Sulfuric acid induced-synthesis coupled with ethanol extraction-water precipitation purification method for orange fluorescent carbon dots with dual-emission: Application for methyl blue detection and cell imaging.

In this work, by adjusting the sulfuric acid content in reaction solvent of ethanol, orange fluorescent carbon dots (O-FCDs) with dual-emission wavelength and blue fluorescent carbon dots (B-FCDs) with single-emission wavelength were successfully prepared using 1,3-dihydroxynaphthalene as precursor. Coupling with ethanol extraction-water precipitation purification method, pure O-FCDs and B-FCDs with yields of 9.0 % and 21.3 %, quantum yields (QYs) of 43.0 % and 13.7 % were obtained, respectively. The structures and optical properties of O-FCDs and B-FCDs were investigated by TEM, AFM, Raman, FT-IR, XPS, UV-vis, fluorescence analysis etc. The results revealed that sulfuric acid promoted the carbonization and the oxidation of precursor in the reaction process. In comparison with the B-FCDs, O-FCDs showed narrower lattice spacing and band gap, demonstrating the important role of sulfur-doping in fluorescence tuning. Additionally, O-FCDs showed good sensitivity for methyl blue with a linear response range of 0.05-100 µM (LOD was 20 nM) and the satisfactory results were obtained when O-FCDs were applied to the detection of methyl blue in real fish sample. Moreover, two FCDs showed good biocompatibility and negligible cytotoxicity proved by MTT experiment, while, O-FCDs showed better cell imaging effects than that of B-FCDs. Therefore, the O-FCDs had a broad application prospect as sensing platform in detection of methyl blue and for imaging in biological field.

Butterfly-Shaped Nanographenes with Excellent Second-Order Nonlinear Optical Properties: The Synergy of B/N and Azulene.

Azulene, a simple polar polycyclic aromatic hydrocarbon with connected electron donor and acceptor (DA), ignites the hope of designing second-order nonlinear optical (NLO) molecular materials from pure nonpolar carbon nanomaterials. In this work, a butterfly-shaped nanographene (π-DA-π) was designed by incorporating azulene between two coronenes. One more electron in a N atom or one electron fewer in a B atom with respect to a C atom can polarize charge distribution in carbon nanomaterials, and further doping of B and N in the designed butterfly-shaped nanographene changes the system from π-DA-π to D-π-A, leading to strong NLO responses. For example, the largest static first hyperpolarizability even reaches 173.89×10-30  esu per heavy atom. The synergetic role of B, N and azulene in the nanographene is scrutinized, and such a doping strategy is found to provide an effective means for the design of carbon-based functional materials. The strong second-order NLO responses of these butterfly-shaped carbon-based nanographenes under external fields, for example, sum frequency generation and difference frequency generation, could inspire future experimental exploration.

Hydroxyl-riched covalent organic framework for solid-phase microextraction of flavonoids aglycones or their metabolites in mice's plasma: Luteolin and quercetagetin as examples.

Herein, a hydroxyl­riched covalent organic framework (named COF-DES-1) was synthesized using 1,3,5-tris(4-aminophenyl)benzene and 2,5-dihydroxyterephthalaldehyde as building blocks and employed as a coating of solid-phase microextraction (SPME) fiber. Ascribed to the advantages (e.g. suitable pore size and rich functional group characteristics) of coating, the SPME fiber showed good adsorption capacities to flavonoids aglycones including luteolin and quercetagetin, and the maximum adsorption capacities for them were 145.31 µg and 84.75 µg, respectively. Due to the size exclusion property of COF-DES-1, SPME fiber showed good protein exclusion effects on seven selected proteins with high exclusion efficiencies (>93%). Accordingly, an attractive strategy of the combination of COF-DES-1 based SPME fiber and HPLC-MS/MS was proposed for the extraction and determination of luteolin, quercetagetin or their metabolites. The results revealed that the fiber can be effectively applied to extract luteolin and its metabolites, and quercetagetin from mice's palsma. Compared with the traditional protein precipitation methods, the extraction effects of SPME fiber based extraction method were much better, indicating the promising applicability of the fiber for the enrichment of flavonoids aglycones or their metabolites in biological samples.

A novel hydroxyl-riched covalent organic framework as an advanced adsorbent for the adsorption of anionic azo dyes.

