Zhisen Shen

piR-823, a novel non-coding small RNA, demonstrates in vitro and in vivo tumor suppressive activity in human gastric cancer cells

Piwi-interacting RNAs (piRNAs), new non-coding small RNAs, are association with chromatin organization, messenger RNA stability and genome structure. However, the roles of piRNA in carcinogenesis are not clearly defined. By using real-time reverse transcription-polymerase chain reaction technology, we found that the expression level of piR-823 in gastric cancer tissues was significant lower than that in non-cancerous tissues. After increase the level of piR-823 in gastric cancer cells, cell growth was inhibited. The results of xenograft nude mice model confirmed its tumor suppressive properties. All of the evidences indicated that piR-823 play a crucial role in the occult of gastric cancer.

MicroRNA-34a affects the occurrence of laryngeal squamous cell carcinoma by targeting the antiapoptotic gene survivin.

Survivin has been shown to be an ideal target for cancer gene therapy because of its strong antiapoptotic effect. MicroRNA-34a (miR-34a) can function as a tumor suppressor in some cancers through negative regulation of gene expression. However, the relationship between miR-34a and survivin in larynx squamous cell carcinoma (LSCC) has not been explored. The abundance of survivin mRNA and miR-34a in LSCC tissues were measured using quantitative real-time polymerase chain reaction. Their expression levels were analyzed and correlated with tumor differentiation, lymphatic metastasis, clinical stages, and survival rates. MiR-34a mimic was transfected using liposomes to increase its level in LSCC cancer cell line, Hep-2. The effects of miR-34a on survivin protein expression were tested using western blot analysis. Cell cycle analyses were performed using flow cytometry. The results showed that transfection of miR-34a mimic significantly suppressed cell proliferation with decreased survivin protein expression, but did not affect mRNA expression level. The results from LSCC tissue samples showed that miR-34a was downregulated, while survivin expression was upregulated. The miR-34a levels were negatively correlated with histologic differentiation and were positively correlated with survival rate. MiR-34a significantly suppressed cell proliferation by arresting cells at G0/G1 phase in Hep-2 cells. These results indicated that miR-34a may affect the occurrence of LSCC by targeting survivin.

Long non-coding RNA profiling in laryngeal squamous cell carcinoma and its clinical significance: potential biomarkers for LSCC.

Long non-coding RNAs (lncRNAs) are novel transcripts that may play important roles in cancer. Our study aimed to resolve the lncRNA profile of larynx squamous cell carcinoma (LSCC) and to determine its clinical significance. The global lncRNA expression profile in LSCC tissues was measured by lncRNA microarray. Distinctly expressed lncRNAs were identified and levels of AC026166.2-001 and RP11-169D4.1-001 lncRNAs in 87 LSCC samples and paired adjacent normal tissue were analyzed by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). The clinical significance of these lncRNAs in laryngeal cancer was analyzed and survival data were estimated by the Kaplan–Meier method and the log-rank test. A receiver operating characteristic (ROC) curve was constructed to check the diagnostic value. In the lncRNA expression profile of tumor samples, 684 lncRNAs were upregulated and 747 lncRNAs were downregulated (fold-change >2.0). Of these, AC026166.2-001 and RP11-169D4.1-001 were distinctly dysregulated, with AC026166.2-001 exhibiting lower expression in cancer tissues and RP11-169D4.1-001 higher expression. We verified that both AC026166.2-001 and RP11-169D4.1-001 were expressed at a lower level in cervical lymph nodes compared with paired laryngeal cancer tissues and paired normal tissues. RP11-169D4.1-001 levels were positively correlated with lymph node metastasis (P = 0.007). From the survival analysis, decreased levels of AC026166.2-001 and RP11-169D4.1-001 were associated with poorer prognosis. The area under the ROC curve was up to 0.65 and 0.67, respectively, and the cut-off point of ΔCt was 11.23 and 10.53, respectively. AC026166.2-001 and RP11-169D4.1-001 may act as novel biomarkers in LSCC and may be potential therapeutic targets for LSCC patients. Both AC026166.2-001 and RP11-169D4.1-001 could be independent prognostic factors for survival in LSCC.

