[LLY-283 inhibits proliferation and metastasis of head and neck squamous cell carcinoma by targeting PRMT5].

PURPOSE: To explore the effect of LLY-283 on the biological behavior of Head and neck squamous cell carcinoma(HNSCC) proliferation and metastasis through protein arginine methyltransferase 5(PRMT5). METHODS: TCGA database was used to analyze the expression level of PRMT5 in HNSCC tissues and cell lines by RT-PCR and Western blot. Lentiviral technology was used to construct PRMT5 knockdown stable cell line, and analyze the effect of PRMT5 on the biological behavior of HNSCC. Drug killing experiment was used to observe the IC50 changes of LLY-283 in cell lines. Nude mouse xenograft experiments were further tested to observe the biological effects of LLY-283 on HNSCC through PRMT5. RESULTS: PRMT5 was highly expressed in HNSCC tissues and cell lines, which promoted the proliferation and metastasis of cell lines, and reduced the IC50 value of LLY-283. LLY-283 could significantly reduce the cell proliferation and metastasis, tumor volume and Ki-67 expression in nude mice in vivo of HNSCC through PRMT5. CONCLUSIONS: LLY-283 inhibits the expression of PRMT5 and Ki-67, thereby decreases the proliferation and metastasis of HNSCC and the ability to form transplanted tumors in nude mice, exerting anti-tumor effects.

[Effect of silencing LASP1 on biological behaviors of oral squamous cell carcinoma cells and IC50 of three anti-tumor drugs].

PURPOSE: This study was designed to investigate the effects of LASP1 on proliferation, metastasis, invasion, and cycle of oral squamous cell carcinoma cells and analyze the changes of IC50 in three antitumor drugs: cisplatin, apatinib and docetaxel. METHODS: The correlation between LASP1 and survival rate and prognosis of patients with head and neck cancer were analyzed on the human protein atlas data. RT-PCR and Western blot were used to detect mRNA and protein expression of LASP1 in oral squamous cell carcinoma cell lines. LASP1 silenced HN30 stable transfectant cell line was constructed by lentivirus. CCK-8 assay was used to detect cell proliferation. Plate colony assay was used to detect cell clone formation ability. Transwell assay was used to detect cell migration and invasion ability. Flow cytometry was used to detect cell cycle changes. Oral squamous cell carcinoma metastases were established in nude mouse, the number of metastatic lung nodules was counted and stained with H-E. CCK-8 method was used to analyze the changes of IC50 in three antitumor drugs: cisplatin, apatinib and docetaxel. Statistical analysis was performed using SPSS 11.0 software package. RESULTS: LASP1 was closely related to the survival rate and prognosis of head and neck cancer. LASP1 promoted proliferation, colony formation, metastasis and invasion of oral squamous cell carcinoma cell line HN30, promoted G2/M phase transition of cell cycle, and significantly reduced the formation of lung metastasis in nude mice after silencing. There was significant correlation with docetaxel IC50 but no significant impact on cisplatin IC50 and aptatinib IC50. CONCLUSIONS: LASP1 enhances cell proliferation, plate cloning, metastasis and invasion, G2/M phase transition of cell cycle, promotes lung metastasis in nude mice and docetaxel resistance of oral squamous cell carcinoma cell line HN30.

[Clinical effect of 3 polishing methods on resin composite restoration in filling wedge-shaped defect].

PURPOSE: To evaluate the clinical effect of 3 polishing methods on resin composite restoration in filling wedge-shaped defect. METHODS: One hundred and fifty patients with wedge-shaped defects were randomly divided into 3 groups. After being filled with Nano composite resin(FILTEK Z350,3M), restorations in group 1 were polished with Sof-lex discs system, restorations in group 2 were polished with Super-snap system and group 3 with diamond bur and rubber cup. Restorations in each group were reexamined and assessed utilizing standards of USPH&Ryge after 0.5, 1 and 2 a. Chi-square test was performed using SPSS17.0 software package. RESULTS: No significant difference of secondary decay, marginal adaption, marginal staining, surface roughness, colour matching, wearing and gingival condition among 3 groups was found after 0.5 a and 1 a. Restorations in group 1 and group 2 showed better performance with regards to secondary decay, marginal adaption, marginal staining, wearing and color matching than group 3 after 2 years of restoration. CONCLUSIONS: Sof-lex discs and Super-snap polishing system after composite filling of wedge-shaped defect was effective in reducing secondary decay, marginal staining and wearing, as well as in maintaining the marginal adaption, which is worthy of wide clinical application.