Considering the highly carcinogenic and mutagenic of anionic azo dyes to the environment and humans, the development of high efficiency adsorbent for them are of great significance. In this study, a novel hydroxyl-riched covalent organic framework (denoted as COF-OH), which can act as an advance adsorbent for anionic azo dyes, was fabricated for the first time. The as-prepared COF-OH demonstrated good dispersion in water, remarkable adsorption performances and good selectivity for anionic azo dyes including eriochrome black T, eriochrome blue black R and congo red. The adsorption capacities of them ranged from 90.71 to 229.12 mg g-1, and the extraction efficiencies of them (>75.91%) were much higher than other dyes (e.g. Methylene blue, direct red 80, 1.46%-39.57%). By optimizing the adsorption conditions (adsorbent dosage, adsorption time, pH, and salt concentration) and desorption conditions (desorption solvent, desorption time and desorption frequency), a dispersive micro solid-phase extraction (D-µ-SPE) method was developed. Further, coupled D-µ-SPE with HPLC-PDA analysis, an effective method was fabricated for the extraction and detection of three selected dyes. The method showed good linearity in the range of 0.1-200 µg mL-1 (R2 > 0.9966), low limits of quantification (0.10 µg mL-1-2.00 µg mL-1), low limits of detection (0.03-1.50 µg mL-1) and good precision. Finally, the COF-OH based D-µ-SPE was successfully applied to extract three selected dyes from water samples (recoveries ranged from 73.90 to 104.00%) and congo red from beverages (recoveries ranged from 81.40 to 111.80%). Besides, by using computer simulation, FT-IR and UV-vis analysis, the adsorption mechanisms of COF-OH to three selected dyes were explored preliminarily.

Preparation of sulphuric acid-mediated N,S-codoped red emissive carbon dots: Applications in food dyes detection, solid-state luminescence and cell imaging.

As diseases such as cardiovascular disease and cancer caused by food problems are more and more frequent, food safety has received great attention. Among them, the safety problem caused by food dyes is more prominent. Thus, it is of great value to develop sensitive detection methods for food dyes. In present study, sulphuric acid-mediated N,S-codoped red emissive carbon dots (namely as R-CDs) had been manufactured by using N-phenyl-o-phenylenediamine as precursor, sulfuric acid as additive for the first time. The structural and fluorescence properties of R-CDs had been systematically studied. The results demonstrated that R-CDs showed uniform spherical morphology and had a graphite-like structure, for which the average diameters size was 5.05 nm. Due to the various functional groups such as hydroxyl, pyridinic N, pyrrolic N and -C-SO4, R-CDs emitted bright red fluorescence. Importantly, because of the interactions between the functional groups of R-CDs with the selected food dyes, three dyes including amaranth, brilliant blue FCF and methylene blue can sensitively quench the fluorescence of R-CDs through IFE and static quenching effects. The linearity ranges of them were separately detected as 0.20 µM -20 µM, 10 nM-1 µM and 60 nM-8 µM. The limits of detection (LODs) of them were 70 nM, 4 nM and 20 nM, respectively. Further, R-CDs was successfully applied to the sensitive detection of three dyes from various food samples. To maximize the fluorescence properties of R-CDs, a R-CDs/PVA composite gel was fabricated to make R-CDs fluoresce in solid state condition. The potential of R-CDs/PVA composite gel for preliminary visualization analysis of three dyes was studied. Finally, ascribed to the low toxicity and good biocompatibility, the potential of R-CDs as probe for cell imaging was explored preliminarily.

Sulfuric-acid-mediated synthesis strategy for multi-colour aggregation-induced emission fluorescent carbon dots: Application in anti-counterfeiting, information encryption, and rapid cytoplasmic imaging.

