Relationship between proteoglycans and proliferation of human salivary adencid cystic carcinoma / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of down-regulated proteoglycans on the proliferation of human salivary adenoid cystic carcinoma (SACC).</p><p><b>METHODS</b>The short hairpin RNA (shRNA) plasmid silencing human xylosyltransferase-I (XT-I) gene was constructed and named shRNA-WJ3. Adenoid cystic carcinoma cells with high metastatic tendency (ACC-M) were transfected by shRNA-WJ3. The plasmid shRNA-HK not targeting any human gene was transfected into ACC-M cells used as negative control. After 48 h of transfection, the positive cells were screened by G418 to isolate the stable transfected cells. Real-time PCR and Western blotting were used to test the gene silence, and the proteoglycans contents of the cells were detected. The stable cell line silenced XT-I was named ACC-M-WJ3. MTT assay was performed to detect the cell proliferation. The cell cycle was analyzed by flow cytometry.</p><p><b>RESULTS</b>ShRNA-WJ3 showed powerful RNA interference and gene silence of XT-I. The inhibition rate was 83.70% of mRNA expression and 79.60% of protein expression respectively. The content of proteoglycans in ACC-M-WJ3 was down-regulated by 49.71%-54.59%. The results of MTT assay showed that the cell growth was inhibited significantly. S phrase decreased and G₁-G₀ phrase increased in group ACC-M-WJ3 compared with that of group ACC-M-HK (P < 0.05).</p><p><b>CONCLUSIONS</b>The down-regulated proteoglycans could inhibit the proliferation of human ACC-M cells.</p>

Effect of shRNA-mediated silence of H-ras gene on proliferation of human SACC-M cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To examine the effects of H-ras gene silence on cell cycle, proliferation and apoptosis of salivary adenoid cystic carcinoma -M (SACC-M) cell lines.</p><p><b>METHODS</b>The plasmid H-ras-shRNA, containing the sequence of shRNA targeting H-ras, and HK-shRNA (without interfering effect) were constructed and transfected into SACC-M cells. The cell line with shRNA plasmid stable expression was isolated by G418. The expression levels of H-ras were detected by RT-PCR and protein immunofluorescent assay; cell cycle and cell apoptosis were analyzed by flow cytometry (FCM). The proliferation of cell was also determined by subcutaneous tumor formation in nude mice.</p><p><b>RESULTS</b>After transfection of H-ras-shRNA plasmid, the mRNA expression of H-ras in SACC-M cells was down-regulated by 61.80% and protein expression of H-ras was inhibited by 62.76%; the cell proliferation was inhibited obviously; the G0G1 phase cells were increased. The cell apoptosis rate of H-ras-shRNA group was significantly higher than that of HK-shRNA group (P <0.05). The volume of subcutaneous tumor in nude mice was significantly smaller in Hras-shRNA group than in control group.</p><p><b>CONCLUSIONS</b>The recombinant plasmid HRAS-shRNA could efficiently down-regulate the expression of H-ras gene and protein, induce apoptosis of SACC-M cells and simultaneously inhibit proliferation of these cells in vitro and in vivo.</p>

The relationship between methylation of p16 INK 4/CDKN2 gene in promoter region and progress of squamous cell carcinoma of buccal mucosa / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To determine the correlation between methylation of p16 gene in promoter region and the carcinogenesis and progression of squamous cell carcinoma (SCC) of buccal mucosa.</p><p><b>METHODS</b>Methylation of pl6 gene in SCC and leukoplakia of buccal mucosa was investigated by MSP and pl6 protein was analyzed by Western blot.</p><p><b>RESULTS</b>The methylation of p16 gene was found in 15 of 30 cases SCC and 1 of 10 cases of leukoplakia of buccal mucosa (P < 0.05). Methylation of p16 gene in SCC of buccal mucosa was not related with age, sex, cell differentiation and clinical stage. But methylation of p16 in the cases with lymph node-metastasis was higher than that in the cases without lymph node-metastasis protein (P < 0.05). Meanwhile Methylation of p16 gene was positively correlated with no-expression of p16 protein (P < 0.01).</p><p><b>CONCLUSIONS</b>The methylation of p16 gene leaded to the inactivation of p16 gene and was related with the carcinogenesis and progress of SCC of buccal mucosa.</p>

The study of apoptosis of salivary adenoid cystic carcinoma in nude mice / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To study the feature of apoptosis of salivary adenoid cystic carcinoma (SACC) induced by recombined human tumor necrosis factor-alpha (rhTNF-alpha) in nude mice, and to evaluate the related genes expression of apoptosis.</p><p><b>METHODS</b>Twelve SPF grade 4 approximately 5 weeks old female Balb/c nude mice were selected in this study. SACC-83 cells were collected to 6 x 10(7) per milliliter and injected subcutaneously. Group A and B were experimental group which was given 100 x 10(4) IU/kg TNF-alpha or 10 x 10(4) IU/kg TNF-alpha respectively. Group C was only given normal saline and used as normal control. The investigations were adopted by using both light and transmission electron microscope (LM and TEM), flow cytometer and In Situ Cell Death Detection Kit. The evaluations of bax and bcl-2 expression were utilized by immunohistochemistry.</p><p><b>RESULTS</b>The percentage of apoptosis of transplanted tumors was much higher than that of the control (P<0.01). Apoptotic cells were calcified and grit bodies were formed. Apoptotic cells expressed and contained significantly higher proportions of both bax and bcl-2 proteins (P<0.05).</p><p><b>CONCLUSIONS</b>It is suggested that calcification may be the obvious feature and the last outcome of the apoptosis of SACC transplanted tumors. Apoptosis induced by TNF-alpha can increase the expressions of bax and bcl-2.</p>