TUC338 Overexpression Leads to Enhanced Proliferation and Reduced Apoptosis in Tongue Squamous Cell Carcinoma Cells In Vitro.

PURPOSE: Long noncoding RNAs are closely related to the development of tumors. In this study, we explored the contribution of the long noncoding RNA TUC338 to cellular processes in tongue squamous cell carcinoma (TSCC). MATERIALS AND METHODS: First, we detected TUC338 expression using quantitative reverse transcription-polymerase chain reaction in 25 patients. Then, we transfected a short hairpin RNA to silence TUC338 expression in the CAL-27 and SCC-9 cell lines. Tumor cell growth was determined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, and apoptosis and cell-cycle analyses were performed via flow cytometry. RESULTS: The results indicated that TUC338 was overexpressed in TSCCs (P < .05). In addition, silencing TUC338 in CAL-27 and SCC-9 cells inhibited cell growth and increased apoptosis significantly in vivo (P < .05). CONCLUSIONS: Long noncoding RNA TUC338 overexpression leads to enhanced proliferation and reduced apoptosis in TSCC.

A study on the inhibition of VEGF expression in salivary gland adenoid cystic carcinoma cells via iNOS gene RNAi in vitro.

In this study, we aimed to explore the effect of inducible nitric oxide synthase (iNOS) on vascular endothelial growth factor (VEGF) expression in salivary gland adenoid cystic carcinoma (SACC). Using RNAi, we transfected chemically synthesised iNOS siRNA into ACC-M cells (a highly metastatic adenoid cystic carcinoma cell line) and detected the change in the gene and protein expression levels of iNOS and VEGF by qRT-PCR and Western blotting. A transwell invasiveness assay was used to examine the changes in invasive ability of ACC-M cells. Cell growth was determined using a CCK-8 assay. Apoptosis and cell-cycle phases were detected by flow cytometry. We found that silencing iNOS down-regulated the expression of VEGF and then inhibited cell growth and invasiveness of SACC cells, while it increased apoptosis. Therefore, we concluded that iNOS can regulate VEGF expression and iNOS may be a therapeutic target.

Potential functions of long non­coding RNAs in the osteogenic differentiation of human bone marrow mesenchymal stem cells.

Long non­coding RNAs (lncRNAs) are a specific group of RNA molecules that do not encode proteins. They have been shown to serve important regulatory functions in various biological and cell differentiation processes. However, the potential functions and regulatory mechanisms of lncRNAs that are associated with the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) remain to be elucidated. The present study aimed to investigate lncRNAs that are differentially expressed during the osteogenic differentiation of hBMSCs, along with the potential functions of those lncRNAs. To this end, three groups of hBMSCs were stimulated to undergo osteogenic differentiation for 7 days. Known lncRNAs, unknown lncRNAs and mRNAs that demonstrated differential expression prior to and following the osteogenic differentiation of hBMSCs were screened using lncRNA high­throughput sequencing. In addition, 12 lncRNAs were selected for reverse transcription­quantitative polymerase chain reaction (RT­qPCR) validation of the accuracy of the sequencing results. The potential functions and possible targets of the differentially expressed lncRNAs were analyzed using bioinformatics technologies (gene ontology, Kyoto Encyclopedia of Genes and Genomes and gene co­expression network analysis). In total, 64 lncRNAs were differentially expressed by at least two­fold in hBMSCs prior to and following osteogenic differentiation; these included seven known lncRNAs (two upregulated and five downregulated lncRNAs) and 57 unknown lncRNAs (35 upregulated and 22 downregulated lncRNAs). In addition, 409 mRNAs (257 upregulated and 152 downregulated mRNAs) were differentially expressed by at least two­fold. The RT­qPCR results obtained for 12 selected differentially expressed lncRNAs were consistent with the sequencing results. The gene co­expression network analysis of lncRNAs and mRNAs demonstrated that four lncRNAs (ENSG00000238042, lnc_1269, lnc_1369 and lnc_1708) may serve important roles in the osteogenic differentiation of hBMSCs. In conclusion, during the osteogenic differentiation of hBMSCs, the lncRNA expression profile changed significantly; certain of the observed differentially expressed lncRNAs may be derived from protein­coding genes and may serve important roles in osteogenic differentiation.

