| Salivary gland carcinoma is the major tumor in oral malignant tumors, because oral cancer pathogenesis is relatively complex, the development mechanism is unclear, early diagnosis and prevention of clinical oral malignant tumor is difficult, and recurrence rate of oral malignant tumors is high and the prognosis is poor, so the correct study of the occurrence of oral malignant tumor and the development mechanism are the vital significance for the prevention and early onset after treatment.Pituitary tumor transforming gene (PTTG) has been identified as human securin, PTTG participates in mitotic spindle checkpoint pathway and inhibits sister chromatid separation to ensure chromosomal stability. Although the PTTG overexpression correlates with metastasis and poor overall survival in these cancers, it is not clear whether the oncogenic molecule contributes to tumor genesis of Salivary gland carcinoma neoplasms (SGMNs). It is popular to study the basic fibroblast growth factor(bFGF) for inducing tumor, and promote the rapid tumor growth, many studies have shown that bFGF over expression on cancer, there are close relationship between the expression levels and cance rapid growth、transfer and prognosis.As results,we evaluated the PTTG overexpression in the human mucousepidermoid carcinoma of submandibular gland by immunohistochemical staining、reverse transcription quantitative polymerase chain reaction(RT-qPCR) and western blot analysis to investigate the oncogenic role of PTTG in SGMNs;then we constructed a PTTG-overexpressing and EGFP marker co-expressed A-253 cell line, which is epidermoid carcinoma cell line from human submaxillary salivary gland, we investigated the influence of PTTG on the proliferation and migration of A-253 cells,it is implied that PTTG plays an important role in SGMN cell migration and can be a potential target for anticancer therapy.we evaluated the bFGF overexpression in the human malignant neoplasms of submandibular glands by immunohistochemical analysis and RT-qPCR, to study the influence to submaxillary salivary gland and the relationship between bFGF with survival time、postoperation and prognotic, to summary basis and value it provides for individualized clinical treatment.Part ⅠOverexpression of PTTG in mucousepidermoid carcinoma of submandibular glandObjective:In present study, we evaluated the PTTG overexpression in the human mucousepidermoid carcinoma of submandibular gland by immunohistochemical staining、reverse transcription quantitative polymerase chain reaction(RT-qPCR)-. western blot analysis and cell count assay and cell migration assay to investigate the oncogenic role of PTTG in SGMNs; then we constructed a PTTG-overexpressing and EGFP marker co-expressed A-253 cell line, which is epidermoid carcinoma cell line from human submaxillary salivary gland, then we investigated the influence of PTTG on the proliferation and migration of A-253 cells. It is implied that PTTG plays an important role in SGMNs cell migration and can be a potential target for anticancer therapy.Methods:19 human mucousepidermoid carcinoma of submandibular gland and 18 control submaxillary salivary gland specimens (as control) were resected by a surgery section before subject to radiotherapy or chemotherapy from SGMN patients or from pleomorphic adenoma patients, with informed consent and agreement. Present study has been approved by the medical ethics committee. Clinico-pathological data of the patients were recorded prospectively, including the age at diagnosis%tumor size、 axillary lymph node metastasis and histological grade. Epidermoid carcinoma cell line from human submaxillary salivary gland, A-253 cell line To generate an A-253 cell line overexpressing PTTG.1.Immunohistochemical stainingrwe had study the expression of PTTG on mucousepidermoid carcinoma and the normal submaxillary salivary gland with immunohistochemical analysis; 2.Total cellular mRNA from tumor specimens or from A-253 cells was isolated using the RT-qPCR was performed to quantify PTTG expression in mRNA level; 3.PTTG in protein level in tumor specimens was analyzed by western blot assay:tumor specimens were homogenized before protein isolation,the homogenazed specimens or cultured cells were collected and lyzed with a lysis reagent, according to the manual, and were supplemented with a protease inhibitor cocktail,protein samples were separated by 