LATS2 induced by TNF-alpha and inhibited cell proliferation and invasion by phosphorylating YAP in oral squamous cell carcinoma.

OBJECTIVE: Many reports indicated LATS2 was a component of the Hippo pathway, could phosphorylate and inactivate YAP, acted as a tumor suppressor in human cancers. But few studies investigated the role of LATS2 in oral squamous cell carcinoma (OSCC) and clarified the mechanisms of regulation of LATS2 expression. DESIGN: The expressions of LATS2 and phosphorylated YAP were detected by Western blotting in HN6 cells treated with TNF-α in different time and different dose. Luciferase reporter assays were performed to detect whether YAP can be phosphorylated by LATS2 in HN6 cells. Cell proliferation, anchorage independent growth in soft agar, transwell cell invasion assay, and nu mice in vivo xenografts growth were performed to study the effects of overexpression of LATS2 on OSCC cells. RESULTS: In this study, we confirmed that YAP can be phosphorylated by LATS2. LATS2 can be dose- and time-dependently induced by TNF-α in HN6 cells. Overexpression of LATS2 inhibited cell proliferation, colony formation, cell invasion, and in vivo xenografts growth in OSCC cells. CONCLUSION: LATS2 could be induced by TNF-alpha and inhibited cell proliferation and invasion by phosphorylating YAP in OSCC cells. LATS2 might play a role in the tumorigenesis of OSCC and might be a potential therapeutic target in OSCC treatment.

[Expression of galectin-1 in carcinogenesis of oral mucosal epithelium].

OBJECTIVE: To investigate the expression of galectin-1 in oral squamous cell carcinoma(OSCC) and its clinical significance. METHODS: Detection of the mRNA and protein expression of galectin-1 in the in vitro cellular carcinogenesis model of OSCC, OSCC cell lines and tissue specimens from 30 primary OSCC patients were performed using real-time polymerase chain reaction (PCR), Western blotting and immunohistochemistry, respectively. RESULTS: The value of galectin-1 mRNA and protein level in human immortalized oral epithelia cell (HIOEC) cell was 0.071 ± 0.023, 0.118 ± 0.046, Compared with the HIOEC, galectin-1 mRNA level and protein expression were increased significantly in all the cell lines (0.141 ± 0.049, 0.504 ± 0.33) (P < 0.01). The levels of mRNA and protein expression of galectin-1 were significantly higher in the cancerous tissue (0.059 ± 0.034, 1.5 ± 0.68) than in the normal adjacent tissues (0.029 ± 0.012, 0.4 ± 0.56) (P < 0.01). CONCLUSIONS: The expression of galectin-1 gene up-regulated in carcinogenesis process of OSCC significantly may be related to the tumorigenesis and development of OSCC, which illustrates its potential clinical application as tumor marker for early diagnosis.

Yes-associated protein promotes cell proliferation by activating Fos Related Activator-1 in oral squamous cell carcinoma.

In our previous study, we established an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC), including a human immortalized oral epithelial cell (HIOEC) and a cancerous cell line (HB96). Microarray analysis showed that the gene encoding Yes-associated protein (YAP) was significantly increased in HB96 cells compared with HIOEC cells. But the underlying mechanism of YAP on oncogenesis, especially its downstream targets, are still not clear. YAP expression in OSCC cell lines and tissue specimens were investigated by using real-time PCR, western blotting and immunohistochemistry staining. YAP put-back plasmid with four mutation sites after YAP-siRNA interference was constructed by site-directed mutagenesis. Cell growth and colony formation were observed after YAP-siRNA interference or YAP put-back again in CAL27 cells. YAP expression was increased in the cellular carcinogenesis models and the clinical samples from primary OSCC patients. Inhibition of YAP by siRNA interference in CAL27 cells significantly inhibited cell proliferation and colony formation in soft agar, but these abilities were rescued when YAP was put-back again. At the same time, Fos Related Activator-1 (Fra-1) was down-regulated when YAP was inhibited by siRNA interference while Fra-1 was rescued when YAP was put-back again. Immunohistochemistry results also indicated that higher levels of YAP were significantly associated with Fra-1 overexpression in OSCC clinical samples. YAP could promote cell proliferation by activating transcription factor Fra-1 in oral squamous cell carcinoma.

