Xinjie Yang

EMMPRIN silencing inhibits proliferation and perineural invasion of human salivary adenoid cystic carcinoma cells in vitro and in vivo

Salivary adenoid cystic carcinoma (SACC) is a frequent subtype of salivary gland malignancy, and it has an important biological behavior for perineural invasion (PNI). Extracellular matrix metalloproteinase inducer (EMMPRIN) is a transmembrane glycoprotein that is involved in the invasive property of many malignancies by stimulating matrix metalloproteinase (MMP) expression. The present study was designed to investigate the role and possible mechanism of EMMPRIN in the PNI of SACC using RNA interference (RNAi) technology. We found that silencing of EMMPRIN expression in the human SACC cell line (SACC-83) suppressed the cell proliferation, adhesion, MMP-2 and MMP-9 secretion, and PNI activity in vitro. EMMPRIN silencing also inhibited the tumor growth and Ki-67 labeled proliferation index in vivo. Using tumor PNI models in nude mice, EMMPRIN silencing inhibited the infiltration, swelling and functional loss of the affected sciatic nerves, as well as the expression of MMP-2 and MMP-9. These results demonstrate that EMMPRIN participates in the PNI of SACC cells by mediating the expression of MMP-2 and MMP-9. Our findings suggest that EMMPRIN is a potential target for anti-PNI treatment in SACC.

BDNF mediated TrkB activation contributes to the EMT progression and the poor prognosis in human salivary adenoid cystic carcinoma.

BACKGROUND The aim of the present study was to investigate whether the expression of Brain-Derived Neurotrophic Factor (BDNF) and its receptor Tropomyosin-related kinase B (TrkB) is correlated with the clinical progression of salivary adenoid cystic carcinoma (SACC) and whether the BDNF/TrkB axis is associated with the induction of epithelial-mesenchymal transition (EMT) in SACC cells. METHOD The expression of BDNF, TrkB, and E-cadherin (an EMT biomarker) in 76 primary SACC specimens and 20 normal salivary gland tissues was analyzed by immunohistochemistry. Additionally, the expression of BDNF, TrkB, and E-cadherin in SACC cell lines (SACC-83 and SACC-LM) was analyzed by RT-PCR and Western blotting. The biological role of the BDNF/TrkB axis in the EMT progression of SACC was evaluated after treatment with increased levels of BDNF and by inhibiting TrkB activity in SACC-83 cell line. The progression of SACC cells through EMT was assessed by RT-PCR, Western blotting, photography, migration and invasion assays. RESULTS Elevated expression of TrkB (92.1%) and BDNF (89.5%), and downregulated expression of E-cadherin (47.4%) was found in SACC specimens, which was significantly correlated with the invasion and metastasis in SACC (P<0.05). The high expression of TrkB and the low expression of E-cadherin was significantly correlated with the poor prognosis of SACC patients (P<0.05). The expression of TrkB was inversely correlated with the expression of E-cadherin in both SACC cases and cell lines (P<0.05). Increasing BDNF levels after treatment with exogenous recombinant human BDNF (rhBDNF) at 100 ng/ml significantly promoted the activation of TrKB and the progression of EMT in SACC cells. While obstruction of TrkB by its inhibitor, k252a (100 nM), significantly inhibited the EMT progression of SACC cells. CONCLUSIONS These results suggest that BDNF-mediated TrkB activation contributes to the EMT progression and the poor prognosis in SACC. The present study demonstrated that the BDNF/TrkB axis promotes the migration and invasion of SACC cells via EMT in vitro. Targeting the inactivation of the BDNF/TrkB axis may be a potential strategy for the treatment of SACC.

EMMPRIN contributes to the in vitro invasion of human salivary adenoid cystic carcinoma cells

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a transmembrane glycoprotein that is involved in tumor invasion by stimulating matrix metalloproteinase (MMP) expression. Our previous immunohistochemical study found that the expression of EMMPRIN in salivary adenoid cystic carcinoma (SACC) was positively correlated with tumor perineural and perivascular invasion. The present study was designed to further investigate the role of EMMPRIN in the invasion of SACC. Western blot results showed that EMMPRIN was upregulated in the highly metastatic SACC cell line SACC-LM, compared to SACC-83, a SACC cell line with low metastatic ability. Blocking of EMMPRIN by its antibody significantly decreased the adhesion, secretion of MMP-2 and MMP-9, and invasion activity of SACC-LM cells in vitro (P<0.01). Co-cultures of SACC-LM cells with fibroblasts significantly produced elevated levels of MMP-2 and MMP-9, and promoted the in vitro invasion activity of SACC-LM cells, compared with cultures of SACC-LM cells alone (P<0.01). These results indicate that EMMPRIN may play an important role in the invasion of SACC by stimulating the expression of MMP-2 and MMP-9 in tumor and stromal cells.