12%SDS-PAGE gel, and were transferred to a nitrocellulose membrane,the PTTG and p-actin were quantified, via successively using anti-PTTG or anti-p-actin polyclone rabbit antibody, a peroxidase-conjugated secondary antibody and the electrochemoluminescence (ECL) detection system following the manufacturer’s instructions; 4.Cell count assay and cell migration assay:briefly,1x103 or 1x104 A-253 PTTG (+) or A-253 PTTG (-) cells seeded in six-well plates and were incubated at 37℃ for various periods of time and then trypsinized, the number of viable cells was counted in a hemocytometer with the use of trypan blue staining, the cell migration was determined by scratch assay, A-253 PTTG (+) or A-253 PTTG (-) cells were cultivated to 90% confluence on the six-well plates, then, cell scrapers was utilized to scratch the confluent cells.48 h later, migrating cells across the baseline were observed and counted, all the experiments were repeated in triplicate. All data were expressed as mean±standard error of the mean (SEM). The difference in PTTG-positivity between two groups was evaluated by chi-square test. The expression of PTTG in mRNA or protein level, or the cell number two groups was analyzed by Student’s t test. A p value of 0.05 or less was considered statistically significant.Results:1.Overexpression of PTTG in SGMN specimens:①there was a higher rate of PTTG-positive cells in SGMN tissues than in control submaxillary salivary gland tissues (14/19 vs 7/18, p=0.0327);②to further reveal the difference in PTTG level between the tumor and non-tumor groups, we analyze the PTTG expression in mRNA level in all specimens in the two groups.Compared to the average level of 1.032± 0.096 (n=18) in control group, the PTTG mRNA in SGMN specimens was 1.772± 0.187 (n=19), and there was a significant difference (p=0.0014);(3)in addition, we re-evaluated the PTTG expression in protein level by western blot amalysis,the protein level of PTTG in SGMN specimens was also significantly higher than in the control specimens (p<0.05).2.construction of A-253 stable cell line overexpressing PTTG:①to identify the oncogenic role of PTTG in SGMN cells, we generate a PTTG and EGFP co-expressing A-253 cell line, a stabilized PTTG overexpression in mRNA level in A-253 PTTG (+) cells were determined by the RT-qPCR method with the PTTG specific primers,a significantly higher level of PTTG mRNA was observed in the A-253 PTTG (+) cells with various passages (p<0.01 respectively, compared to the control cells, A-253 cells);②and the PTTG overexpression in protein level was stabilized in the A-253 PTTG (+) cells higher than A-253 PTTG (-) cells.3.PTTG overexpression promotes the growth and migration of A-253 cells:①the growth of A-253 PTTG (+) or A-253 PTTG (-) cells in vitro was assessed by cell count assay, which was performed for cell counting for 12,24, or 48 h post cell inoculation, A-253 PTTG (+) cells grew more efficiently than A-253 PTTG (-) cells at 48 h post inoculation, with a significant difference (4.51±0.38 x 104/mL for A-253 PTTG (+) cells and 3.733±0.32 x 104/mL for A-253 PTTG (-) cells at 48 h post inoculation, p < 0.05);②the growth difference was reconfirmed with an inoculated with 103/mL, the growth of A-253 PTTG (+) cells was also significantly more efficient than A-253 PTTG (-) cells;③cell migration contributed to the tumor metastasis of scratch, significantly more A-253 PTTG (+) cells migrated across the baseline than A-253 PTTG (-) cells (92.67±7.51 vs 41±4.62, p<0.01).Conclution:1.PTTG are over expression in mucousepidermoid carcinoma of submandibular gland specimens.2.PTTG improve proliferation and migration of mucousepidermoid carcinoma of submandibular gland.3.PTTG can be a notable marker for SGMN diagnosis and can be a potential target for anticancer therapy.Part Ⅱ bFGF upregulate metastasis in malignant neoplasms of the human submandibular glandObjective:we evaluated the basic fibroblast growth factor (bFGF) overexpression in the malignant neoplasms of the human submandibular gland,to study the oncogenic role of the bFGF influenced to malignant neoplasms of submandibular gland and the relationship between bFGF with survival time、postoperation and prognotic, in order to summary basis and value of it which provides for individualized clinical treatment.Methods:32 cases of complete surgical resection after pathologic diagnosis of Submaxillary SGMN