Surface modification of polyurethane towards promoting the ex vivo cytocompatibility and in vivo biocompatibility for hypopharyngeal tissue engineering

Polymeric substrates with good biocompatibility have been widely employed to create a living construct with the complexities of tissue histology and function in the field of tissue engineering. In this study, poly(ester-urethane) (58213, NAT022) was used to be substrate due to its good physical and chemical properties. Proteins like gelatin or silk fibroin were covalently bonded on its surface using method of diamine aminolysis and glutaraldehyde crosslinking, which had been setup in our group in order to improve poly(ester-urethane)’s hydrophilicity and biocompatibility. The modification was proved by the measurements of static and dynamic contact angles and fluorescence detection. The biological properties were evaluated as in vitro cell culture and in vivo transplantation via cell number counting, morphology observation, immunohistochemistry analysis, etc. The results showed that gelatin or silk fibroin grafted membrane displayed good cytocompatibility, i.e. good proliferation and differentiation of human hypopharynx fibroblast and skeletal muscle cell though the control poly(ester-urethane) indicated low toxicity to cells and good biocompatibility, which was also verified in in vivo experiment. After poly(ester-urethane)–silk fibroin was implanted subcutaneously in rat back, it exhibited a better compatibility to peripheral tissue and faster biodegradation than the control poly(ester-urethane) did. This information supplied us valuable knowledge for poly(ester-urethane) to be used as matrix in situ hypopharynx regeneration study.

Skeletal Muscle Regeneration on Protein-Grafted and Microchannel-Patterned Scaffold for Hypopharyngeal Tissue Engineering

In the field of tissue engineering, polymeric materials with high biocompatibility like polylactic acid and polyglycolic acid have been widely used for fabricating living constructs. For hypopharynx tissue engineering, skeletal muscle is one important functional part of the whole organ, which assembles the unidirectionally aligned myotubes. In this study, a polyurethane (PU) scaffold with microchannel patterns was used to provide aligning guidance for the seeded human myoblasts. Due to the low hydrophilicity of PU, the scaffold was grafted with silk fibroin (PU-SF) or gelatin (PU-Gel) to improve its cell adhesion properties. Scaffolds were observed to degrade slowly over time, and their mechanical properties and hydrophilicities were improved through the surface grafting. Also, the myoblasts seeded on PU-SF had the higher proliferative rate and better differentiation compared with those on the control or PU-Gel. Our results demonstrate that polyurethane scaffolds seeded with myoblasts hold promise to guide hypopharynx muscle regeneration.

Growth inhibitory effects of DJ-1-small interfering RNA on laryngeal carcinoma Hep-2 cells.

Cancer of the larynx is the commonest head and neck squamous cell carcinoma. The DJ-1 gene is a novel mitogen-dependent oncogene. Survivin is a structurally unique member of the inhibitors of apoptosis proteins. DJ-1 and survivin play important roles in carcinogenesis. The function of DJ-1, and the relationship between DJ-1 and survivin in laryngeal carcinoma, has never been explored. Small interfering RNAs (siRNAs) directed against the DJ-1 gene were initially transfected into laryngeal carcinoma Hep-2 cells with liposome. The viability of Hep-2 cells was then detected by the MTT assay. The changes in cell-cycle distribution were monitored by flow cytometry. Finally, changes in DJ-1 and survivin genes mRNA and protein levels were evaluated by semi-quantitative RT-PCR and Western blotting, respectively. Blocking expression of the DJ-1 gene with DJ-1-siRNA significantly suppressed the viability of Hep-2 cells. Treatment with DJ-1-siRNA resulted in a G2/M accumulation. Expressions of DJ-1 and survivin gene mRNA and protein levels were suppressed by DJ-1-siRNA in a dose-dependent manner. These data indicate, for the first time, that the DJ-1 gene may have an important role in the carcinogenesis of laryngeal carcinoma.

Promoter hypermethylation of miR-34a contributes to the risk, progression, metastasis and poor survival of laryngeal squamous cell carcinoma

MiR-34a is a direct transcriptional target of p53, which induces cell cycle arrest, senescence, and apoptosis. Recently, we and others identified abnormal expression of miR-34a in laryngeal squamous cell carcinoma (LSCC). The aim of our present study was to investigate the contribution of miR-34a promoter methylation to LSCC. Bisulfite pyrosequencing technology was applied to measure DNA methylation levels of six CpG sites in the miR-34a promoter from 104 LSCC tumor tissues and their corresponding adjacent tissues. Our results showed that the methylation levels of the miR-34a promoter were significantly higher in cancer tissues compared with the adjacent tissues (adjusted P=5.05E-10). A breakdown analysis for cigarette smoking behavior indicated a significantly elevated tendency of miR-34a methylation level in LSCC patients with smoking behavior but not in LSCC patients without smoking behavior (Smoking: Tumor vs Normal, adjusted P=3.12E-9; Non-smoking: Tumor vs Normal, adjusted P=0.073). In addition, miR-34a promoter methylation frequency remarkably increased in the advanced stage patients (adjusted P=0.003) and advanced T classified tumors (adjusted P=0.015). Moreover, significant association of miR-34a promoter hypermethylation with LSCC lymph metastasis was observed (adjusted P=0.002). Meanwhile, Kaplan-Meier survival curves results showed that high methylation of miR-34a promoter were associated with poor overall survival (log-rank test, P=0.023). Our study revealed that miR-34a promoter hypermethylation was a risk factor for LSCC, played a critical role in the disease progression and metastasis, and could serve as a poor prognostic factor for LSCC.