Decreased expression of S100A6 in oral squamous cell carcinoma.

We previously established an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC) with a line of human immortalized oral epithelia cells (HIOECs), a line of cancerous HB96 cells, and other cells (HB56 cells) at the early stage of carcinogenesis. In this study, comparative proteomic analysis identified a panel of differentially expressed proteins among these cells, and S100A6 was shown as one of the significantly down-regulated proteins accompanying cellular transformation. S100A6 was further validated for its expression in the three cell lines and in the clinical samples of cancerous and paracancerous tissues from 30 primary OSCC patients. Western blot analysis and real-time PCR revealed the decreased S100A6 protein and mRNA levels in the cancerous HB56 and HB96 cells over HIOECs. Immunohistochemistry and real-time PCR also showed decreased S100A6 protein and mRNA levels in the cancerous tissues compared to the paracancerous tissues from OSCC patients. The results presented here suggest that the expression of S100A6 decreases along with the cancerization in OSCC both in vitro and in vivo.

Overexpression of Galectin-1 is negatively correlated with pathologic differentiation grade in oral squamous cell carcinoma.

PURPOSE: To determine the Galectin-1 protein expression in oral squamous cell carcinoma (OSCC). METHODS: Comparative proteomic analysis of an in vitro cellular carcinogenesis model of OSCC we previously established was performed to identify differentially expressed proteins. Galectin-1 was further validated in vitro (human immortalized oral epithelia cell line and OSCC lines) and in vivo (tissue samples from OSCC patients) by Western blotting and immunohistochemistry, respectively. RESULTS: Increased Galectin-1 protein expression was identified in the cancerous cell line compared with the immortalized oral epithelial cell line in the in vitro cellular carcinogenesis model, and then validated in the OSCC lines and cancerous tissues. Galectin-1 protein expression was negatively correlated with the tumor pathologic differentiation grades, a higher Galectin-1 protein expression indicating a poorer pathologic differentiation grade. CONCLUSIONS: Galectin-1 protein expression level increases in OSCC, it may serve as a candidate marker for pathologic differentiation grade of OSCC.

Fos-related activator-1 is overexpressed in oral squamous cell carcinoma and associated with tumor lymph node metastasis.

BACKGROUND: Previously, we established an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC), including the human immortalized oral epithelia cells (HIOECs) and its derived cancerous HB cells. Then, expression microarray analysis showed that the gene encoding fos-related activator-1 (Fra-1) was significantly upregulated in the cancerous HB cells compared with HIOECs. METHODS: To confirm the expression of Fra-1 at mRNA and protein levels by real-time PCR and western blotting analysis in the carcinogenesis model of OSCC and CAL27 cells. To investigate Fra-1 expression in clinical samples from 30 primary OSCC patients by immunohistochemistry. RESULTS: Fra-1 expression was increased both at mRNA and protein levels in this carcinogenesis model of OSCC and CAL27 cells. Nuclear and cytoplasmic Fra-1 protein expressions both increased in the cancerous tissues compared with those in the paired adjacent non-malignant epithelia (nuclear: P < 0.001, cytoplasmic: P = 0.003). A higher level of nuclear Fra-1 expression was seen in the tumor samples of patients with lymph node metastasis than those without lymph node metastasis (5.07 +/- 1.33 vs 3.81 +/- 1.33, P = 0.023). Higher level of Fra-1 expression was also found in the tumor invasive margin than tumor center. CONCLUSIONS: Fra-1 is a positive gene of OSCC development and progression, Fra-1 can be used as a potential therapeutic target gene and an additional marker for evaluation of lymph node metastasis.

Decreased expression of Annexin A1 correlates with pathologic differentiation grade in oral squamous cell carcinoma.

