Tumor-associated Fibroblasts Promoted Proliferation And Metastasis Of Gastric Cancer Cells By Stimulating CircFAM188A/mir-708-5p/PD-L1

Background:Gastric cancer is a common malignant tumor of the digestive system.Metastasis of gastric cancer is an important cause of death in patients with gastric cancer.The invasion and metastasis of gastric cancer are very complicated,involving many complicated processes such as extracellular matrix and angiogenesis.These pathological changes related to tumor metastasis can induce changes in the tumor microenvironment,which in turn can promote tumor Cell transfer.The tumor microenvironment is composed of extracellular matrix,interstitial cells,etc.TAFs are important interstitial cells,which can not only induce tumor metastasis through cell-cell interactions,but also induce tumor cells by secreting multiple cytokines invasion and migration.circRNA is a kind of non-coding RNA with special functions,and its function has not been fully elucidated yet.CircRNA is known to be expressed in mammals and can affect life processes through complex regulatory networks circRNA can regulate miRNA expression by binding to miRNA,and miRNA can affect the expression of downstream target mRNA.circRNA can affect cell growth,invasion and other processes through this regulatory mechanism.Previous studies have found that circFAM188A is upregulated in the plasma of gastric cancer patients,and circFAM188A may be involved in gastric cancer progression.In this experimental study,the role and mechanism of circFAM188A in the growth and metastasis potential of gastric cancer cells co-cultured with TAFs were explored,and the role of circFAM188A was verified by a nude mouse transplantation tumor model.Objective:In this study,the effects of circFAM188A regulation of miR-708-5p on the expression of PD-L1 on the proliferation,cloning,cell cycle,invasion,migration and EMT of gastric cancer cells co-cultured with TAFs were investigated,and the growth of transplanted tumor in nude mice was confirmed to clarify the mechanism of circFAM188A in the development and metastasis of gastric cancer.Methods:1.Isolated and cultured TAFs and NFs,and detected the expression of ?-SMA by immunofluorescence method.2.TAFs and NFs were co-cultured with gastric cancer cells,cell proliferation was detected by MTT method,cell clone formation was detected by plate cloning,cell cycle distribution was detected by PI single staining,cell migration and invasion were detected by Transwell Chambers,and expression changes of EMT-related proteins Vimentin,n-cadherin and e-cadherin were detected by Western blot.3.The expression changes of circFAM188A,miR-708-5p and PD-L1 in gastric cancer tissues and gastric cancer cells and after co-culture with TAFs were detected by qRT-PCR.4.CircFAM188A siRNA and miR-708-5p inhibitor,CircFAM188A siRNA and inhibitor control,mir-708-5p mimics and negative control vector,mir-708-5p mimics and PD-L1 overexpression vector were transfected into gastric cancer cells,respectively.After co-culture with TAFs,the changes of each indicator were detected5.StarBase v2.0 predicted that circFAM188A and miR-708-5p,PD-L1 and miR-708-5p were mutually targeted.The RIP experiment and the dual luciferase reporting system identified their relationships,and the RNA pull down experiment verified their interactions.6.PD-L1 protein changes were detected by Western blot after gastric cancer cells were co-cultured with TAFs after transfection with circFAM188A siRNA and inhibitor control,circFAM188A siRNA and miR-708-5p inhibitor.7.CircFAM188A shRNA lentiviral vector was transfected into gastric cancer cells and inoculated subcutaneous into nude mice to observe the changes in tumor growth volume and weight.The tumor tissues of nude mice were taken and the mRNA expression levels of circFAM188A,miR-708-5p and PD-L1 in the transplanted tumor tissues were detected by qRT-PCR,and the protein expression levels of PD-L1 in the transplanted tumor tissues were detected by Western blot.Results:1.Compared with NFs,the expression level of a-SMA in TAFs is higher,the proliferation,clone formation,invasion and migration of gastric cancer cells increased after co-culture with TAFs,and the proportion of G0/G1 stage cells decreased,the expression levels of Vimentin and n-cadherin proteins increased,and the expression levels of e-cadherin proteins decreased.2.CircFAM188A and PD-L1 are highly expressed in gastric cancer tissues and cells.Patients with high expression of circFAM188A have poor prognosis.The expression level in gastric cancer cells was increased after co-culture with TAFs.3.MiR-708-5p was down-regulated in gastric cancer tissues and cells,and down-regulated in gastric cancer cells co-cultured with TAFs4.CircFAM188A and miR-708-5p are mutually targeted.MiR-708-5p and PD-L1 are mutually targeted.5.Gastric cancer cells transfected with circFAM188A siRNA were co-cultured with TAFs,the cell proliferation,cloning,invasion and migration abilities were reduced,the cell G0/G1 phase ratio was increased,the expression levels of Vimentin and n-cadherin proteins were decreased,the expression levels of e-cadherin proteins were increased,and the expression levels of PD-L1 proteins were decreased.6.Compared with circFAM188A siRNA and inhibitor control,gastric cancer cells co-transfected with circFAM188A siRNA and mir-708-5p inhibitor after co-culture with TAFs showed increased proliferation,cloning,invasion and migration,decreased cell G0/G1 phase ratio,increased Vimentin and n-cadherin protein expression levels,decreased e-cadherin protein expression levels,and increased PD-L1 protein expression levels.7.Compared with the negative control vectors that were co-transfected with miR-708-5p mimics,gastric cancer cells that were co-transfected with mir-708-5p mimics and PD-L1 overexpression vectors were co-cultured with TAFs,with increased cell proliferation,cloning,invasion and migration,decreased cell G0/G1 phase ratio,increased Vimentin and n-cadherin protein expression levels,and decreased e-cadherin protein expression levels.8.After transfection with circFAM188A shRNA lentiviral vector,the growth volume and weight of transplanted tumor in nude mice with gastric cancer cells decreased,the levels of circFAM188A and PD-L1 decreased and the level of miR-708-5p increased in the transplanted tumor tissues of nude mice with gastric cancer cells.Conclusion:CircFAM188A regulated the proliferation,cloning,migration,invasion,cell cycle changes and epithelial mesenchymal transformation of gastric cancer cells co-cultured with TAFs by regulating miR-708-5p/PD-L1.CircFAM188A acts by targeting miR-708-5p,so it can be seen that circFAM188A can be used as a new therapeutic target for gastric cancer and a potential biological marker for early gastric cancer screening.At the same time,the detection results of clinical samples in this study showed that patients with gastric cancer with high circFAM188A expression had poor prognosis,and circFAM188A is expected to be used as an important indicator to monitor the prognosis of gastric cancer patients in the future.