specimens were collected, with informed consent and agreement by all the patients. Present study has been approved by the medical ethics committee. There are 19 cases of men and 13 cases of women, mean age was (45.3±4.8) years old,period I were 8 cases, Period Ⅱ were 12cases, Period Ⅲ were 10 cases, 2 cases were IV period,the spatients1 average survival period was (39,2±4.5) months.1.Determination of bFGF expression with immunohistochemical method, integrating the histologic grade were used for calculation, the integral was 0 points in (-),1-3 points were represented as weak positive, expressed by (+),3-8 were divided into moderate positive (++),9-12 were divided into strong positive, expressed with (+ ++), on the basis of the experimental section, HE stain and negative for photos werre judged the result, the final review, above (++) is defined as the positive expression; 2.total cellular bFGF mRNA from tumor specimens or from Submaxillary SGMN specimenswas isolated using RT-qPCR was performed to quantify PTTG expression in mRNA level, PTTG expression was normalized to P-actin and was expressed as the fold change over control and calculated.3.Clinical stage,pathology type,age,gender,prognosis were compared.Results:1.The expression of bFGF:① bFGF dyeing position was mainly in the cytoplasm and cytomembrane composed yellow grain were been observed, and dyeing degree were higher than the background,but negative control has no expressioin, the integral wasO points in (-) was 8 cases(25%),1-3 points were represented as weak positive, there was 6 cases (18.75%)expressed by (+),3-8 were divided into moderate positive (++), there were 16 cases(50%),9-12 were divided into strong positive, there were 2 cases(6.25%)expressed with (+++); ② bFGF expression on human mucoepidermoid carcilnoma specimens in submaxillary salivary gland were detected by RT-PCR,bFGF of carcilnoma specimens (0.82±0.21)were higher than normal tissue(0.11±0.08),there is significant difference between two groups(p=0.010).2.The relationship between the expression of bFGF and clinical characteristic:①There were no relationship between the expression of bFGF and sex and age (p>0.05); ②8 cases were adenoid cystic carcinoma,the positive expression rate was 61.53%, mucoepidermoid carcinoma were7 cases,the positive expression rate was 53.84%,3cases were other (50.00%),there were no difference between three types (P>0.05);③The expression of bFGF on different clinical stage, Ⅰ period were 8 cases,the positive expression rate was 37.50%, Ⅱ period were 12 cases, the positive expression rate was 50.00%,Ⅲ period were 10 cases, the positive expression rate was 60.00%, Ⅳ period were 2 cases, the positive expression rate was 100.00%, there were significance difference among difference clinical stage (P< 0.05).3The relationship of bFGF expression with survival time:①survival of patients were 9-86 months, average was (39.2±4.5) months, the positive expression was 18 cases,average survival of patients were (27.8±2.6) months,the negtive expression was 14 cases,average survival of patients were (54.3±4.7) months,there were signification difference between negative group and positive group(p=0.0I0);②positive expression group,the survival time of male were longer than female (P=0.05),there were no signification difference between gender in negative expression (P>0.05);③32 cases were divided into two groups,one group was older than 60 years, the other was younger 60 years, the survival time of bFGF negative expression were longer than positive; ④in different pathological type, the survival time of bFGF negative expression were longer than positive (P<0.05) ⑤in bFGF positive expression group,in early clinical stage of TNM,The survival time was longer (P<0.05)Conclution1. bFGF are over expression in malignant neoplasms of the human submandibular gland specimens, there are no signification between gender、age and pathology, while there are signification between clinical stage and survival time.2.1n malignant neoplasms of submandibular gland,the average survival time of those with bFGF negative expression were longer than positive expression, bFGF was one of independent factor to identify malignant neoplasms of the human submandibular gland specimens;survival time were longer for male and Ⅰ-Ⅱ stage of TNM clinical stage in bFGF positive expression. |
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