Study On The Cell Proliferation Regulated By Annexin A7 Via P21-Cyclin/CDK-PCNA-E2F Signal Axis In Gastric Cancer

Gastric cancer?GC?is the second most common malignant tumor in the world and poses a serious threat to human health.According to the latest cancer data released by the National Cancer Center in February 2017,gastric cancer is the fourth highest incidence of cancer in China and the second highest in mortality.Chemotherapy,as a traditional treatment for tumors,has problems such as low efficiency,large adverse reactions,and acquired resistance.Because of its good molecular selectivity,molecular targeted therapy can effectively kill tumor cells and reduce damage to normal tissues,thus becoming one of the main treatment methods for cancer patients.Therefore,finding the gene target with high positive expression rate and exploring its mechanism of action has become an urgent need for molecular targeted therapy of gastric cancer.Annexin?ANX?is a family of Ca²⁺-dependent phospholipid-binding proteins.The abnormal expression of multiple members of this family is closely related to the malignancy,metastasis and clinical progression of tumor cells.A number of studies have shown that the abnormal expression of annexin A7?ANXA7?is closely related to the occurrence and development of gastric cancer.Many scholars have used ANXA7 as a molecular target to explore its development and treatment of tumors from various angles.effect.p21 is a cyclin-dependent kinase inhibitor.As a representative of the KIP family,it not only inhibits the binding of CDK4/6-cyclinD and CDK2-cyclin E complexes,but also blocks the G1?S phase of cells;it also can inhibit DNA proliferation by inhibiting DNA polymerase extension DNA by interacting with PCNA.A number of studies have confirmed that the loss of expression of p21 is involved in the development of gastric cancer.However,the correlation between ANXA7and p21 in gastric cancer has not been reported yet.This study intends to detect the expression of ANXA7 and p21 in gastric cancer and analyze the correlation between them.Then,changing the expression of ANXA7 in vitro to detect the effecting on gastric cancer cells and its mechanism was explored to preliminarily clarify that ANXA7 affects the development of gastric cancer through p21-cyclin/CDK-PCNA-E2F signal axis.At the same time,it provides a new theoretical basis for ANXA7 to become a potential target for targeted therapy of gastric cancer,and has important clinical significance.Objective:1 Tissue level:To explore the relationship between ANXA7 and p21,and investigate the relationship between the expression of ANXA7 and p21 in gastric cancer and the clinicopathological features of gastric cancer and their correlation.2 Cell level:To investigate the effect of low expression of ANXA7 on the proliferation of human gastric cancer cells and its mechanism by down-regulating the expression of ANXA7 in gastric cancer cells.Methods:1 Tissue level:From September 2016 to May 2018,50 specimens of gastric cancer paraffin tissue and 20 specimens of fresh gastric cancer were collected from the Affiliated Hospital of Chengde Medical College and Chengde Central Hospital.Each specimen contained gastric cancer tissue and corresponding adjacent normal tissues.Gastric mucosal tissue.The expressions of ANXA7and p21 in 50 paraffin-embedded tissues were detected by immunohistochemical SP method.The expression of ANXA7 and p21 in fresh tissues was detected by Western blot.2 Cell level:?1?Human gastric adenocarcinoma well-differentiated MKN-28 cells,human gastric adenocarcinoma differentiated SGC-7901 cells,human gastric adenocarcinoma poorly differentiated MGC-803 cells and human normal gastric mucosal GES-1 cells were cultured in vitro.Western blot was used to detect the highest expression of ANXA7 in three human gastric adenocarcinoma cells.?2?The gastric cancer cell with the highest expression of ANXA7 was divided into si-ANXA7 group,negative control group and blank control group.siRNA interference group transfected with siRNA targeting to ANXA7,negative control group transfected with scrambled siRNA and blank group without any treatment.After 48h of transfection,the inhibition efficiency was determined by qRT-PCR and Western blot at mRNA and protein levels.?3?MTT and plate colony formation assays were used to detect cell proliferation ability.?4?Flow cytometry was used to detect cell cycle distribution.?5?qRT-PCR and Western blot were used to detect the expression of p21,PCNA,cyclinD1,cyclin E and E2F1 at mRNA and protein levels.Results:1 Tissue level:?1?Immunohistochemistry results showed that the positive expression of ANXA7 in gastric cancer tissues was significantly higher than that in adjacent normal gastric mucosa tissue,the difference was statistically significant?P<0.05?.The positive expression of p21 in gastric cancer tissues was significantly lower than that in adjacent normal gastric mucosa tissues,the difference was statistically significant?P<0.05?;The differential expression of ANXA7 in gastric cancer was related to tumor differentiation,depth of invasion and lymph node metastasis,the difference was statistically significant?P<0.05?;The differential expression of p21 in gastric cancer was related to the depth of tumor invasion,the difference was statistically significant?P<0.05?.There were10 cases?24%?with positive expression of p21 in 42 cases of ANXA7 positive gastric cancer,and 32 cases?76%?with negative expression of p21;There were5 cases?63%?with positive expression of p21 in 8 cases of gastric cancer with negative expression of ANXA7,and 3 cases?37%?with negative expression of p21.Spearman correlation analysis showed a negative correlation between ANXA7 and p21 expression in gastric cancer tissues??²=4.79,P<0.05,r_s=-0.31?.?2?The results of Western Blot showed that the expression of ANXA7protein in gastric cancer tissues was significantly higher than that in normal gastric mucosa tissues?P<0.05?,the expression of p21 in gastric cancer tissues was significantly lower than that in normal gastric mucosa tissues?P<0.05?.2 Cell level:?1?The expression of ANXA7 in gastric cancer cells was significantly higher than that in normal gastric mucosa GES-1 cell,and ANXA7was the highest in gastric cancer poorly differentiated MGC-803 cells,the difference has statistically significant?P<0.05?.?2?siRNA targeting ANXA7significantly inhibited the expression of ANXA7,the difference has statistically significant?P<0.05?.?3?After inhibition of ANXA7 expression,cell proliferation was significantly inhibited in the si-ANXA7 group compared with the negative control group and the blank control group,the difference has statistically significant?P<0.05?.?4?After the expression of ANXA7 was inhibited,the G1 phase block of the si-ANXA7 group was significantly higher than that of the negative control group and the blank control group,the difference has statistically significant?P<0.05?.?5?After ANXA7 expression was inhibited,compared with the negative control group and the blank control group,the expressions of PCNA,cyclinD1,cyclin E and E2F1 in the si-ANXA7group were significantly down-regulated?P<0.05?;p21 expression Significantly up-regulated,the difference was statistically significant?P<0.05?.Conclusions:1 ANXA7 is highly expressed in gastric cancer,and its differential expression in gastric cancer is closely related to tumor differentiation,depth of invasion and lymphatic metastasis;while p21 is significantly low in gastric cancer,and its expression in gastric cancer differential expression is associated with tumor infiltration depth.2 ANXA7 and p21 were negatively correlated in gastric cancer.3 ANXA7 can promote cell proliferation by regulating the cell cycle by regulating the p21-cyclin/CDK-PCNA-E2F signal axis.