Aggregation-induced emission fluorescent carbon dots (AIE-CDs) have applications in the fields of multi-colour anti-counterfeiting, information encryption, and imaging. In this study, four AIE-CDs (B-AIE-CDs, G-AIE-CDs, Y-AIE-CDs, and O-AIE-CDs) with blue, green, yellow, and orange fluorescence at high concentrations were fabricated using crystal violet as a precursor, solutions with different sulfuric acid concentrations as solvents under different temperatures and reaction times for the first time. The structural properties and fluorescence behaviour of the AIE-CDs were investigated. The results revealed that the sulfuric acid concentration had a significant effect on the fluorescence colour of the AIE-CDs because sulfuric acid can affect the degree of carbonisation, the type and content of nitrogen. Moreover, the reaction temperature and time affected the surface-defect state and the degree of carbonisation of the AIE-CDs, which affected the emission wavelength and quantum yield (QY) of the AIE-CDs. Furthermore, to exploit the unique characteristics (polychromatic aggregation fluorescence and acid-sensitive properties) of the obtained-AIE-CDs, anti-counterfeiting and information encryption methodologies (i.e., acid-stimuli-response producing multi-colour fluorescence) were preliminarily developed. Finally, B-AIE-CDs with a high QY of 43.5% were successfully used for rapid cytoplasmic imaging, demonstrating their applicability in biological fields.

N,Cl-Codoped Carbon Dots from Impatiens balsamina L. Stems and a Deep Eutectic Solvent and Their Applications for Gram-Positive Bacteria Identification, Antibacterial Activity, Cell Imaging, and ClO- Sensing.

In this study, we first synthesized metal-free N,Cl-doped carbon dots (N,Cl-CDs) using Impatiens balsamina L. stems as green precursors in a deep eutectic solvent (DES). The obtained N,Cl-CDs were characterized through transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), Fourier-transform infrared (FT-IR) spectroscopy, fluorescence (FL) spectroscopy, and ultraviolet (UV) spectroscopy. In addition to the common features of carbon dots (CDs), such as high light stability, small size, low toxicity, good aqueous solubility, and favorable biocompatibility, these N,Cl-CDs exhibited excellent recognition and selectivity for Gram-positive bacteria by doping with N and Cl elements using DES. The N,Cl-CDs with positive charge cannot only differentiate Gram-positive bacteria by selective fluorescence imaging but also have antibacterial effects on Gram-positive bacteria. Through potential, ROS, and morphological analyses of bacteria before and after treatment with N,Cl-CDs, the antibacterial mechanisms of bacteriostasis, Enterococcus faecium, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Salmonella were explored. In addition, N,Cl-CDs demonstrated low cytotoxicity and good cell imaging ability in cancer and normal cells. Moreover, they can be used as a fluorescence sensor for the detection of ClO- with a detection range from 100 nM to 40 µM and a limit of detection (LOD) of 30 nM. In summary, the prepared N,Cl-CDs could be applied as environmentally friendly Gram-positive bacterial identification and antibacterial agents. Additionally, their cell imaging and ClO- detection abilities were outstanding.

Highly Crystalline Covalent Organic Frameworks Act as a Dual-Functional Fluorescent-Sensing Platform for Myricetin and Water, and Adsorbents for Myricetin.

Selective detection of active ingredients in complex samples has always been a crucial challenge because there are many disturbing compounds, especially structural analogues that interfere with the detection. In this work, a fluorescent covalent organic framework (named COF-TD), which can be used for the selective fluorescence detection and enrichment of myricetin from complex samples, was reported for the first time. The highly crystalline COF-TD with bright blue fluorescence was formed through a solution polymerization method by the condensation reaction between 4,4',4″-(1,3,5-triazine-2,4,6-triyl)trianiline and 2,5-dihydroxy-1,4-benzenedicarboxaldehyde. Due to spatial size selectivity, multisites hydrogen bonding, and π-π interaction, myricetin can quench the fluorescence of COF-TD with an inner filter effect (IFE) and static quenching mechanisms as well as can be enriched on COF-TD. Myricetin can observably eliminate the interference of other compounds and selectively quench the fluorescence of COF-TD with a limit of detection (LOD) of 0.30 µg·mL-1. The high adsorption ability of COF-TD (Q = 124.6 mg·g-1) to myricetin was also obtained. Finally, a sensing platform based on COF-TD for myricetin was successfully developed and applied for the detection of myricetin from vine teas. In addition, COF-TD also showed good water sensing ability and could be used effectively to detect water content in organic solvent (1-18% water in acetone, 0.5-5% water in acetonitrile, 1-4.5% water in ethyl acetate, v/v). To the best of our knowledge, this is the first report where COF-TD was used to detect water in a relatively wide concentration range. In all, this work provided dual-functional fluorescent COFs with the properties of an adsorbent, opening up new methodologies for the simple, selective, and enrichment detection method for myricetin.