[Effect of macrophage inflammatory protein-1ß on proliferation and apoptosis of human tongue squamous cell carcinoma CAL-27 cells in vitro].

OBJECTIVE: To detect CCR5 protein expression in different human tongue squamous cell carcinoma cells and observe the effect of macrophage inflammatory protein-1ß (MIP-1ß) on the proliferation and apoptosis of CAL-27 cells. METHODS: Western blotting and immunofluorescence staining were used to detect the expression of the CCR5, the receptor of MIP-1ß, in 3 human tongue squamous cell carcinoma cells UM-1, CAL-27, and Tca-8113. CCK-8 assay was used to assess the proliferation of CAL-27 cells stimulated with 10, 20, and 40 ng/mL MIP-1ß for 12, 24, or 48 h. The apoptosis of the cells stimulated with MIP-1ß (10, 20, and 40 ng/mL) for 24 h was analyzed using flow cytometry with Annexin V/PI double staining. RESULTS: CCR5 expression was detected both on the membrane and in the cytoplasm in all the 3 tongue squamous cell carcinoma cell lines. At the concentrations of 10, 20, and 40 ng/mL, MIP-1ß stimulation for 12 and 24 h significantly promoted the proliferation of CAL-27 cells (P<0.05); MIP-1ß stimulation for 48 h at the concentrations 10 and 20 ng/mL, but not at 40 ng/mL, promoted the proliferation of CAL-27 cells (P<0.05). MIP-1ß stimulation at 40 ng/mL for 24 produced the most obvious apoptosis-inducing effect in CAL -27 cells (P<0.05), while MIP-1ß at 10 or 20 ng/mL did not induce obvious apoptosis in the cells (P>0.05). CONCLUSION: CCR5 is expressed in all the 3 human tongue squamous cell carcinoma cells. MIP-1ß can promote the proliferation of CAL-27 cells but high concentrations of MIP-1ß also induced cell apoptosis. Prolonged stimulation of the cells with a high concentration of MIP-1ß shows attenuated effect in promoting cell proliferation probably as a result of cell apoptosis induced by MIP-1ß.

[Application of quantum dots fluorescent probes in tissue of human tongue squamous cell carcinoma].

OBJECTIVE: To study if quantum dots fluorescent probes can be applied to detect P53 protein and Bcl-2 protein in tissue of human tongue squamous cell carcinoma. METHODS: By indirect immunofluorescence assay the same particle size quantum dots fluorescent probes were applied to detect P53 protein and Bcl-2 protein respectively. Different particle size quantum dots fluorescent probes were applied to detect P53 protein and Bcl-2 protein simultaneously in paraffin-embedded tissue section of human tongue squamous cell carcinoma under fluorescent microscope. RESULTS: P53 protein and Bcl-2 protein can be combined with quantum dots fluorescent probes and specific fluorescene can be observed with ultraviolet light excited. P53 protein was mainly distributed in the nucleus, and Bcl-2 protein major in the cytoplasm. P53 protein and Bcl-2 protein can be combined with different particle size quantum dots fluorescent probes respectively in the same paraffin-embedded tissue section of human tongue squamous cell carcinoma and two kinds of fluorescene can be observed. CONCLUSION: Quantum dots fluorescent probes can be applied to detect two kinds of specific protein in paraffin-embedded tissue section of human tongue squamous cell carcinoma.

Double labeling and comparison of fluorescence intensity and photostability between quantum dots and FITC in oral tumors.