Incorporation of osteogenic and angiogenic small interfering RNAs into chitosan sponge for bone tissue engineering

Engineered bone substitutes are being extensively explored in response to growing demand. However, the angiogenesis that occurs during bone formation is often overlooked in scaffold design. In this novel study, we incorporated two small interfering RNAs (siRNAs), ie, small interfering RNA targets casein kinase 2 interaction protein 1 (siCkip-1) and small interfering RNA targets soluble VEGF receptor 1 (siFlt-1), which can promote osteogenesis and angiogenesis, into a chitosan sponge. This scaffold could maintain siRNAs for over 2 weeks in neutral phosphate-buffered saline and degraded rapidly in the presence of lysozyme. The chitosan sponge with siCkip-1 and siFlt-1 in vitro bioactivity was investigated using mesenchymal stem cells. Target genes were significantly suppressed, and osteocalcin, alkaline phosphatase, and vascular endothelial growth factor were significantly upregulated. Alizarin Red staining revealed that mineralization of the extracellular matrix was markedly enhanced by dual transfection. Further analysis by immunofluorescence confirmed that the siRNA-modified scaffold simultaneously improved the expression of osteocalcin and von Willebrand factor. In vivo testing in a skull critical-size defect model showed marked bone regeneration in rats treated with siCkip-1 and siFlt-1. In conclusion, chitosan sponge containing osteogenic and angiogenic siRNAs may be used as a scaffold for bone regeneration. The dual siRNA concept may also be useful in the biofunctionalization of other materials.

Schwann cells promote EMT and the Schwann-like differentiation of salivary adenoid cystic carcinoma cells via the BDNF/TrkB axis

Perineural invasion (PNI) is a striking biological behavior observed in salivary adenoid cystic carcinoma (SACC). The present study was designed to establish a co-culture model of SACC cells with Schwann cells (SCs), and then study epithelial-mesenchymal transition (EMT) and the Schwann-like differentiation of SACC cells to investigate the likely molecular mechanism of PNI. The co-culture models of SCs with tumor cells (SACC-83, SACC-LM and MEC-1) were established using a Transwell system. An elevated concentration of brain-derived neurotrophic factor (BDNF) was detected by ELISA assay in the co-cultured medium of the SACC-83 group and SACC-LM group rather than the MEC-1 group. The EMT process and Schwann-like differentiation in SACC-83 cells were analyzed by RT-PCR, western blotting, immunofluorescence, photography, and migration and perineural invasion assays. The SACC-83 cells under the co-culture condition with SCs changed to a mesenchymal morphology and had higher migration and invasion capabilities compared with the solely cultured SACC-83 cells, accompanied by the downregulation of E-cadherin and upregulation of N-cadherin and vimentin. The co-cultured SACC-83 cells also developed Schwann-like differentiation with increased expression of SC markers, S100A4 and GFAP. However, inhibition of tropomyosin-related kinase B (TrkB) by K252a markedly blocked these effects. Additionally, the expression and correlation of TrkB, E-cadherin and S100A4 were analyzed by immunohistochemistry in 187 primary SACC cases. The levels of TrkB and S100A4 expression were both positively associated with PNI in the SACC cases, while E-cadherin expression was negatively associated with PNI. Elevated expression of TrkB was significantly correlated with the downregulated expression of E-cadherin and the upregulated expression of S100A4 in the SACC cases. Our results suggest that SCs play a pivotal role in the PNI process by inducing the EMT process and the Schwann-like differentiation of SACC cells via the BDNF/TrkB axis. Interruption of the interreaction between SACC cells and SCs by targeting the BDNF/TrkB axis may be a potential strategy for anti-PNI therapy in SACC.