Downregulation of iASPP Expression Suppresses Proliferation, Invasion and Increases Chemosensitivity to Paclitaxel of Head and Neck Squamous Cell Carcinoma / 中国医学科学杂志(英文版)

Objective Our previous study has revealed that iASPP is elevated in human head and neck squamous cell carcinoma (HNSCC) and iASPP overexpression signifcantly correlates with tumor malignant progression and poor survival of HNSCC. This study investigated the function of iASPP playing in proliferation and invasion of HNSCC . Methods HNSCC cell line Tu686 transfected with Lentiviral vector-mediated iASPP-specific shRNA and control shRNA were named the shRNA-iASPP group and shRNA-NC group, respectively. The non-infected Tu686 cells were named the CON group. CCK-8 assay, flow cytometry, transwell invasion assay were performed to detect the effects of iASPP inhibition . Results Our results demonstrated that the proliferation of shRNA-iASPP cells at the time of 72 h (=32.459, =0.000), 96 h (=51.407, =0.000), 120 h (=35.125, =0.000) post-transfection, was significantly lower than that of shRNA-NC cells and CON cells. The apoptosis ratio of shRNA-iASPP cells was 9.42% ± 0.39% (=299.490, =0.000), which was significantly higher than that of CON cells (2.80% ± 0.42%) and shRNA-NC cells (3.18% ± 0.28%). The percentage of shRNA-iASPP cells in G0/G1 phase was 74.65% ± 1.09% (=388.901, =0.000), which was strikingly increased, compared with that of CON cells (55.19% ± 1.02%) and shRNA-NC cells (54.62% ± 0.88%). The number of invading cells was 56 ± 4 in the shRNA-iASPP group (=84.965, =0.000), which decreased significantly, compared with the CON group (111 ± 3) and the shRNA-NC group (105 ± 8). The survival rate of shRNA-iASPP cells administrated with paclitaxel was highly decreased, compared with CON cells and shRNA-NC cells (=634.841, =0.000). Conclusion These results suggest iASPP may play an important role in progression and aggressive behavior of HNSCC and may be an efficient chemotherapeutic target for the treatment of HNSCC.

Establishment of a SCID beige mouse model bearing transplanted human choriocarcinoma using JAR cell line / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a mouse model bearing human choriocarcinoma xenograft in severe combined immuno-deficient (SCID) beige mice and investigate the disease course and biological behaviors of the tumors.</p><p><b>METHODS</b>Human choriocarcinoma JAR cells were injected in female SCID beige mice (3-5 weeks old) either subcutaneously (group A, n=6) or via the tail vein (group B, n=6). Morphological studies, radioactive immunoassay, in vivo tumor imaging and histopathological examinations were performed to confirm JAR cell engraftment at the subcutaneous injection site and in the lungs of the mice.</p><p><b>RESULTS</b>On day 28 after tumor cell inoculation, the mice in group A showed palpable subcutaneous nodules, and HE staining revealed morphological features of the nodules consistent with choriocarcinoma cells; in vivo imaging in group B showed single or multiple solid tumor masses in the lungs, and tissue biopsy examination demonstrated varying degrees of tumor cell infiltration. Compared with the control mice, peripheral blood β-HCG levels in both groups A and B increased significantly on day 14 after cell inoculation (P<0.05), and the increment was more conspicuous in group B (P<0.05).</p><p><b>CONCLUSION</b>Mouse models bearing human choriocarcinoma xenograft can be successfully established by injecting JAR cells either subcutaneously or via the tail vein to mimic the characteristics of epithelial solid tumors and lung metastasis of human choriocarcinoma.</p>

