Studies On The Expression And Mechanisms Of Annexin A11 In Human Gastric Cancer

With the improvement of diagnosis and treatment of precancerous lesions and gastric cancer,its morbidity and mortality have declined.However,gastric cancer is still one of the most frequent malignant tumors in the world,especially in Southeast Asian countries.So far,the occurrence and development of gastric cancer are believed to correlate with the accumulation of oncogenes activated and tumor suppressor genes silenced.Based on this theory,finding and validating new oncogenes can not only clarify the possible pathogenesis of gastric cancer,but also help to discover new tumor markers for early diagnosis.Annexins(ANXs)are Ca2+-regulated phospholipid-binding proteins widely expressed in all eukaryotic cells except yeasts.They exhibit diverse biological functions by mediating the interactions between proteins in cell membranes with other proteins in cells,in the nuclear membrane,and in the extracellular matrix.The annexins have been classified into five groups.Group A includes 12 members(annexins A1-A11 and A13)and be found in vertebrates(mammalian);Group B,C,D and E annexins are found in non-vertebrates,fungi/molds,plants and protists,respectively.Recent studies had revealed the relationship between ANXA11 and cancers.ANXA11 play an important role in biological function related cancer such as cell proliferation,migration and invasion.The abnormal expression of ANXA11 has been reported in ovarian cancer,head and neck squamous cell carcinoma,breast cancer,colorectal cancer,chronic myelocytic leukemia,gastric cancer and liver cancer.However,papers of deep investigation of ANXA11 in the occurrence and development of cancer is rather small,and its specific mechanism in gastric cancer is not clear.In this study,firstly we determined the ANXA11 expression of paired gastric cancer tissue and adjacent mucosa by quantitative real-time PCR(q PCR),Western Blot,immunehistochemistry(IHC),and analyzed the relationship between ANXA11 expression and clinicopathological parameters of patients with gastric cancer;Then we investigated the biological effects of ANXA11 knockdown on gastric cancer cell lines(cell proliferation,cell cycle,migration and invasion),In addition,we testify the effect of ANXA11 genes on the proliferation of subcutaneous xenografts in vivo.Part one The expression of ANXA11 in gastric cancer tissues and cell lines and the relationship between ANXA11 expression and clinicopathological parameters of patients with gastric cancerObjective: To investigate the expression of ANXA11 in gastric cancer tissues and cell lines and the relationship between ANXA11 expression and clinicopathological parameters of patients with gastric cancer.Methods: 1 Expression levels of ANXA11 in 63 paired GC samples and normal gastric tissue was determined by q PCR,Western blot and IHC.2 Relationship between ANXA11 expression and clinicopathological parameters of patients with gastric cancer was analyzed.3 Expression levels of ANXA11 in a panel of gastric cancer cell lines(n=4)and human gastric mucosa epithelial cell(GES-l)were examined by q PCR and Western Blot analysis.Results: ANXA11 expression was significantly up-regulated in gastric cancer tissues,and its expression was high in AGS and SGC-7901 cells.Increased expression of ANXA11 was significantly associated with tumor size,tumor infiltration,local lymph node metastasis,TNM staging,and vascular invasion.1.Results of q PCR,Western Blot and immunohistochemistry showed that ANXA11 expression was significantly up-regulated in cancer tissue compared to normal gastric tissue.2.ANXA11 expression was related to tumor size,tumor infiltration,local lymph node metastasis,TNM staging,and vascular invasion,but was not correlated with gender,age,differentiation,Borrmann classification,distant metastasis and location.3.Compared to GES-1,the ANXA11 expression in AGS and SGC-7901 cells were significantly high,while ANXA11 expression was no significant difference between GES-1 and other two common gastric cancer cell lines MGC-803 and BGC-823.Summary: 1.ANXA11 expression was significantly up-regulated in cancer tissue compared to normal gastric tissue.2.ANXA11 expression was related to tumor size,tumor infiltration,local lymph node metastasis,TNM staging,and vascular invasion,and may be one of the tumor markers of gastric cancer.3.ANXA11 m RNA and protein were highly expressed in AGS and sgc-7901 gastric cancer cell lines,which were suitable for in vitro construction to inhibit ANXA11 cell model.Part two The biological effects of ANXA11 knockdown in AGS and SGC-7901 cell linesObjective: The inhibitory effect of ANXA11 on the biological behavior of AGS and SGC-7901 gastric cancer cell lines.Methods: 1 Gastric cancer cell lines with most effective ANXA11 knockdown were established.2 Cell proliferation by ANXA11 knockdown was confirmed by plate colony formation and CCK-8 assay.3 Cell cycle by ANXA11 knockdown was assessed by flow cytometry.4 Cell migration and invasion by ANXA11 knockdown were investigated by Wound-healing experiments and Transwell invasion assay and the expression of migration and invasion related proteins were determined by Western Blot;5 Effect of down-regulation of ANXA11 on the AKT/GSK-3?