2A, had antimicrobial activity against V. alginolyticus. Moreover, LcLEAP-2C treatment at low concentrations was evaluated and found to improve survival rate in V. alginolyticus-infected large yellow croaker, resulting in a decrease in bacterial load and expression of inflammatory cytokines. These results suggest that LcLEAP-2 isoforms play an important role in innate immunity by killing bacteria and inhibiting early inflammatory response in large yellow croaker.

Change of fucosylated IgG2 Fc-glycoforms in pancreatitis and pancreatic adenocarcinoma: a promising disease-classification model.

The fixed constant (Fc) region of IgG is subject to changes in glycosylation state in response to diseases. On the basis of sera from 75 healthy controls, 75 pancreatitis (PT) patients, and 75 pancreatic adenocarcinoma (PAC) patients, we analyzed six fucosylated glycoforms of IgG2 (G0F, G1F, G2F, G0FN, G1FN, and G2FN), by matrix-assisted laser desorption/ionization-Fourier-transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS), to evaluate their use as biomarkers for pancreatic diseases. Compared with healthy controls, significant increases in agalactosylated glycoforms and decreases in galactosylated glycoforms were observed for PT and PAC patients. Logistic regression analysis suggested that truncation of the sugar chain was prone to occur in PT and, especially, PAC patients. After participants were stratified by sex and age, receiver operating characteristic curve analysis revealed good overall sensitivity and specificity for discrimination of PAC and PT patients from healthy controls. A combination of G0F and galactosylation also had acceptable power for differentiating PAC patients from PT patients.

Change in IgG1 Fc N-linked glycosylation in human lung cancer: age- and sex-related diagnostic potential.

The interactions of IgG Fc region with Fc receptors are optimized by the tailoring of a single-conserved N-linked glycosylation site at Asn-297. Our previous study has demonstrated that the age-related Fc-glycosylation change is featured by sex specificity and that the Fc-glycosylation has the potential for disease discrimination. Here, we conducted a Fourier transform ion cyclotron resonance MS-based profiling study involving 410 control individuals and 259 lung cancer (LC) patients. As compared to healthy controls, the marked increase in IgG1 Fc-agalactosylation and decrease in galactosylation were observed in LC patients. The binary logistic regression in combination with the receiver operating characteristic curve was used to determine the diagnostic ability of IgG1 Fc-glycosylation. It was found that this diagnostic ability was both sex and age dependent. Additionally, the change in Fc-glycosylation upon many different physiological and pathological conditions was retrospectively discussed. The data furthered the understanding of the immune-associated change in human LC, and also might be useful in the future attempts for Fc-glycosylation-associated diagnostic evaluations and clinical assays.

The effect of proteoglycans inhibited on the neurotropic growth of salivary adenoid cystic carcinoma.

OBJECTIVE: To evaluate the relationship between inhibition of proteoglycans which secreted by salivary adenoid cystic carcinoma cell line (SACC-83) and the neurotropic ability of the tumor cells. METHODS: The expression vector of short hairpin RNA (shRNA-WJ4) targeting xylosyltransferase-I gene was constructed and transfected into SACC-83 cells (group SACC83-WJ4), shRNA-HK used as negative control was transfected into SACC-83 cells (group SACC-83-HK), SACC-83 cells without transfection was used as black control (group SACC-83). The xylosyltransferase-I gene expression was measured by real-time PCR. The content of proteoglycans was detected by Blyscan Assay Kit. The effect of down-regulated proteoglycans on the perineural invasion of nude mice was observed. All data were analyzed by the software spss 13.0. RESULTS: The results showed that the transfected efficiency of shRNA was 43.3%. The expression of xylosyltransferase-I was inhibited by 43.0% 48 h after transfection of shRNA-WJ4. The content of proteoglycans was down-regulated by 30.25% 48 h after transfection. In the neurotropic experiment in vivo of nude mice, the rate of perineural invasion of group SACC-83-WJ4 was 33.33%, significantly lower than that of the negative control (100%) and the black control (100%) (P < 0.05). CONCLUSION: Xylosyltransferase-I gene of SACC cells was silenced by RNA interference technology, proteoglycans secretion was reduced and neurotropic invasion behavior of SACC was inhibited obviously.

