Expression Of Annexin A7 And Its Clinical Significance In The Development Of Gastric Cancer

Gastric cancer(GC) is a major threat to health worldwide. The treatment of GC includes a combination of surgery, chemotherapy and radiation therapy. Surgical resection of the primary tumor and regional lymph nodes is still the treatment of first line choice for GC. Despite the developments of surgical techniques and improvements of anticancer drugs in recent years, it remains the first common malignancies in China and the second leading cause of cancer death worldwide. Therefore, at present the urgent problems needed to be solved are to investigate the pathogenesis, pathology and new treatments.Gastric carcinogenesis is a multifactorial and multistep process that involves activating oncogenes and inactivating tumor suppressor genes in different stages of GC progression.Annexin A7, also named as synexin, belongs to the group A annexin family. The annexin A superfamily consists of 13 calcium- or calcium- and phospholipid-binding proteins with significant biological and structural homology(40-60%). Altered expression of annexins has been associated with cell transformation, tumor progression, and metastasis. Previous studies have found that Annexin A7 specifically functions as a cancer promoter candidate in majority of gastrointestinal cancers. However, effects of Annexin A7 on the development of GC were still unknown.In the present study, clinical samples of GC tissue and cultured gastric cancer cell lines were selected as the research object. The methods including pathology, immunohistochemistry, western blot, quantitative real-time PCR(q RT-PCR), MTT colorimetric method, flow cytometry and Transwell were used to investigate the relationship of the expression of Annexin A7 and iological behaviour of GC. The expression of Annexin A7 was examined by immunohistochemistry, western blot and q RT-PCR. The effects of Annexin A7 on proliferation, and invasion of GC cells were detected after silencing of endogenous Annexin A7 using si RNA technique, for clarifying the important roles of Annexin A7 in the process of development of GC. The inhibitory effect of si RNA-Annexin A7 on growth, migration and invasion of transplanted gastric cancer in nude mice was also explored in this study. All the five parts of studies were aimed to investigate the effects of Annexin A7 on the generation, migration and invasion of GC, and to provide evidence as well as target for diagnosis and treatment of GC.The detailed methods and results of this study are as follows: Part I Expression of annexin A7 and its clinical significance related to the differentiation and metastasis of gastric cancerObjectives: To investigate the expression and clinical significance of Annexin A7 in the differentiation and lymphatic metastasis of gastric cancer(GC).Methods: The clinical and pathological data were recorded for analysis.Immunohistochemical staining and Western blot were performed to analyze the expression of Annexin A7 in primary GC tissues. Logistic regression analyses were conducted to evaluate the associations between annexin A7 expression levels and differentiations of GC. Analyses of the ROC were conducted to determine the cut-off value of the ratio of pixel density of annexin A7 for predicting lymphatic metastasis of GC.Result: A total of 162 GC patients were enrolled in this study. The expression rate of annexin A7 was 65.4% in GC. The survival rate of patients with positive expression of annexin A7 was lower than that in patients with negative expression(p < 0.001). The results of COX regression showed that the positive expression of annexin A7, submucosal confinement and pathological stage of GC were associated with poor clinical outcomes. The ratio of pixel density value of primary GC tissues with PN1-3 lymphatic spread was significantly higher than those in tissues with PN0 lymphatic spread(0.56 ± 0.09 vs. 0.42 ± 0.07, p < 0.05). ROC analysis showed a high area under the curve for the ratio of pixel density value of annexin A7 in primary GC tissues. At a cut-off level of > 0.505, the ratio of pixel density value of annexin A7 exhibited 76.7% sensitivity and 88.3% specificity for detecting lymphatic metastasis of GC.Summary:1 The expression of Annexin A 7 in GC tissues is higher than that in paracancerous tissue;2 The survival rate of patients with positive expression of Annexin A7 is low;3 High annexin A7 expression is associated with poor differentiation in GC patients;4 Positive expression of Annexin A7 it may be a predictor for lymphatic metastasis of GC. Part II Expression of Annexin A7 in gastric cancer cell lines with different differentiationObjective: To explore the expression of Annexin A7 in different differentiation of gastric cancer cell lines.Methods: Human gastric adenocarcinoma cell lines with different differentiations(MKN-28, SGC-7901, and