/ Cyclin D1 signal pathway was detected by Western Blot.Results: ANXA11 knockdown significantly inhibited proliferation,induced cell cycle arrest and impaired migration and invasion of AGS and SGC-7901 cells lines in vitro.ANXA11 acts as an oncogene in gastric cancer and exerts its role at least partly via the AKT/GSK-3?/ Cyclin D1 pathway.1.The results of RT-PCR and q PCR showed that the expression level of ANXA11 m RNA in the experimental group was significantly lower than that in the NC group,and the effect was most obvious at the 40 n M concentration of si RNA1.Western Blot results showed that the expression level of ANXA11 protein in the experimental group was significantly lower than that in the NC group.Moreover,the effect of si RNA1 at 40 n M was the most obvious also.2.The experimental results showed that the number of clones in the transfection group was significantly lower than that of the NC group.CCK-8 results showed that there was no significant difference in the absorbance value(OD value)between the NC group and the transfection group at 0h and 24 h,while the OD value of the transfection group was significantly lower than that of the NC group at 48 h,72h and 96 h.3.The results of flow cytometry showed that after inhibiting ANXA11 expression,the G0/G1 phase cells in the transfection group were significantly increased compared with the NC group.4.The results of wound healing assay were shown compared with the NC group,the planar migration activity of transfected cells was significantly inhibited.5.The results of Transwell assay were shown after inhibition of ANXA11,transfection group cells were significantly less than the NC group cells that passed through the cell matrix adhesive.6.The expression level of MMP-2 and MMP-9 in transfection group was significantly lower than that in NC group.7.Compared with the NC group,the expression levels of AKT1,p-AKT1/2/3,GSK-3?,p-GSK-3?,PCNA and Cyclin D1 proteins were significantly reduced.Summary: 1.AGS and SGC-7901 gastric cancer cell lines with knockdown of ANXA11 were successfully established;2.Knockdown of ANXA11 significantly inhibited the proliferation and colony formation capacity of AGS and SGC-7901 cell lines compared with the NC group and led to significantly cell accumulation in the G0/G1 phase.3.Knockdown of ANXA11 significantly suppressed AGS and SGC-7901 cell migration and invasion and may be related to the down-regulation of MMP-2 and MMP-9 levels.4.Western blot results showed that the protein expression levels of AKT1,p-Akt1/2/3,GSK-3?,p-GSK-3?,Cyclin D1 and PCNA were significantly decreased at 48 h after transfect with ANXA11-si RNA in AGS and SGC-7901 cell lines in comparison with the NC groups.Part three Effect of knockdown ANXA11 on subcutaneous xenografts in nude miceObjective: To study the effect of ANXA11 on the proliferation of subcutaneous transplanted tumor in nude mice.Methods: 1 Gastric cancer cell lines with stable ANXA11 knockdown were established.Nude mice were inoculated subcutaneously in the right flank with AGS cells,Tumor size was measured by a slide caliper and tumor volume was calculated as(length×width2)/2.2 Western Blot and immunohistochemistry were used to detect the expression of ANXA11 and Ki-67 in the implanted tumor tissues of nude mice.Results: Knockdown of ANXA11 significantly inhibits the proliferation of subcutaneous transplanted tumor in nude mice.1.Under fluorescence microscope,the positive rates of AGS and SGC-7901 cells were over 80% when MOI was 50 and 100,respectively.RT-PCT and q PCR proved that the expression level of AXNA11-m RNA in the transfected group was significantly lower than that in the NC group.2.The growth curve showed that the growth of the subcutaneous transplanted tumor in the transfection group was significantly slower than that of the NC group,and the difference was significant at 3 weeks.The tumor weight of the NC group was 1.52 ± 0.23 g,the transfection group was 0.66 ±0.19 g,and the weight of the transplanted tumor in the NC group was higher than that in the transfection group.3.Western Blot and immunohistochemical results showed that the expression of ANXA11 and Ki-67 in the subcutaneous transplanted tumor cells of the transfection group was significantly reduced.Summary: 1.AGS and SGC-7901 cell lines with stable ANXA11 knockdown were established.2.Subcutaneous nude mouse xenograft model has been successfully constructed.3.Knockdown of ANXA11 could significantly inhibit the growth of subcutaneous transplanted tumor and reduce the weight of the tumor.4.Decline of Ki-67 expression in ANXA11 knockdown xenografts tissue indicates the proliferation activity of gastric cancer cells is inhibited.Conclusions: 1.ANXA11 expression was significantly up-regulated in gastric cancer tissues,and its expression was high in AGS and SGC-7901 cells.Increased expression of ANXA11 was significantly associated with tumor size,tumor infiltration,local lymph node metastasis,TNM staging,and vascular invasion.2.Knockdown of ANXA11 significantly inhibited the proliferation and colony formation capacity of AGS and SGC-7901 cell lines and led to significantly cell accumulation in the G0/G1 phase.3.Knockdown of ANXA11 significantly suppressed AGS and SGC-7901 cell migration and invasion and may be related to the down-regulation of MMP-2 and MMP-9 levels.4.ANXA11 regulates cell proliferation possibly through the AKT/ GSK-3? /Cyclin D1 signaling pathway.5.Knockdown of ANXA11 could significantly inhibit the proliferation of subcutaneous transplanted tumor in nude mice.