| Objective Salivary gland adenoid cystic carcinoma is a common malignant tumor of salivary gland. It has biological characteristics of the invasion of nerve and its gradual diffusion and metastasis, thus, the metastasis rate and recurrence rate of SACC patients after operation were high, and the SACC patients have a poor prognosis. However, at present, the mechanism of the high invasiveness and metastasis of SACC is still lack of knowledge. Dual specificity phosphatase 1 (DUSP1). a bi-directional specific Sue/tyrosine phosphatase, through dephosphorylation to regulate the activity of MAPK signal pathway, and play a very important role in cell growth cycle and cell proliferation. In recent years, the concept of epigenetic in cancer research has been paid more and more attention, DNA methylation is a common biological phenomenon in malignant tumors. Methylation is to regulate in transcriptional level, and the protein production can be reduced by inhibiting the production of the mRNA. At present, hypermethylation of tumor suppressor gene has been reported in SACC, but the expression of DUSP1 and methylation status in SACC has not been reported.Methods1. Immunohistochemisty was used to determine the expression of DUSP1 in 48 patients with salivary adenoid cystic carcinoma and 20 samples of normal gland tissue, and correlative clinical data were collected, and then the relationship between DUSP1 expression and clinicopathological features was analyzed.2. RT-PCR method was used to detect the RNA level of DUSP1 in 32 pairs of SACC and adjacent tissues of cancer.3. Kaplan-Meier method and Cox regression analysis were used to examine the relationship between the expression of DUSP1 and the prognosis of patients.4. Real-time methylation specific PCR (qMSP) was used to detect the methylation status of DUSP1 gene promoter region in SACC cancer cells and adjacent cells, and the salivary adenoid cystic carcinoma cell lines, SACC-83 and SACC-LM were treated with different concentrations of 5-Aza-dc, and then the expression of DUSP1 gene was detected by western blot.5. The DUSP1 overexpression cell line (SACC-83-DUSP1, SACC-LM-DUSP1) and negative control cell lines (SACC-83-vector, SACC-LM-vector) were established by using the lentiviral transfection technique.6. Colony formation assay, transwell insert cell migration assay, wound healing assay and cell proliferation assay was used to determine the effect of the up-regulation of DUSP1 on the proliferation, invasion and migration of salivary adenoid cystic carcinoma cells.7. Human whole genome microarray sequencing was used to detect the different downstream pathways of the two cell lines of SACC-83-DUSP1 and SACC-83-vector cell lines.8. The effect of DUSP1 expression on the tumorigenesis and metastasis of salivary adenoid cystic carcinoma was detected by nude mouse tumorigenesis test.Results1. Immunohistochemistry showed that the DUSP1 expression was positive in 20 tumor adjacent tissue, the positive rate was 100%; and in 48 salivary gland ACC samples,18 cases (37.5%) were DUSP1 positive, while 30 (62.5%) were DUSP1 negative, the difference was statistically significant (P<0.05). There were significant differences in expression of DUSP1 among patients with different T stages, lymph node metastasis, nerve invasion and local invasion (P<0.05), but no significant differences among patients with different gender, age, tumor location, histological types and distant metastasis (P>0.05)2. RT-PCR result revealed that the expression of DUSP1 was significantly lower in SACC tissues compared to pericancerous tissues, the difference was statistically significant(P<0.05)3. Univariate survival curves (Kaplan-Meier) showed that:30 patients with DUSP1 negative expression, tumor recurrence occurred in 21 cases (70%); 18 patients with DUSPl positive expression, tumor recurrence occurred in 4 cases (22.2%), the difference was statistically significant (P=0.028), but multivariate survival analysis (Cox regression analysis) showed that DUSPI expression was not the independent indicator of the prognosis of the patients.4. Real-time methylation specific PCR (qMSP) showed that:the methylation of DUSP1 gene promoter in SACC cancer cells was significantly higher than that in the adjacent cells, and the western blot was used to detect the expression level of DUSP1 protein after the treatment of 5-Aza-dc, and found that the expression level of DUSP1 protein was significantly increased.5. The lentiviral transfection technique was used in SACC-83 and SACC-LM cells lines, and the expression levels of DUSP1 protein in the two cell lines were obviously increased, the DUSP1 overexpression cell line (SACC-83-DUSP1, SACC-LM-DUSP1) and negative control cell lines (SACC-83-vector. SACC-LM-vector) were successfully established.6. Colony formation assay, transwell insert cell migration assay, wound healing assay and cell proliferation assay results showed that:the clone forming ability, invasion, migration and proliferation ability of the DUSP1 overexpression cell lines (SACC-83-DUSP1, SACC-LM-DUSP1) were weaker than the negative control cell lines (SACC-83-vector, SACC-LM-vector).7. Human whole genome microarray sequencing results showed that:in the GO function enrichment analysis, the differentially expressed genes in SACC-83 cells after DUSP1 were mainly enriched in the important processes, such as olfactory transduction, metabolic pathways, and neuroactive ligand-receptor interaction. KEGG pathway enrichment analysis results indicated that the over expression of DUSP1 affect the activation of cytoskeleton, positive regulation of gene expression, integrin-mediated signaling pathway, and so on.8. The nude mouse tumorigenesis test results showed that:in the 10 nude mice of SACC-83-DUSP1 group, tumors were formed in 3 nude mice, but they did not occur metastasis; in the 10 nude mice of SACC-83-Vector group, tumors were formed in 10 nude mice, and they all occured metastasis.ConclusionThe expression of DUSP1 in SACC was significantly lower than that in the adjacent tissues, and the low expression of DUSP1 was correlated with T stage, lymph node metastasis, neural invasion, local invasion and tumor recurrence; DUSP1 gene promoter was found to have a high level of methylation in SACC, and the expression level of DUSP1 was negatively correlated with the methylation of DUSP1 promoter; DUSP1 overexpression can weaken the clone forming ability, invasion, migration and proliferation ability of SACC cells, and the tumor formation and metastasis ability in nude mice. Furthermore, it is closely related to many signal transduction pathways. These results suggest that DUSP1 plays as a tumor suppressor in SACC, the up-regulation of DUSP1 could obviously inhibit the occurrence and development of SACC. It is expected to be an effective biomarker compounds in the prognostic evaluation, clinical diagnosis and treatment of SACC. |
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