BGC-823) were cultured. Immunocytochemistry, western blot and q RT-PCR were used to observe the expression of Annexin A7 in the cell lines. The relationship between the expression of Annexin A7 and the differentiated stage of gastric cancer cell lines was analyzed.Results: The results of Immunocytochemistry staining showed that the expression of Annexin A7 in BGC-823 cell was higher than those in MKN-28 cells and SGC-7901 cells. The ratio of pixel densities of the protein was 0.287±0.030, 0.421±0.047 and 0.676±0.048, respectively each for MKN-28, SGC-7901 and BGC-823 cell line. The result of q RT-PCR showed that the expression Annexin A7 m RNA from low to high as follows: MKN-28, SGC-7901, and BGC-823 cell lines.Summary:1 In vitro cultured gastric cancer cell lines, the Annexin A7 expression is lowest in MKN28 cells and highest in BGC823 cells;2 The intensity of Annexin A7 expression is associated with the differentiated stages of GC, the higher of Anneixn A7 expression, the lower of differentiation of GC. Part III The Effect of annexin A7 si RNA on generation capability of gastric cancer BGC823 cell lineObjectives: To investigate the effect of annexin A7 si RNA on generation capability of gastric cancer BGC823 cell line.Methods: Synthesized si RNA targeting Annexin A 7 was transfected into gastric cancer BGC 823 cell by Lipofectamine 2000 as the transfecting agent. Western blot was used to observe the transfection efficiency.MTT was used to make up the time-dose curve of gastric cancer cells. Western blot and q RT-PCR were used to evaluate the generation capability of BGC 823 cells in vitro.Results: The expression of Annexin A7 was reduced much more by Annexin A7- si RNAs transfection in both protein and m RNA levels compared with the control group, suggesting Annexin A7 si RNAs efficiently inhibited the transcription and translation of endogenous Annexin A7. The most prominent of Annexin A 7-si RNA was 80 n M, on which concentration Annexin A7 expression was inhibited compared to the control group significantly. The results of flow cytometry showed that the BGC 823 cells in stage S were decreased significantly, and the cells in G0/G1 stage were increased after 48 hours of Annexin A7- si RNAs transfection compared with cells in NS-control group(p < 0.01).The results of MTT assay showed that the expression of Annexin A7 inhibited time-dependently by Annexin A7-si RNA-3 treatments. Th results of western blot analysis and q RT-PCR showed that expressions of p16, p21 and p27 were significantly up-regulated, while expressions of PCNA, Cyclin D1, Cyclin E1 and Cyclin A were significantly down-regulated in BGC823 cells transfected with 80 n M of Annexin A7-si RNA-3, compared with cells treated with control si RNA.Summary:1 Three sequences of Annexin A7-si RNA was successfully constructed, and the sequence in the 80 n M concentration for 48 h has the highest transfection efficiency and the best inhibition of Annexin A7;2 After inhibition of Annexin A7 expression, the activity of BGC823 cell decreased;3 After inhibition of Annexin A7 expression, BGC823 cell proliferation was inhibited. The cells in S phase was significantly reduced, and cells in G0/G1 phase was significant increased;4 Annexin A 7 plays an important role in the generation of gastric cancer through down-regulating the expressions of PCNA, Cyclin D1, Cyclin E1 and Cyclin A, as well as up-regulating the expressions of p16, p21 and p27. Part IV Inhibitory effects of Annexin A7 si RNA on the migration and invasion of BGC 823 cellObjective: To explore the inhibitory effects of Annexin A7 si RNA on the migration and invasion of BGC823 cell.Methods: Synthesized si RNA targeting Annexin A 7 were transfected into gastric carcinoma BGC 823 cells by Lipofectamine 2000 as the transfecting agent. Scratch test and Transwell Chambers model were used to analyze the effects of Annexin A7 on migration and invasion in BGC 823 cells. q RT-PCR and western blot were use to observe the expressions of TIMP-1, TIMP-2, ICAM-1, MMP-1, MMP-2, and MMP-9, in order to detect the migration and invasion of BGC 823 cells after Annexin A7-si RNA transfection.Results: The migration inhibition rate of BGC823 cells increased significantly by Annexin A7-si RNA-3 transfection(p < 0.05). The invasion inhibition rate of BGC823 cells was increased significantly by Annexin A7-si RNA-3 transfection(p < 0.05). However, the control si RNA had no significant effect on the cell migration and invasion compared with those treated with Lipofectamine alone. The expressions of ICAM-1, MMP-2 and MMP-9 were significantly down-regulated in BGC823 cells transfected with si RNA-3, compared with cells treated with control si RNA. No differences were found in the expressions of TIMP-1, TIMP-2, and MMP-1.Summary:1 After inhibition of Annexin A7 expression, the migration activity of BGC823 cell decreased;2 After inhibition