Expression of mucin 15 in hepatocellular carcinoma and its clinical implications / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expression of mucin 15 (MUC15) in hepatocellular carcinoma and explore its association with the prognosis of patients.</p><p><b>METHODS</b>The expression of MUC15 was detected by quantitative RT-PCR and Western blotting in liver cell line L02, liver cancer cell lines HepG2, MHCC-97H, and SMMC-7721, and in 122 HCC and corresponding adjacent non-tumor liver tissues. The association of MUC15 expression in HCC tissues with the clinical parameters and the patients' survival was analyzed.</p><p><b>RESULTS</b>The 1iver cell line L02 showed significantly higher MUC15 expression level than the liver cancer cell lines HepG2, MHCC-97H, and SMMC-7721 (P<0.05). The expression level of MUC15 was markedly lower in the HCC tissues than in the adjacent non-tumor liver tissues (P<0.05). MUC15 expression in the HCC tissues was significantly correlated with the tumor TNM stage, intrahepatic or lymphatic metastasis, portal vein thrombosis and tumor differentiation (P<0.05). Kaplan-Meier survival analysis suggested that a low MUC15 expression was associated with a poor clinical prognosis of the patients.</p><p><b>CONCLUSION</b>The expression of MUC15 is correlated with the clinicopathological features and prognosis of HCC patients, and may potentially serve as a novel prognostic marker for HCC.</p>

Irradiation induced epithelial-mesenchymal transition in nasopharyngeal carcinoma in vitro / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the irradiation induced epithelial-mesenchymal transition (EMT) in nasopharyngeal carcinoma (NPC) in vitro.</p><p><b>METHODS</b>NPC CNE-2 cells with radioresistance (CNE-2-Rs) were established by exposure to gradiently increased dose of irradiation. CCK-8 cell viability kits, colony formation assay and fluorescence-activated cell sorting analysis were used to confirm the capacity of radioresistance of CNE-2-Rs cells. Invert microscope was used to monitor the morphological changes and western blot was applied to detect the expression of epithelial cell marker E-cadherin and mesenchymal cell marker Vimentin during the phase of CNE-2 exposure to irradiation.</p><p><b>RESULTS</b>Irradiation exposure successfully induced the radioresistance of CNE-2 cells. After exposed to irradiation, the survival rate in CNE-2-Rs was higher than that in CNE-2 by CCK-8 assays. No significant difference of proliferation ability was observed between the CNE-2 and CNE-2-Rs pre-radiotherapy, but a higher proliferation ability in the CNE-2-Rs post-radiotherapy. By using the colony forming assay, the parameters of CNE-2 and CNE-2-Rs in multi-target single-hit and linear quadratic model were obtained and the data demonstrated that parameters mean lethal dose (D0) , quasi-thres hold dose (Dq) , surrival fraction in 2Qy (SF2) and mean inctivation dose (MID) value increased, α and α/β value decreased (P < 0.05) . At the same time, the CNE-2-Rs cells showed higher percentage of cells in S and G2 phase (P < 0.05) . In terms of biomorphology, CNE-2-Rs cells were more narrow, long strips or fusiform shapes, stretched out tentacles, and the contacts between them were loosened. When radiation dose accumulated to 24 Gy, an over-expression of Vimentin was observed in treated cells, while E-cadherin was down-regulated (P < 0.01) .</p><p><b>CONCLUSIONS</b>NPC cells present with typical morphorlogical and biomolecular changes of EMT during exposure to irradiation, indicating the potential critical roles of EMT in the malignant behavior of radioresistance in NPC.</p>

Expression of astrocyte elevated gene-1 protein and its clinical significance in laryngeal squamous cell carcinoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To assess the protein expression of astrocyte elevated gene-1 (AEG-1) in tissue specimens of laryngeal squamous cell carcinoma (LSCC), and to correlate its expression with clinicopathological parameters and prognosis in patients with LSCC.</p><p><b>METHODS</b>RT-PCR was used to assay the expression of AEG-1 mRNA in 13 pairs of LSCC tissues and their corresponding noncarcinoma epithelia. Immunohistochemistry was performed on paraffin-embedded tissue specimens to investigate the protein expression of AEG-1 in 88 cases of LSCC specimens and 15 cases of adjacent epithelial samples.</p><p><b>RESULTS</b>The expression of AEG-1 mRNA was significantly increased in LSCC tissues compared to adjacent noncarcinoma epithelial tissues (0.81 ± 0.17 vs. 0.23 ± 0.10;t = 10.337, P < 0.001). Meantime, the positive rate of AEG-1 protein in 88 cases of LSCC was 87.5% (77/88). However, 15 cases of adjacent noncarcinoma epithelial merely demonstrated negative or mild expression of AEG-1 protein. AEG-1 overexpression was closely correlated with T stage (χ(2) = 6.289, P = 0.018), clinical stage (χ(2) = 11.049, P < 0.01), metastasis (χ(2) = 20.859, P < 0.01) and recurrence(χ(2) = 13.459, P < 0.01). The overall survival rates of patients with AEG-1 overexpression and low expression were 35.9% and 86.4%, respectively (χ(2) = 23.409, P < 0.01). Multivariate Cox regression analysis revealed that AEG-1 expression was an independent prognostic factor (P = 0.016).</p><p><b>CONCLUSION</b>AEG-1 protein may play a critical role in the initiation and progression of LSCC, implicating its predictive value in prognosis.</p>

