Expression Of BIRC5 In Gastric Cancer And Its Role And Mechanism In Gastric Cancer Proliferation,Invasion And Sensitivity To Chemotherapy

Objective:Gastric cancer(GC)is the leading cause of cancer-related death worldwide,with limited treatment options and poor patient survival.With the improvement of molecular understanding of gastric cancer,we gradually realize that gastric cancer is a heterogeneous group of diseases with unique pathogenic mechanisms and active oncogenic pathways.It is this molecular diversity and the common activation of certain oncogenic driver genes that contribute to poor treatment outcomes for gastric cancer.Therefore,it is of special importance and urgency to deeply study the molecular mechanism of the occurrence and development of gastric cancer,establish genetic markers that can be used for early diagnosis and monitoring of high-risk individuals,and provide new targets for the clinical treatment of gastric cancer.Inhibitor of apoptosis proteins(IAPs)are a family of proteins known primarily for their anti-apoptotic activity and ability to directly bind and inhibit cysteine-containing aspartate proteolytic enzymes(caspase).Studies have confirmed that IAPs can regulate ubiquitin(Ub)-dependent signaling pathways through their E3 ligase activity and affect the activation of nuclear factor kappa B(NF-κB).IAPs play a broad role in the control of many cellular processes,but more studies have shown that IAPs play an important role in the occurrence and development of cancer.Baculovirus inhibitor of apoptosis protein repeat 5(BIRC5)is one of the important members of IAPs,and it is also the smallest member of IAPs.The unique structural feature of BIRC5 is the presence of a baculovirus repeat(BIR)domain.BIRC5 is an evolutionarily conserved eukaryotic protein that is essential for cell division and inhibits cell death.Under normal conditions,BIRC5 is rarely expressed in healthy adult tissues,but is abnormally upregulated in most cancer tissues.Therefore,BIRC5 has received great attention as a potential cancer therapeutic target.In this study,we designed and carried out a series of in vitro and in vivo functional experiments by applying molecular biology and bioinformatics methods,aiming to explore the function and regulation of BIRC5 gene in gastric cancer related signaling pathways,and provide new strategies for gastric cancer treatment.Methods:1.Expression and clinical significance of BIRC5 in gastric cancer tissue and gastric cancer cells:The GEPIA database was used to analyze the expression of BIRC5m RNA in gastric cancer tissue.The expression of BIRC5 protein in 637 cases of gastric cancer and paired adjacent non-cancer tissue chips was detected by immunohistochemistry.The expression of BIRC5 in 10 cases of gastric cancer and paired adjacent noncancerous tissues was detected by western blot assay.The expression of BIRC5 in gastric cancer cells and human normal gastric epithelial cells was detected by western blot assay.The correlation between BIRC5 and clinicopathological features was analyzed by clinical follow-up information.The Kaplan-Meier curve and Kaplan-Meier plotters database were used to analyze the correlation between the expression level of BIRC5 and the prognosis of gastric cancer patients.2.To explore the function of BIRC5 in gastric cancer in vitro and in vivo:use CCK8 assay to detect the effect of BIRC5 on gastric cancer cell proliferation ability and chemotherapeutic drug sensitivity in vitro;use nude mouse subcutaneous tumorigenesis assay to detect the sensitivity of BIRC5 to gastric cancer proliferation and chemotherapeutic drugs in vivo sexual influence.The effect of BIRC5gene on the migration and invasion ability of gastric cancer cells was detected by Transwell assay in vitro;the effect of BIRC5 on gastric cancer metastasis was detected by the lung metastasis assay by tail vein injection of nude mice in vivo.The effect of BIRC5 on apoptosis and cell cycle of gastric cancer cells was detected by flow cytometry.The effect of BIRC5 on the expression of epithelial-mesenchymal transition(EMT)pathway-related molecules was detected by western blot