of Annexin A7 expression, the migration rate and migration activity of BGC823 cells were inhibited;3 Annexin A 7 plays an important role in the migration and invasion of gastric cancer through down-regulating the expressions of ICAM-1, MMP-2 and MMP-9. Part V Inhibitory effect of si RNA-Annexin A7 on growth, migration and invasion of transplanted gastric cancer in nude miceObjective: To explore the inhibitory effect of si RNA-Annexin A7 on growth, migration and invasion of transplanted gastric cancer in nude mice.Methods: The si RNA sequence targeting to human Annexin A7 gene was designed, based on which a pair of complementary oligonucleotides were synthesized, annealed and cloned into plasmid p Genesil-1.1 to construct recombinant plasmid si RNA-Annexin A7. Transplanted gastric cancer model was established by injecting s.c. nude mice with human gastric cancer BGC823 cells, and si RNA-Annexin A7 was injected into the tumors formed. The nude mice were observed for clinical manifestation relying on the size and weight of transplanted tumors. The tumor tissue and angiogenesis were examined by pathological sections. Flow cytometry was used to detect the changes of cell cycle. Western blot and q RT-PCR were used to analyze the expressions of PCNA, P27, MMP-2, and TIMP-2.Results: Both the size and weight of transplanted tumors of nude mice injected with si RNA-Annexin A7 were less than those of control groups(p < 0.05). The examination of pathological sections showed that, compared with that in control group, obvious necrosis of tumor cells was observed in si RNA-Annexin A7 group. The cells at stage S were less in si RNA-Annexin A 7 group than those in the other two groups, while the cells at stage G0/G1 were much more in si RNA-Annexin A 7 group. The results of western blot and q RT-PCR confirmed that the expressions of PCNA and MMP-2 were down-regulated,whereas the expressions of p27 was up-regulated.Summary:1 Transplanted gastric cancer model was successfully established by injecting s.c. nude mice with human gastric cancer BGC823 cells;2 The volume and weight of tumor were decreased after inhibition of Annexin A7 expression in BGC823 cells;3 Tumor cells arranged sparsely after inhibition of Annexin A7 expression in BGC823 cells;4 The si RNA-Annexin A7 inhibited the Annexin A7 expression in transplanted gastric cancer of nude mice, and influenced the growth, migration and invasion of tumors by down-regulating the expressions of PCNA and MMP-2, as well as up-regulating the expression of P27.Conclusion:1 The expression of Annexin A 7 in GC tissues is higher than that in paracancerous tissue; The survival rate of patients with positive expression of Annexin A7 is low; High annexin A7 expression is associated with poor differentiation in GC patients; Positive expression of Annexin A7 it may be a predictor for lymphatic metastasis of GC.2 In vitro cultured gastric cancer cell lines, the Annexin A7 expression is the lowest in MKN28 cells and the highest in BGC823 cells; The intensity of Annexin A7 expression is associated with the differentiated stages of GC, and the higher the Anneixn A7 expression is, the lower the differentiation of GC is.3 Three sequences of Annexin A7-si RNA was successfully constructed, and the sequence in the 80 n M concentration for 48 h has the highest transfection efficiency and the best inhibition of Annexin A7; After inhibition of Annexin A7 expression, the activity of BGC823 cell decreased; After inhibition of Annexin A7 expression, BGC823 cell proliferation was inhibited. The cells in S phase was significantly reduced, and cells in G0/G1 phase was significant increased; Annexin A 7 plays an important role in the generation of gastric cancer through down-regulating the expressions of PCNA, Cyclin D1, Cyclin E1 and Cyclin A, as well as up-regulating the expressions of p16, p21 and p27.4 After inhibition of Annexin A7 expression, the migration activity of BGC823 cell decreased; After inhibition of Annexin A7 expression, the migration rate and migration activity of BGC823 cells were inhibited; Annexin A 7 plays an important role in the migration and invasion of gastric cancer through down-regulating the expressions of ICAM-1, MMP-2 and MMP-9.5 Transplanted gastric cancer model was successfully established by injecting s.c. nude mice with human gastric cancer BGC823 cells; The volume and weight of tumor decreased after inhibition of Annexin A7 expression in BGC823 cells; Tumor cells arranged sparsely after inhibition of Annexin A7 expression in BGC823 cells; The si RNA-Annexin A7 inhibited the Annexin A7 expression in transplanted gastric cancer of nude mice, and influenced the growth, migration and invasion of tumors by down-regulating the expressions of PCNA and MMP-2, as well as up-regulating the expression of P27.6 Annexin A7 is a key molecular target in diagnosis and treatment of gastric cancer.