Engineered Hypopharynx from Coculture of Epithelial Cells and Fibroblasts Using Poly(ester urethane) as Substratum

Porous polymeric scaffolds have been much investigated and applied in the field of tissue engineering research. Poly(ester urethane) (PEU) scaffolds, comprising pores of 1–20 μm in diameter on one surface and ≥200 μm on the opposite surface and in bulk, were fabricated using phase separation method for hypopharyngeal tissue engineering. The scaffolds were grafted with silk fibroin (SF) generated from natural silkworm cocoon to enhance the scaffolds hydrophilicity and further improve cytocompatibility to both primary epithelial cells (ECs) and fibroblasts of human hypopharynx tissue. Coculture of ECs and fibroblasts was conducted on the SF-grafted PEU scaffold (PEU-SF) to evaluate its in vitro cytocompatibility. After co-culture for 14 days, ECs were lined on the scaffold surface while fibroblasts were distributed in scaffold bulk. The results of in vivo investigation showed that PEU porous scaffold possessed good biocompatibility after it was grafted by silk fibroin. SF grafting improved the cell/tissue infiltration into scaffold bulk. Thus, PEU-SF porous scaffold is expected to be a good candidate to support the hypopharynx regeneration.

Synthesis and Characterization of Biodegradable Polyurethane for Hypopharyngeal Tissue Engineering

Biodegradable crosslinked polyurethane (cPU) was synthesized using polyethylene glycol (PEG), L-lactide (L-LA), and hexamethylene diisocyanate (HDI), with iron acetylacetonate (Fe(acac)3) as the catalyst and PEG as the extender. Chemical components of the obtained polymers were characterized by FTIR spectroscopy, 1H NMR spectra, and Gel Permeation Chromatography (GPC). The thermodynamic properties, mechanical behaviors, surface hydrophilicity, degradability, and cytotoxicity were tested via differential scanning calorimetry (DSC), tensile tests, contact angle measurements, and cell culture. The results show that the synthesized cPU possessed good flexibility with quite low glass transition temperature (T g, −22°C) and good wettability. Water uptake measured as high as 229.7 ± 18.7%. These properties make cPU a good candidate material for engineering soft tissues such as the hypopharynx. In vitro and in vivo tests showed that cPU has the ability to support the growth of human hypopharyngeal fibroblasts and angiogenesis was observed around cPU after it was implanted subcutaneously in SD rats.

Analysis of factors in successful nasal endoscopic resection of nasopharyngeal angiofibroma

Abstract Conclusions: Endoscopic resection of nasopharyngeal angiofibroma is less traumatic, causes less bleeding, and provides a good curative effect. Using pre-operative embolization and controlled hypotension, reasonable surgical strategies and techniques lead to successful resection tumors of a maximum Andrews-Fisch classification stage of III. Objective: To investigate surgical indications, methods, surgical technique, and curative effects of transnasal endoscopic resection of nasopharyngeal angiofibroma, this study evaluated factors that improve diagnosis and treatment, prevent large intra-operative blood loss and residual tumor, and increase the cure rate. Methods: A retrospective analysis was performed of the clinical data and treatment programs of 23 patients with nasopharyngeal angiofibroma who underwent endoscopic resection with pre-operative embolization and controlled hypotension. The surgical method applied was based on the size of tumor and extent of invasion. Curative effects were observed. Results: No intra-operative or perioperative complications were observed in 22 patients. Upon removal of nasal packing material 3–7 days post-operatively, one patient experienced heavy bleeding of the nasopharyngeal wound, which was treated compression hemostasis using post-nasal packing. Twenty-three patients were followed up for 6–60 months. Twenty-two patients experienced cure; one patient experienced recurrence 10 months post-operatively, and repeat nasal endoscopic surgery was performed and resulted in cure.