Previously, we established an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC), including the human immortalized oral epithelia cells (HIOECs) and its derived cancerous HB96 cells. In this study, comparative proteomic analysis identified that Annexin A1 was one of the significantly down-regulated genes in the cancerous HB96 cells. To investigate Annexin A1 down-regulation and its potential usefulness as a molecular marker in OSCC, we further screened Annexin A1 expressions with a panel of OSCC lines, and clinical samples of cancerous and the paired adjacent normal tissues from primary OSCC patients. By Western blot analysis and real-time PCR, we showed that both Annexin A1 mRNA and protein expressions decreased in OSCC cell lines except in two cell lines for the mRNA levels. Immunohistochemistry and real-time PCR also showed that both Annexin A1 mRNA and protein expressions decreased in the cancerous tissues from OSCC patients compared with those in the paired adjacent non-malignant epithelia. More importantly, both Annexin A1 mRNA and protein expressions negatively correlated with the pathologic differentiation grades of cancerous tissues. The lower Annexin A1 mRNA or protein expressions correlated with the poorer pathologic differentiation grades. These results suggest that decreased expression of Annexin A1 contributes to the cancerous progression of OSCC, and Annexin A1 may be a potential biomarker for pathologic differentiation grade of OSCC.

Increased expression of Annexin A2 in oral squamous cell carcinoma.

Previously, in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC) was established with a line of human immortalized oral epithelia cells (HIOECs), a line of cancerous HB96 cells, and another kind of cells (HB56 cells) at the early stage of carcinogenesis. In this study, comparative proteomic analysis identified a panel of differentially expressed proteins among these cells, and Annexin A2 shown as one of the significantly up-regulated proteins accompanying cellular transformation. Annexin A2 was further validated for its expression in the three kinds of cells and in the clinical samples of tumour tissues and their adjacent normal epithelia from primary OSCC patients. Western blot analysis and real-time PCR detected increased Annexin A2 protein and mRNA levels in cancerous HB56 and HB96 cells over HIOECs. Immunohistochemistry showed elevated Annexin A2 protein expression in tumour tissues over the adjacent non-malignant epithelia from OSCC patients; however, the mRNA levels between tumour and normal tissues did not change significantly. Interestingly, levels of Annexin A2 protein expression negatively correlated with the tumour differentiation grades. The results presented here suggest that Annexin A2 protein may play important roles in carcinogenesis of OSCC, and it may also serve as a candidate biomarker for pathologic differentiation grade of OSCC.

Comparative proteomic analysis of differentially expressed proteins in an in vitro cellular carcinogenesis model of oral squamous cell carcinoma.

In vitro cellular model is an important tool to be used to investigate the cellular events related to pathophysiological conditions in humans. We have developed an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC). In this study, we performed comparative proteomic analysis using 2-DE and LC-tandem mass chromatography to separate and identify differentially expressed proteins. Forty-five proteins were identified, including 24 proteins with decreased expression and 19 proteins with increased expression during carcinogenesis from immortalized oral epithelial cells to squamous cancerous cells. The identified known proteins were classified into three ontologies of cellular component, molecular function, and biological process. Further validation of five identified proteins (ANXA1, ANXA2, CTSB, KRT17, and S100A6) in the cellular carcinogenesis model and cancerous tissues from OSCC patients confirmed the comparative proteomic results. Moreover, Annexin A1 and A2 expression levels correlated with the pathological differentiation grade of cancerous tissues. Thus, this work provides a dynamic protein file of differentially expressed proteins in oral squamous carcinoma cells, which could provide clues to study the mechanisms of OSCC carcinogenesis and possibly be developed as potential biomarkers for clinical diagnosis or prognostic monitoring.

[Application of serum tumor markers and support vector machine in the diagnosis of oral squamous cell carcinoma].

