The Molecular Mechanism Of YAP1-FSTL1 Positive Feedback Loop In Promoting The Progression Of Gastric Cancer

Background:Follistatin-like protein 1(FSTL1)is an extracellular secreted glycoprotein of the BM-40 / SPARC / Osteonectin gene family.It is located in the 3q13.33 region of human chromosome and consists of 308 amino acids,including 20 amino acid signal sequences,one is called a half Multiple studies have shown that FSTL1 is closely related to the occurrence and development of tumors.The influence of gastric cancer proliferation and metastasis ability,and in-depth study of the molecular mechanism and clinical significance.Methods:1.Predict and analyze the target genes of Hippo-YAP1 signaling pathway through gene chip;2.Analyze the expression of FSTL1 in gastric cancer and its prognosis through the TCGA database,and analyze the relationship between FSTL1 and key downstream target genes of the Hippo-YAP1 signaling pathway;3.Detect the expression of FSTL1 protein in paraffin samples and serum of gastric cancer patients by immunohistochemical methods and ELISA methods;4.Concentrate the FSTL1 protein in the gastric cancer cell culture medium by using an ultrafiltration centrifuge tube;5.Use real-time fluorescent quantitative PCR(q RT-PCR)and Western blot to detect the expression of FSTL1 m RNA and protein in fresh gastric cancer tissues,and analyze the regulatory relationship between FSTL1 and YAP1 and its key target genes;6.Use immunofluorescence test(IF)to detect the change of YAP1 in the nucleus of gastric cancer cells;7.Use CCK-8 experiment and plate cell cloning experiment to detect the effect of FSTL1 on gastric cancer proliferation;use Transwell chamber experiment to detect the effect of FSTL1 on gastric cancer cell invasion;8.Use nude mouse subcutaneous tumor model and nude mouse abdominal cavity metastasis model to verify the effect of FSTL1 on gastric cancer cell proliferation and abdominal cavity metastasis in vivo;9.Use luciferase report experiment,chromatin immunoprecipitation experiment,Western blot experiment and q RT-PCR experiment to verify the regulatory relationship between FSTL1 and YAP1.Result:1.Compared with the adjacent tissues of gastric cancer,the protein expression level of FSTL1 in the tissues of gastric cancer patients is significantly increased,and the high expression of FSTL1 in gastric cancer tissues is associated with larger tumor volume(p<0.001),lymph node metastasis(p<0.001))And TNM staging(p=0.001)are significantly related;at the same time,the high expression of FSTL1 protein in the tissues of gastric cancer patients is related to poor prognosis(p<0.001);2.Compared with healthy people,the protein expression level of FSTL1 in the serum of gastric cancer patients was significantly increased,and the FSTL1 protein in the serum of gastric cancer patients after treatment was significantly decreased;the expression of FSTL1 protein in the ascites of gastric cancer patients was significantly higher than that of serum;3.In gastric cancer cell lines,the expression of FSTL1 in BGC-823 is relatively low,and the expression of FSTL1 in MGC-803 is relatively high;4.Interfering with the expression of FSTL1 significantly inhibits the proliferation and invasion of BGC-803 cells,overexpression of FSTL1 significantly promotes the proliferation and invasion of BGC-823 cells;the collected media of BGC-823 cells overexpressing FSTL1 and the in vitro synthesis Recombinant FSTL1 protein can significantly promote the proliferation and invasion ability of untransfected BGC-823 cells,while FSTL1 neutralizing antibody shows the opposite result;5.Overexpression of FSTL1 can promote the proliferation and peritoneal metastasis of gastric cancer cells in nude mice,while silencing FSTL1 can inhibit the proliferation and peritoneal metastasis of gastric cancer cells in nude mice;6.The expression of FSTL1 and YAP1 in gastric cancer cells is positively correlated.At the same time,transcription co-activator YAP1/TEADs binds to the promoter region of FSTL1 gene to transform and induce the expression of FSTL1;in addition,FSTL1 can promote the expression of FSTL1 by binding to the TLR4 receptor on gastric cancer cells.The YAP1 agonist IRF3 phosphorylates and retains YAP1 in the nucleus to enhance YAP1/TEADs transcriptional activity.Conclusion:1.The expression of FSTL1 is significantly related to the characteristics of malignant clinical cases,treatment effects and prognosis of gastric cancer patients.FSTL1 may become a potential new target for treatment effects and prognosis of gastric cancer patients;2.FSTL1 may be a new target gene downstream of the Hippo-YAP1 signaling pathway;3.FSTL1 promotes YAP1/TEADs transcriptional activity by activating the TLR4-IRF3 axis,forming a YAP1-FSTL1 positive feedback regulation pathway.