assay.3.BIRC5 regulates the downstream signaling pathway of gastric cancer:Western blot was used to detect the effect of BIRC5on the expression of TGF-β/Smad signaling pathway-related molecules.The specificity of BIRC5 in activating TGF-β/Smad signaling pathway was detected by western blot rescue assay.Results:1.Public database analysis the expression of BIRC5 m RNA was increased in gastric cancer(P<0.05).2.Western blot experiments confirmed that the expression of BIRC5 protein was increased in gastric cancer cells.3.Western blot experiments were performed to verify the expression of BIRC5 protein in 10 pairs of gastric cancer and paired adjacent non-cancerous tissues.The results confirmed that the expression of BIRC5protein in gastric cancer tissues was significantly higher than that in adjacent non-cancerous tissues(P<0.001).4.Immunohistochemical experiments verified the expression of BIRC5 protein in 637 gastric cancer tissue microarray.It was found that the expression of BIRC5 protein in gastric cancer tissue was significantly higher than that in adjacent non-cancerous tissues(P<0.0001),and the high expression of BIRC5 was associated with gastric cancer N stage(P=0.001)and TNM stage(P=0.019)were significantly correlated.5.Kaplan-Meier curve(P=0.031)and Kaplan-Meier plotters database(OS:P=0.00013;PPS:P=0.0063;FP:P=0.00021)analysis confirmed that BIRC5 was an independent prognostic factor for gastric cancer.6.CCK8 experiments confirmed that knockdown of BIRC5 inhibited the cell viability of gastric cancer cells.7.Flow cytometry confirmed that knockdown of BIRC5 promoted gastric cancer cell apoptosis and promote gastric cancer cell cycle G0/G1 arrest.8.In vitro drug sensitivity experiments confirmed that knockdown of BIRC5 increased the drug sensitivity of gastric cancer cells.9.Subcutaneous tumor formation in nude mice and in vivo drug sensitivity experiments confirmed that the tumor formation in nude mice in the knockdown BIRC5 group was significantly smaller than that in the nude mice in the control group(volume:741.8±52.3mm~3vs 972.0±89.6mm~3,P=0.004;mass:0.769±0.106g vs 1.137±0.104g,P=0.009),and compared with the nude mice in the intraperitoneal injection of PBS group,the degree of tumor formation in the nude mice in the intraperitoneal injection of 5-FU group was more significant(volume:81.6±17.2mm~3vs 233.6±37.5mm~3,P<0.001;mass:0.173±0.039g vs 0.503±0.051g,P<0.001).10.Transwell assay confirmed that knockdown of BIRC5 inhibited gastric cancer cell migration and invasion.11.Western blot experiments confirmed that knockdown of BIRC5 can significantly reduce the expression of mesenchymal cell markers N-cadherin and Vimentin,and promote the expression of epithelial cell marker E-cadherin.12.The lung metastasis experiment by tail vein injection of nude mice confirmed that knockdown of BIRC5 inhibited the metastasis of gastric cancer cells(Control:16.0±1.7;BIRC5-sh RNA:4.0±1.0,P=0.009).13.Western blot experiments confirmed that knockdown of BIRC5 could significantly inhibit the phosphorylation of Smad2 and Smad3,and reduce the expression of transcription factor Snail.14.Western blot rescue experiments confirmed that overexpression of BIRC5 can significantly promote the phosphorylation of Smad2 and Smad3,and at the same time promote the expression of transcription factor Snail.After adding TGF-β/Smad signaling pathway inhibitor,the phosphorylation of Smad2 and Smad3 by BIRC5 was antagonized,and the expression of transcription factor Snail was also decreased.Conclusion:1.BIRC5 is up-regulated in gastric cancer,and its expression level is significantly correlated with N stage,TNM stage and survival rate of gastric cancer.2.Decreased BIRC5 expression inhibits the proliferation,migration and invasion of gastric cancer in vivo and in vitro,and promotes gastric cancer cell apoptosis and G0/G1 phase arrest of the cell cycle in vitro.3.Decreased expression of BIRC5 increased the sensitivity of gastric cancer to chemotherapeutic drugs in vivo and in vitro.4.BIRC5 promotes the EMT process.5.BIRC5 regulates the development of gastric cancer by activating the TGF-β/Smad signaling pathway.