Sympathetic Denervation-Induced MSC Mobilization in Distraction Osteogenesis Associates with Inhibition of MSC Migration and Osteogenesis by Norepinephrine/adrb3

The sympathetic nervous system regulates bone formation and resorption under physiological conditions. However, it is still unclear how the sympathetic nerves affect stem cell migration and differentiation in bone regeneration. Distraction osteogenesis is an ideal model of bone regeneration due to its special nature as a self-engineering tissue. In this study, a rat model of mandibular distraction osteogenesis with transection of cervical sympathetic trunk was used to demonstrate that sympathetic denervation can deplete norepinephrine (NE) in distraction-induced bone callus, down-regulate β3-adrenergic receptor (adrb3) in bone marrow mesenchymal stem cells (MSCs), and promote MSC migration from perivascular regions to bone-forming units. An in vitro Transwell assay was here used to demonstrate that NE can inhibit stroma-derived factor-1 (SDF-1)-induced MSC migration and expression of the migration-related gene matrix metalloproteinase-2 (MMP-2) and downregulate that of the anti-migration gene tissue inhibitor of metalloproteinase-3 (TIMP-3). Knockdown of adrb3 using siRNA abolishes inhibition of MSC migration. An in vitro osteogenic assay was used to show that NE can inhibit the formation of MSC bone nodules and expression of the osteogenic marker genes alkaline phosphatase (ALP), osteocalcin (OCN), and runt-related transcription factor-2 (RUNX2), but knockdown of adrb3 by siRNA can abolish such inhibition of the osteogenic differentiation of MSCs. It is here concluded that sympathetic denervation-induced MSC mobilization in rat mandibular distraction osteogenesis is associated with inhibition of MSC migration and osteogenic differentiation by NE/adrb3 in vitro. These findings may facilitate understanding of the relationship of MSC mobilization and sympathetic nervous system across a wide spectrum of tissue regeneration processes.

Mesenchymal stem cells modified with nerve growth factor improve recovery of the inferior alveolar nerve after mandibular distraction osteogenesis in rabbits.

Distraction osteogenesis is widely used in the treatment of bony deformities and defects. However, injury to the inferior alveolar nerve is a concern. Our aim was to investigate the feasibility of using lentiviral-mediated human nerve growth factor beta (hNGFβ) of the inferior alveolar nerve in mandibular distraction osteogenesis in rabbits. To achieve this, mesenchymal stem cells (MSC) from the bone marrow of rabbit mandibles were isolated and genetically engineered using recombinant lentiviral vector containing hNGFβ. Twenty New Zealand white rabbits underwent mandibular distraction osteogenesis, and 5 million MSC transduced with hNGFβ-vector or control vector were transplanted around the nerve in the gap where the bone had been fractured during the operation (n=10 in each group). After gradual distraction, samples of the nerve were harvested for histological and histomorphometric analysis. We found that the genetically engineered MSC transduced by the lentiviral vector were able to secrete hNGFβ at physiologically relevant concentrations as measured by ELISA. Histological examination of the nerve showed more regenerating nerve fibres and less myelin debris in the group in which hNGFβ-modified MSC had been implanted than in the control group. Histomorphometric analysis of the nerve showed increased density of myelinated fibres in the group in which hNGFβ-modified MSC had been implanted than in the control group. The data suggest that implantation of hNGFβ-modified MSC can accelerate the morphological recovery of the inferior alveolar nerve during mandibular distraction osteogenesis in rabbits. The use of lentiviral-mediated gene treatment to deliver hNGFβ through MSC may be a promising way of minimising injury to the nerve.

A systematic investigation of the association between HPV and the clinicopathological parameters and prognosis of oral and oropharyngeal squamous cell carcinomas

Human papillomavirus (HPV), the causal factor of cervical cancers, was closely linked to the etiology and prognosis of oropharyngeal squamous cell carcinoma (OPSCC), but its role in oral squamous cell carcinoma (OSCC) was unclear. In addition, few researches based on Chinese population were documented. Hence, we sought to investigate the relationship of HPV marker P16 protein to the clinicopathological parameters and survival of OPSCC and OSCC patients systematically to assess the influence of ethnic, regional difference on HPV susceptibility. Specimens from 93 OPSCC patients and 95 OSCC patients were recut, and P16 immunohistochemistry (IHC) was performed. Moreover, survival analysis was conducted to confirm the independent factors that influenced the prognosis. The P16 results were positive in 25.8% and 9.5% of patients with OPSCC and OSCC, respectively. The overall survival (OS) of HPV‐positive OPSCC patients was significantly longer than that of HPV‐negative OPSCC patients (P = 0.004). Conversely, statistical significance was not observed regarding the OS of OSCC patients (P = 0.343). Cox regression analysis indicated that T stage and P16 status were independent factors that affected the prognosis of OPSCC patients, and the smoking index influenced the prognosis of OSCC patients. Among OPSCC patients who received radiochemotherapy (RCT), HPV‐positive patients had a better survival rate than their HPV‐negative counterparts (P = 0.015). Conversely, no significant difference was observed between HPV‐positive and HPV‐negative OSCC patients who received RCT (P = 0.237). P16 is a credible surrogate by which to define HPV status. HPV expression had a favorable effect on OPSCC patients as opposed to their OSCC counterparts in this single center population‐based study.

