The Role And Mechanism Of CircNFATC3 In The Occurrence And Development Of Gastric Cancer

Objectives:The purpose of this study was to reveal the mechanism by which circNFATC3(hsa＿circ＿0039930)regulates retinoic acid induced protein 14(RAI14)through the adsorption of micro RNA-23b-3p(miR-23b-3p)to promote the occurrence and development of gastric cancer.circNFATC3 is expected to become a new diagnostic marker and prognostic factor for gastric cancer(GC).It will provide the theoretical basis for exploring the pathogenesis of gastric cancer and researching new therapeutic methods.Methods:1.To verify the carcinogenic effect of RAI14 and reverse the anti-tumor effect of miR-23b-3p.Cell transfection was used to up-regulate and down-regulate miR-23b-3p in GC cells,q RT-PCR and Western Blot were used to determine the expression of RAI14,in order to verify the regulatory relationship between miR-23b-3p and RAI14.By upregulating miR-23b-3p and RAI14 gene in MKN-28 cell line with low expression of miR-23b-3p,miR-23b-3p and RAI14 were up-regulated separately,and downregulating miR-23b-3p and RAI14 in AGS cell line with high expression of miR-23b-3p,miR-23b-3p and RAI14 were separately down-regulated to determine whether miR-23b-3p promote the proliferation,invasion and migration of GC cells through the regulation of RAI14,further confirming that RAI14 promote cell growth and invasion in GC and reverse the anti-tumor effect of miR-23b-3p.Luciferase assay was used to determine whether RAI14 gene is a direct target of miR-23b-3p in GC.2.To verify the relationship between circNFATC3 and the survival/prognosis of the patients with GC.The miRbase and circ RNA expression profile were used to predict that the circ RNA of sponge miR-23b-3p was hsa＿circ＿0039930(circNFATC3).The enrichment levels of circNFATC3 and NFATC3 in MKN-28 and AGS cells exposed to RNase R were analyzed by q RT-PCR.The expression level of circNFATC3 and the localization of circNFATC3 in GC cells were determined.SPSS 20.0 was used to analyze the relationship between circNFATC3 and the survival and prognosis of GC patients.3.Study on the relationship between circNFATC3 and miR-23b-3p/RAI14 axis.circNFATC3 was knocked down in MKN-28 cells and AGS cells.The expression of miR-23b-3p of the two cell lines were measured by q RT-PCR and the effects on proliferation,invasion and migration of the two types of GC cells were measured by MTT,Transwell,clone formation.FISH was used to analyze the localization of circNFATC3 and miR-23b-3p in cells,and RIP-PCR was used to determine whether circNFATC3 was associated with miR-23b-3p.The binding site of circNFATC3 and miR-23b-3p was verified by luciferase reporting assay.MKN-28 cells were transfected with si-circNFATC3 and were injected subcutaneous into the lateral abdominal wall of nude mice,and the animals were killed 7-10 days later to observe the tumor growth.IHC was used to determine the expression of RAI14 and Ki67 in gastric tumor tissues to ascertain whether circNFATC3 can promote the occurrence of GC and the relationship between RAI14 and circNFATC3.Results:1.RAI14 can promote cancer and reverse the anti-tumor effect of miR-23b-3p.The results showed that the expression level of miR-23b-3p was higher in AGS cell line,but lower in MKN-28 cell line.The overexpression efficiency of RAI14 plasmid in MKN-28 and the knockout efficiency of si-RAI14 in AGS were both good,and the treatment efficiency of miR-23b-3p mimic in MKN-28 or its inhibitor in AGS was normal.The ectopic expression of RAI14 promoted proliferation,migration,and cell invasion of GC cells,and reversed the inhibitory effect of miR-23b-3p mimic in the MKN-28 cell line,whereas the effect of RAI14 knockout was reversed in the AGS cell line.Luciferase activity assay verified that RAI14 was the direct target of miR-23b-3p in GC.2.The upregulation of circNFATC3 is associated with poor prognosis in patients with GC.circNFATC3 has the potential to bind to miR-23b-3p.circNFATC3 is mainly located in the cytoplasm of GC and normal tissue cells.The expression of circNFATC3 was increased in GC tissues compared with adjacent normal tissues.Survival analysis showed that high circNFATC3 expression was associated with shorter survival than low circNFATC3 expression in patients.3.circNFATC3 can regulate miR-23b-3p/RAI14 axis to promote the occurrence and development of GC.q RT-PCR showed that si-circNFATC3 increased the expression level of miR-23b-3p.FISH analysis showed that circNFATC3 and miR-23b-3p co-located in the cytoplasm of GC tissue cells,and luciferase assay analysis showed that miR-23b-3p could bind to circNFATC3 3’UTR,reducing its WT activity in MKN-28 cells.It was found that circNFATC3 and miR-23b-3p were highly enriched in Ago2 particles by using RIP-PCR technology.In MKN-28 cells,the expression of RAI14 in MKN-28 cells transfected with si-circNFATC3+NC group was lower than that in the transfected si-circNFATC3+miR-23b-3p inhibitor group.In vivo tests showed that GC tumors in the si-circNFATC3 group were smaller in volume and lighter in average tumor weight.IHC analysis showed that RAI14 and Ki67 protein levels in the si-circNFATC3 group were significantly reduced compared with the si-NC group.Conclusions:RAI14 gene can promote the growth,invasion and migration of GC cells.Up-regulation of miR-23b-3p can inhibit the carcinogenic effect of RAI14,while down-regulation of miR-23b-3p can enhance the carcinogenic effect of RAI14.In GC,circNFATC3 has binding sites with miR-23b-3p,and the circNFATC3 is significantly increased in GC tissue.The higher the circNFATC3,the shorter the survival time of patients.Downregulation of circNFATC3 inhibits the carcinogenic effect of RAI14 by enhancing miR-23b-3p in GC.Therefore,it was concluded that circNFATC3 can regulate RAI 14 and promote the occurrence and development of GC by adsorption of miR-23b-3p.