PURPOSE: To investigate the clinical application value of serum tumor markers detection combined with support vector machine (SVM) model in the diagnosis of oral squamous cell carcinoma. METHODS: Serum levels of neuron-specific enolase (NSE), cancer antigen 242 (CA242), cancer antigen 19-9 (CA199), carcinoembryonic antigen (CEA), tissue polypeptide antigen (TPA), cancer antigen 72-4 (CA724), cancer antigen 21-1 (CA211) and alpha fetoprotein (AFP) were detected with enzyme-linked immunosorbent assay (ELISA) and time-resolved fluoroimmunoassay (TRFIA) in 163 oral squamous cell carcinoma patients and 160 healthy persons. All the data was analyzed with SVM; the SVM models for diagnosis of oral squamous cell carcinoma were created, trained and validated by cross validation. RESULTS: Among the 163 oral squamous cell carcinoma patients, there were 128 males and 35 females with the male-to-female ratio of 3.66:1; the age ranged from 30 to 85 years old with a mean age of 59.3 years old; according to the primary site of tumor, 72 cases in tongue, 34 in gingiva, 22 in buccal mucosa, 15 in palatal mucosa, 13 in floor of mouth, 4 in lip and 3 in retromolar region; according to the TNM-UICC classification, there were 33 patients at stage T1, 72 at T2, 44 at T3, 14 at T4, 119 at N0, 42 at N1, 2 at N2, 159 at M0, 4 at M1, 27 at clinical stage I, 51 at stage II, 52 at III, and 33 at IV; according to the pathological differentiation grade, 109 tumors were well differentiated, 42 were moderately differentiated and 12 were poorly differentiated. Five serum tumor markers of CA211, CA199, TPA, CA724 and NSE were selected optimally to create the optimal SVM model for diagnosis of oral squamous cell carcinoma. The accuracy, specificity, sensitivity and positive predictive value of the optimal SVM model were 88.54%, 93.13%, 84.05% and 92.57%, respectively. CONCLUSION: From the results, SVM model combined with 5 optimal serum tumor markers is suggested to be used in the diagnosis of oral squamous cell carcinoma. Supported by Shanghai Leading Academic Discipline Project (Grant No.Y0203).

Overexpression of insulin-like growth factor binding protein 3 in oral squamous cell carcinoma.

Previously, we established an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC), including a human immortalized oral epithelial cell (HIOEC) line and its derived cancerous HB96 cell line. Further cDNA microarray analysis showed a significant up-regulated gene, insulin-like growth factor binding protein 3 (IGFBP3), accompanying with in vitro cancerization from HIOEC to HB96. In order to investigate IGFBP3 up-regulation and its potential usefulness as a molecular marker in OSCC, we detected the IGFBP3 expression with a panel of OSCC lines, and clinical samples of cancerous tissues and paired adjacent non-malignant epithelia from primary OSCC patients. Western blotting and real-time PCR showed increased IGFBP3 mRNA level and protein expression in OSCC cell lines compared with HIOEC in vitro; immunohistochemistry and real-time PCR also showed increased IGFBP3 mRNA level and protein expression in cancerous tissues compared with adjacent non-malignant epithelia from OSCC patients. Positive correlations were found between the IGFBP3 protein-positive grade in cancerous tissue and the tumor size as well as lymph node metastasis, a larger tumor size and positive lymph node metastasis indicating a higher level of IGFBP3 protein-positive grade. Based on these results, IGFBP3 may be used as a positive biomarker for OSCC development and progression.

[The experimental study of facial nerve regeneration through E-PTFE tube with nervous homogenates].

PURPOSE: To investigate the effect of using e-PTFE containing nervous homogenates to repair the rabbit facial nerve defect. METHODS: Thirty-six adult New-Zealand rabbits weighing 2000-2500 g were randomly selected. The right sides were selected as e-PTFE containing nervous homogenates group, 8 mm defect of facial never were made, then the defect was bridged with e-PTFE tube, and the nervous homogenates were implanted into the e-PTFE tube. The left sides were selected as e-PTFE group, 8 mm defect of facial never were made, the proximal and distal stumps were simply bridged with e-PTFE tube. Regeneration of the nerve was assessed by gross observation, nerve electrophysiological test and optical microscope observation. RESULTS: The electrophysiological examination showed that in the e-PTFE group, there was no muscular contraction; while in the homogenates group, the electric stimulus could get through the nerve defect at 4th week. The nerve conduction velocity of the homogenates group recovered better than that of the e-PTFE group at 8th, 12th week. Optical microscope observation showed that at 4th week, in the e-PTFE group, the proximal and distal nerve stumps denervated widely;while in the homogenates group, all the proximal nerve stumps regenerated through the chamber and reached the distal end. At 8th week, in the e-PTFE group, the regenerating nerve fiber got through the nerve defect, the nerve fibers were sparse and arranged in disorder throughout the tube.In the homogenates group, the tube was filled with regenerating nerve fibers which arranged in order. At 12th week, in the e-PTFE group, the nerve fibers lined up in orders, but there existed the phenomena that the nerve fibers were confused. In the homogenates group, regenerating nerve fibers were mature. CONCLUSIONS: From this experiment, we found that the effect of e-PTFE containing nervous homogenates to repair the facial never was better than that of e-PTFE group.