EphA2 mediated vascular endothelial growth factor expression via the p38 MAPK signaling pathway in squamous cell carcinoma of the head and neck / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the regulatory effect of erythropoietin-producing hepatocellular receptor (EphA2) on the expression of VEGF protein, a pro-angiogenic factor, via p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway in squamous cell carcinoma of the head and neck(SCCHN) in vitro.</p><p><b>METHODS</b>SCCHN Tu686 cells were transfected with EphA2 overexpression vector pEGFP-N1-EphA2. Western blot was used to detect the expression of p38 MAPK and enzyme-linked immunosorbent assay (ELISA) was applied to assay of VEGF. SB203580 as a inhibitor of p38 MAPK signaling pathway was used.</p><p><b>RESULTS</b>The expression of VEGF protein was significantly up-regulated in Tu686 cells transfected with EphA2 overexpression vector (535.31 ± 45.71) pg/ml, when compared with Tu686 cells transfected with empty vector (400.99 ± 33.50) pg/ml and Tu686 cells with no transfection (385.30 ± 33.50) pg/ml (F = 17.091, P < 0.01). The expression of phosphorylated p38 MAPK was obviously increased in Tu686 cells with EphA2 overexpression. SB203580 inhibited the expressions of VEGF and phosphorylated p38 MAPK proteins in Tu686 cells with EphA2 overexpression.</p><p><b>CONCLUSION</b>EphA2 can regulate the expression of VEGF protein and stimulate p38 MAPK signaling pathway.</p>

EphA2 promotes angiogenesis and metastasis of head and neck squamous cell carcinoma in vivo / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the effects of EphA2 on the angiogenesis and cervical lymph node metastasis of squamous cell carcinoma of the head and neck (SCCHN) in vivo.</p><p><b>METHODS</b>EphA2 short hairpin (shRNA) lentiviral particles were used to knockdown the expression of EphA2 in SCCHN cell line M2 with high lymph nodes metastasis rate. Stable clones, obtained by puromycin screening, were assayed by RT-PCR and Western blot to validate the gene silencing efficiency and were used to establish SCCHN metastatic xenograft mouse model. Hematoxylin-eosin staining was applied to identify cervical lymph node metastasis of SCCHN in xenografted tumors. Immunohistochemistry was used to observe microvessel density. Western blot was used to investigate the protein expressions of EphA2 and vascular endothelial, growth factor (VEGF).</p><p><b>RESULTS</b>EphA2 shRNA lentiviral particles efficiently decreased the mRNA and protein expressions of EphA2 in SCCHN cell line M2, which were further successfully utilized to establish SCCHN metastatic xenograft mouse model. Compared with xenografted tumors in control group, xenografted tumors in M2EphA2RNAi(+) group decreased significantly tumor volume [(430.7 ± 190.0) mm(3) (x(-) ± s) vs (1179.0 ± 289.4) mm(3)] and weight [(0.26 ± 0.10) g vs (0.54 ± 0.12) g] (both P < 0.05). More importantly, bilateral cervical lymph node metastasis rate in M2EphA2RNAi(+) was also greatly declined (Mann-Whitney U = 10.0, P < 0.05). Decreased protein expressions of EphA2 and VEGF and microvessel density were observed in M2EphA2RNAi(+) group (t = 26.751, P < 0.01).</p><p><b>CONCLUSIONS</b>Knockdown of EphA2 expression led to the inhibition of tumor growth and metastasis in SCCHN nude mouse model. More importantly, SCCHN angiogenesis was also impeded, which might be associated with the decreased expression of VEGF.</p>