The aim of this study was to evaluate the application of quantum dots (QDs) and the FITC labeling technique in Tca8113 cells, and to compare the fluorescence intensity and photostability of these techniques. First, using one-colour confocal microscopy, QDs and FITC were applied separately to tag HSP70 in Tca8113 strains with indirect immunofluorescence. The expression of HSP70 in Tca8113 cells was observed using confocal laser microscopy, and the fluorescence intensity between QDs and FITC was compared. Using two-colour confocal microscopy, QDs and FITC were applied to tag survivin and HSP70, respectively, in order to compare the fluorescence photostability between QDs and FITC. Using a laser scanning microscope, survivin and HSP70 expression was clearly detected in the Tca8113 cells. The strength of the fluorescence signal from QDs was higher than that from FITC. After 40 min of continuous laser exposure, there was no observable decline in the QD fluorescent markers, whereas the FITC fluorescent signals faded very quickly and became undetectable. QDs have better fluorescence intensity and photostability than FITC, and are more appropriate for studies that require a long dynamic observation of physiological changes in cells.

[Clinical research of immediate restoration implant with mini-implants in edentulous space].

OBJECTIVE: The purpose of this study was to investigate the clinical effective of immediate restoration with Osstem MS mini-implant in the edentulous space of 5-6 mm. METHODS: The sample consisted of 36 consecutively treated partially edentulous patients who had a total of 36 Osstem MS mini-implants, which were 2.5 mm or 3.0 mm in diameter and placed in 5-6 mm gap. The chair-side-made or laboratory-made provisional crowns for implants were fabricated at the time of fixtures placed. The final restorations were fabricated with gold alloy-fused-porcelain crown 3 to 5 months later. During the mean 21.3 months (12-37 months) follow-up time since fixtures placement, all implants were examined clinically and radiologically. RESULTS: No implant failed before restoration. One implant led an adjacent tooth pulp necrosis after the implantation, but the natural tooth and implant were successfully retained by root canal therapy. 36 implants in 36 patients who were followed-up were successful and their aesthetic results were satisfactory. CONCLUSION: Immediate loaded implant with Osstem MS mini-implant has good clinical prosthetic effects in the edentulous space of 5-6 mm.

[Research and development of computer aided design and rapid prototyping technology for complete denture].

OBJECTIVE: To explore a computer aided design (CAD) and rapid prototyping (RP) approach for fabrication of complete denture and to develop relevant programs for implementing it. METHODS: Automatic crossing section scanner was used to scan artificial teeth and 3D graphic database of artificial teeth that could be aligned with parameters was established. A 3D laser scanner was used to scan upper and lower edentulous jaw casts and rims made in clinic. The vertical and horizontal relations were recorded before scanning with a patient instrument. Based on Imageware 11, tooth-arrangement curves, coordinate system, and landmark points for positioning were created, and construction cure and shape-controlling curve for base plate were constructed as well. Three-dimensional integrated design of complete denture, including artificial tooth automatic arrangement, aesthetic and individualized design of base plate, and artificial gingival, were finished. The programs were developed following the approach and the CAD platform was established. The virtual molds of complete dentures were constructed according to the above data in design and RP technology was used to make the plaster molds. Finally, the teeth were inserted and the complete denture was finished by dental technician. RESULTS: The approach for the complete denture CAD/RP was confirmed and the CAD software platform was developed. A complete denture was manufactured. CONCLUSIONS: The rules for complete denture in textbooks were expressed in design process with the CAD program developed by researcher. The 3D data of rims were utilized in design so that the digital, intelligentized and individualized design and manufacture process for complete denture was implemented.

[The inlay computer aided design based on reverse engineering technology].

OBJECTIVE: This study was to explore a CAD method for constructing occlusal and proximal surfaces of inlays using reverse engineering (RE) software and to develop a CAD software for the inlay fabrication. METHODS: The dentition model of a volunteer with individual normal occlusion was scanned with a 3D mechanical scanner. The scanned objects were prepared teeth with G. V. Black class I, class II and class VI (MOD) cavities, neighbor teeth, intercuspal position (ICP) record, and functional bite record (or functional generate path, FGP). Inlays were constructed based on RE technology using the database of Chinese normal teeth. RESULTS: The technique guideline of inlay CAD was developed based on RE softwares. Inlays designed with CAD technique showed smooth and continuous contours. The occlusal and proximal contacts of inlay met physiologic demands. CONCLUSIONS: It is a feasible method to develop a CAD software for inlay fabrication based on RE software.