The mechanically activated p38/MMP-2 signaling pathway promotes bone marrow mesenchymal stem cell migration in rats

OBJECTIVE The aim of the present study was to investigate the effect of static strain on bone marrow mesenchymal stem cell (BMMSC) migration and whether the p38/matrix metalloproteinase-2 (MMP-2) axis plays a role in induction of BMMSC migration under mechanical strain. DESIGN Both in vivo and in vitro investigations were performed. Twelve adult male Sprague-Dawley rats were randomly divided into 2 groups (n=6 per group). Rats in the experimental group underwent right mandibular distraction osteogenesis, whereas rats in the control group were subjected to osteotomy in the mandible without distraction. Immunohistochemistry and immunofluorescence were performed to evaluate phospho-p38 (p-p38) and Nestin expression. BMMSCs were isolated from rat mandibles. BMMSCs in the experimental group were subjected to static mechanical strain for 2h, whereas those in the control group underwent no strain. The biological roles of static strain and the p38/MMP-2 axis in BMMSC migration were evaluated by Transwell assays and western blotting by inhibiting p38 phosphorylation. RESULTS There were significantly more Nestin+ cells in the bone calluses of the experimental group than in those of the control group. In addition, Nestin+/p-p38+ cell numbers were significantly higher in the experimental group than in the control group, indicating that static strain activated p38 signaling in BMMSCs in vivo. In accordance with in vivo results, static strain in vitro stimulated phosphorylation of p38 in BMMSCs. Furthermore, expression of MMP-2 was elevated in BMMSCs under static strain compared with the control, and strain-induced MMP-2 expression was abolished by inhibition of p38 phosphorylation in BMMSCs. Moreover, Transwell assay results showed that static strain promoted BMMSC migration, which was abolished by inhibition of p38 phosphorylation. CONCLUSIONS The present study demonstrated that static strain can promote the migration ability of BMMSCs via p38/MMP-2 signaling. To the best of our knowledge, this study is the first report demonstrating that the p38/MMP-2 axis governs BMMSC migration under static mechanical strain.

The CCL5/CCR5 axis contributes to the perineural invasion of human salivary adenoid cystic carcinoma

Salivary adenoid cystic carcinoma (SACC) has a unique tendency for perineural invasion (PNI), which results in tumor recurrence and poor prognosis. Recent studies have shown that the chemokine CCL5 and its receptor CCR5 play important roles in tumor invasion and metastasis. However, the role of the CCL5/CCR5 axis in the PNI of SACC has not been studied to date. In the present study, we evaluated the expression of CCL5 and CCR5 in SACC cases and nerve tissues, and performed a series of in vitro assays with the SACC cell line, SACC-83, to indicate the role of the CCL5/CCR5 axis in the PNI of SACC. We found that CCL5 (35.9%; 23/64) and CCR5 (70.3%; 45/64) were positively expressed in SACC cases, and the expression of CCR5 was significantly associated with the PNI of SACC (P<0.05). We also found that SACC-83 cells expressed the functional receptor, CCR5, for the chemokine CCL5, as demonstrated by calcium mobilization and actin polymerization assays. Furthermore, we found that exogenous CCL5 significantly facilitated the migration, invasion and PNI activity of SACC-83 cells in vitro (P<0.05). Further study showed that the CCR5 inhibitor (maraviroc) effectively blocked the migration, invasion and PNI activity of SACC-83 cells with or without CCL5 stimulation (P<0.05). These results indicate that the CCL5/CCR5 axis plays a critical role in the PNI of SACC, and that antagonists against CCR5 may be an effective anti-PNI agent for SACC therapy.