EGCG regulates TGF-β1-induced epithelial mesenchymal transition in squamous cell carcinoma of head and neck / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To study the effect and molecular mechanism of epigallocatechin-3-gallate (EGCG) on epithelial-mesenchymal transition (EMT) in vitro induced by human recombinant TGF-β1 protein in squamous cell carcinoma of the head and neck.</p><p><b>METHODS</b>EMT morphological changes of Tu686 cells were observed after sequential treatment of 5 ng/ml TGF-β1 and 20 µmol/L EGCG. Tu686 cells were collected after the treatment of 5 ng/ml TGF-β1 for 24 h and EGCG with different concentrations (0, 10, 20, 30 µmol/L) for another 24 h or 20 µmol/L EGCG treatment for different time phase (6, 12, 24 h). Then RT-PCR and Western-blot were applied to detect mRNA and protein expression level of epithelial cell marker E-cadherin, mesenchymal cell marker Vimentin and Smad7, an inhibit molecule of TGF-β1 mediated pathway in Tu686 cells.</p><p><b>RESULTS</b>TGF-β1 successfully induced characterized EMT morphological and molecular changes in Tu686 cells, in which expression of E-cadherin decreased, Vimentin increased and Smad7 declined. However, EGCG could reverse the TGF-β1 mediated process of EMT by downregulating the expression of Vimentin and upregulating the expression of E-cadherin and Smad7.</p><p><b>CONCLUSION</b>EGCG significantly inhibits TGF-β1-mediated EMT inTu686 cell lines of SCCHN, which maybe associated with the upregulated-expression of Smad7, an inhibitor in TGF-β1 signaling pathway.</p>

Expression of HMGB1 protein in laryngeal squamous cell carcinoma and its clinical significance / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To evaluate the expression of HMGB1 protein in tissue specimens of laryngeal squamous cell carcinoma (LSCC) and adjacent normal mucosa, and explore the correlation of HMGB1 protein expression with clinicopathologic features and prognosis in LSCC.</p><p><b>METHODS</b>Ninty-three cases of LSCC and 5 cases of adjcent mucosal tissue samples were included in this study. Immunohistochemical staining was performed on paraffin-embedded tissue specimens to examine the HMGB1 protein expression. The data were futher correlated with the clinicopathological features and prognosis of the LSCC patients.</p><p><b>RESULTS</b>The positive rates of HMGB1 expression in LSCC specimens was 87.1%, significantly higher than that in the adjcent normal mucosa samples (46.7%, P = 0.001), and its overexpresion was closely correlated with T stage (Chi2 = 10.878, P = 0.004), clinical stage (Chi2 = 21.115, P < 0.01), metastasis (Chi2 = 28.298, P < 0.01) and recurrence (Chi2 = 14. 923, P = 0.001) in patients with LSCC. Patients with HMGB1 overexpression had both poorer disease-free survival and poorer overall survival compared with that in patients with low HMGB1 expression (Chi2 = 13.815, Chi2 = 11.912; Both P < 0.01). Univariate and multivariate Cox regression analyses revealed that HMGBI expression is an independent prognostic factor for patients with LSCC.</p><p><b>CONCLUSIONS</b>The results of this study demonstrate that HMGB1 protein expression is significantly increased in LSCC tissues, and HMGB1 protein overexpression is associated with a poorer prognosis in patients with LSCC. These results suggest that HMGB1 may play a critical role in the initiation and progression of LSCC, implicating HMGB1 may become a valuable marker for the prediction of prognosis in patients with LSCC.</p>