[Relationship between proteoglycans and proliferation of human salivary adencid cystic carcinoma].

OBJECTIVE: To investigate the effect of down-regulated proteoglycans on the proliferation of human salivary adenoid cystic carcinoma (SACC). METHODS: The short hairpin RNA (shRNA) plasmid silencing human xylosyltransferase-I (XT-I) gene was constructed and named shRNA-WJ3. Adenoid cystic carcinoma cells with high metastatic tendency (ACC-M) were transfected by shRNA-WJ3. The plasmid shRNA-HK not targeting any human gene was transfected into ACC-M cells used as negative control. After 48 h of transfection, the positive cells were screened by G418 to isolate the stable transfected cells. Real-time PCR and Western blotting were used to test the gene silence, and the proteoglycans contents of the cells were detected. The stable cell line silenced XT-I was named ACC-M-WJ3. MTT assay was performed to detect the cell proliferation. The cell cycle was analyzed by flow cytometry. RESULTS: ShRNA-WJ3 showed powerful RNA interference and gene silence of XT-I. The inhibition rate was 83.70% of mRNA expression and 79.60% of protein expression respectively. The content of proteoglycans in ACC-M-WJ3 was down-regulated by 49.71%-54.59%. The results of MTT assay showed that the cell growth was inhibited significantly. S phrase decreased and G1-G0 phrase increased in group ACC-M-WJ3 compared with that of group ACC-M-HK (P < 0.05). CONCLUSIONS: The down-regulated proteoglycans could inhibit the proliferation of human ACC-M cells.

The effect of proteoglycans inhibited by RNA interference on metastatic characters of human salivary adenoid cystic carcinoma.

BACKGROUND: Salivary adenoid cystic carcinoma (SACC) is one of the most common malignancies of salivary gland. Recurrence or/and early metastasis is its biological properties. In SACC, neoplastic myoepithelial cells secrete proteoglycans unconventionally full of the cribriform or tubular and glandular structures of SACC. Literatures have demonstrated that extracellular matrix provided an essential microenvironment for the biological behavior of SACC. However, there is rare study of the effect of proteoglycans on the potential metastasis of SACC.In this study, human xylosyltransferase-I (XTLY-I) gene, which catalyzes the rate-limited step of proteoglycans biosynthesis, was knocked down by RNA interference (RNAi) to inhibit the proteoglycans biosynthesis in SACC cell line with high tendency of lung metastasis (SACC-M). The impact of down-regulated proteoglycans on the metastasis characters of SACC-M cells was analyzed and discussed. This research could provide a new idea for the clinical treatment of SACC. METHODS: The eukaryotic expression vector of short hairpin RNA (shRNA) targeting XTLY-I gene was constructed and transfected into SACC-M cells. A stably transfectant cell line named SACC-M-WJ4 was isolated. The XTLY-I expression was measured by real-time PCR and Western blot; the reduction of proteoglycans was measured. The invasion and metastasis of SACC-M-WJ4 cells were detected; the effect of down-regulated proteoglycans on the potential lung metastasis of nude mice was observed, respectively. RESULTS: The shRNA plasmid targeting XTLY-I gene showed powerful efficiency of RNAi. The mRNA level of target gene decreased by 86.81%, the protein level was decreased by 80.10%, respectively. The silence of XTLY-I gene resulted in the reduction of proteoglycans significantly in SACC-M-WJ4 cells. The inhibitory rate of proteoglycans was 58.17% (24 h), 66.06% (48 h), 57.91% (72 h), 59.36% (96 h), and 55.65% (120 h), respectively. The reduction of proteoglycans suppressed the adhesion, invasion and metastasis properties of SACC-M cells, and decreased the lung metastasis of SACC-M cells markedly either. CONCLUSION: The data suggested that the silence of XTLY-I gene in SACC-M cells could suppress proteoglycans biosynthesis and secretion significantly. The reduction of proteoglycans inhibited cell adhesion, invasion and metastasis of SACC-M cells. There is a close relationship between proteoglycans and the biological behavior of SACC.