| ObjectiveTo investigate the expression of Annexin A1(ANXA1) protein in the tumor tissue and resume of patients with esophageal squamous cell cancer(ESCC), and study the relationship between the expression levels of ANXA1 and the metastasis and prognosis. To investigate the effect of ANXA1 expression on cell proliferation and invasion, and to explore the modulation role of mi RNA196 a in ANXA1 expression.Methods1. Tissue microassay experiment: The paraffin specimens of resected tissue of patients with squamous cell carcinoma of esophageal were collected, then microassay were acquired from those paraffin specimens. The expression of ANXA1 in the paraffin specimens was detected by immunohistochemical staining. The relevance analysis of the expression of ANXA1 in tumor tissue and clinicopathological characters and prognosis of those patients were performed.2. Clinical experiment: 46 non-surgery patients with squamous cell carcinoma of esophageal who received chemoradiotherapy were recruited, their serum were collected before and after treatmen. The expression levels of ANXA1 protein and micro RNA196 a of the serum were detected. The relevance analysis of the expression of ANXA1 in serum and clinicopathological characters and prognosis of those patients were performed. The correlation of mi RNA196 a and ANXA1 expression was discussed.3. ANXA1 transfer experiment in vitro: Esophageal squamous cell cancer cell lines(Eca109) were cultured and plasmid p CMV5-myc-ANXA1 was constructed. Then, p CMV5-myc-ANXA1 was transferred into Eca109 cell. The effect of ANXA1 on cell proliferation and invasion was assessed by MTT assay and Transwell migration/invasion experiment. Furthermore, mi RNA196a-mimic and mi RNA196a-inhibitor were transferred into Eca109 cells, the ANXA1 m RNA and protein was evaluated by RT-PCR assay and Western blotting. The expression of Snail and E-cadherin were assessed by Western blotting.Results1. Immunohistochemical staining revealed that expression of ANXA1 was significantly decreased in ESCC tumor tissue. In comparison with the expression of ANXA1 in normal epithelium tissues, those in both plasm and nuclear of cancer cell were decreased. The expression of ANXA1 in ESCC cells nuclear correlated significantly with pathologic type, there was no relationship between the expression level of ANXA1 and other clinicalpathological feature.2. The patients carried lower level of ANXA1 expression in nuclear had better prognosis than those carried higher expression.3. The expression of ANXA1 in serum of patients with ESCC was significantly lower than it in serum of healthy people, but it would increase significantly after treatment.4. There was no correlation between the baseline ANXA1 level in patient’s serum before treatment and the response of chemoradiotherapy and prognosis. The patients with higher baseline ANXA1 level(>0.3ng/ml) were responsible for poorer 1-y PFS than those patients with lower ANXA1 level,but the difference was no significant. The increase amplitude of serum ANXA1 after treatment was correlated with prognosis. The patients with higher amplitude(>2 folds of baseline) presented with poorer 1-y PFS than those with slighter ANXA1 increase amplitude, and the difference was statistically significant.5. The expression of mi RNA196 a in serum of patients with ESCC decreased significantly after chemoradiotherapy, there was inverse trends between the expression of mi RNA196 a and the expression of ANXA1 in serum.6. p CMV5-myc-ANXA1 plasmid was constructed successfully and transferred into Eca109 cells, ANXA1 protein was detected by Western blotting assay. The proliferation, migration and invasion of p CMV5-myc-ANXA1 group cells were significantly higher than those of p CMV5-myc(vector) group cells.7. The expression of ANXA1(both m RNA and protein levels) were significantly decreased in Eca109 cells transferred with mi RNA196a-mimic and increased in cells transferred with mi RNA196a-inhibitor. The proliferation, migration and invasion of mi RNA196a-mimic group cells were significantly decreased, co-transferring ANXA1 into the mi RNA196a-mimic group cells would overturn this inhibition.8. The expression of Snail was stimulated and the expression of E-cadherin was suppressed in Eca109 cells transferred with ANXA1, while the conflicting results were observed in cells transferred with mi RNA196a-mimic.Conclusions1. In comparison with the expressions of ANXA1 in healthy people’s serum and normal epithelium tissues, those both in patients’ serum and cancer tissues were decreased. The expression of ANXA1 in serum of patients with ESCC increased after treatment.2. The expression of ANXA1 in ESCC cells nuclear correlated with prognosis, the lower expression of ANXA1, the better prognosis of patient. The increase amplitude of ANXA1 in serum after chemoradiotherapy was associated with prognosis, the higher increase, the poorer outcome.3. In vivo, the expression of ANXA1 was negatively regulated by mi RNA196 a. It implied that ANXA1 maybe the target gene of mi RNA196 a.4. ANXA1 could promote the proliferation of esophageal cancer cells in vitro, and the inhibition of E-cadherin caused by ANXA1 was responsible for the migration and invasion.5. In vitro study demonstrated that ANXA1 is the target gene of mi RNA196 a. mi RNA196 a could suppress the cell proliferation, and inhibit migration and invasion through Snail-E-Cadherin pathway. |
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