Expressions and clinical significance of high mobility group box-1 mRNA and protein in laryngeal squamous cell carcinoma tissues and serum / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the mRNA and protein expressions of high mobility group box-1 (HMGB1) in the tumor tissues and sera of patients with laryngeal squamous cell carcinoma (LSCC) and their clinical significance.</p><p><b>METHODS</b>Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were used to detected the expressions of HMGB1 mRNA and protein in the tumors and adjacent normal epithelial tissues in 30 patients with LSCC. Serum HMGB1 protein levels in the patients with LSCC and in 10 healthy volunteers were detected with enzyme-linked immunosorbent adsorption experiment (ELISA).</p><p><b>RESULTS</b>RT-PCR demonstrated that the mean relative mRNA expression levels of HMGB1 (HMGB1/GAPDH) in LSCC tissues and in adjacent normal epithelial tissues were 1.25 ± 0.12 and 0.32 ± 0.04, respectively (t = 40.27, P < 0.05). Western blot revealed that the mean relative protein expression levels of HMGB1 (HMGB1/β-actin) were 1.29 ± 0.10 and 0.34 ± 0.03 (t = 49.84, P < 0.05), respectively. Both mRNA and protein expression levels of HMGB1 were associated with T stage, clinical stage, lymph node metastasis status and smoking (all P < 0.05), but no significant correlation with age, alcohol consumption and primary tumor grade and location (all P > 0.05). Mean serum HMGB1 protein levels in patients with LSCC and healthy volunteers were (24.80 ± 14.08) ng/ml and (23.58 ± 14.69) ng/ml (t = 0.37, P > 0.05).</p><p><b>CONCLUSIONS</b>Both mRNA and protein expressions of HMGB1 were obviously elevated in LSCC, which were associated closely with T stage, clinical stage and lymph node metastasis.</p>

Correlation of EphA2 protein expression with clinicopathological characteristics and prognosis in laryngeal squamous cell carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To evaluate the expression of EphA2 protein in tissue specimens and cell lines of laryngeal squamous cell carcinoma (LSCC), and to further study the correlation of EphA2 protein expression with clinicopathological characteristics and prognosis in LSCC.</p><p><b>METHODS</b>Western blot was applied to assess the EphA2 protein expression in LSCC cell line Hep-2 cells and the head and neck immortalized epithelial cell line NP-69 cells. Immunohistochemical staining was performed on paraffin sections of 88 cases of LSCC specimens and 16 cases of adjcent normal tissue samples to investigate the EphA2 protein expression, and to futher elucidate its correlation with clinicopathological characteristics.</p><p><b>RESULTS</b>Compared with the NP-69 cells, EphA2 expression in LSCC cell line Hep-2 cells was upregulated. The positive rates of EphA2 expression in LSCC and adjcent normal tissues samples were 80.7% and 43.8%, respectively, with a significant difference between the two groups (P < 0.001). EphA2 overexpresion was closely correlated with clinical stage (I + II/III + IV, P = 0.005), metastasis (P = 0.025) and recurrence (P = 0.021) in LSCC. Furthermore, patients with EphA2 overexpression had poorer tumor-free survival and 5-year overall survival compared with that in patients with low EphA2 expression (33.3% vs. 63.2%, P = 0.003; 46.7% vs. 81.6%, P = 0.002). EphA2 expression combined with clinical stage provided a better predictive value in prognosis. Univariate and multivariate Cox regression analysis revealed that EphA2 expression is an independent prognostic factor for patients with LSCC (P = 0.019).</p><p><b>CONCLUSIONS</b>The results of this study demonstrate that EphA2 protein expression is significantly increased in LSCC tissues and cell lines, and EphA2 protein overexpression is associated with tumor recurrence, metastasis and poorer prognosis in LSCC patients. These results suggest that EphA2 may play a critical role in the initiation and progression of LSCC, implicating EphA2 as a valuable marker for the prediction of recurrence, metastasis and prognosis in LSCC.</p>

[Cloning of hsa-miR-148a and construction of its retroviral expression vector].

OBJECTIVE: To clone hsa-miR-148a and construct its retroviral expression vector. METHODS: The pre-miR-148a amplified by PCR was inserted to pMSCV to construct the recombinant retroviral expression plasmid pMSCV-miR-148a, which was confirmed by restriction endonuclease analysis and DNA sequencing. The retroviral expression vector pMSCV-miR-148a and PIK packaging plasmid were cotransfected into 293FT packaging cells by calcium phosphate-mediated transfection to produce the retrovirus, and the retrovirus titer was measured by infection of NIH3T3 cells. RESULTS: Restriction enzyme digestion and DNA sequencing demonstrated that the retroviral vector pMSCV-miR-148a was constructed successfully, and the virus titer was 5x10(8) CFU/ml after infection of NIH3T3 cells. CONCLUSION: The successful construction of the retroviral expression vector MSCV-miR-148a allows the production of high-titer retrovirus to facilitate further study of the molecular functions of miR-148a.