PRKAR2A-AS1 Regulates The Proliferation Of Gastric Cancer Through MiR-34a-5p And PKA Pathway-Mediated Expression And Nuclear Translocation Of FOXP3

Objective: There were about 1 million new cases of gastric cancer and about 700,000 deaths worldwide in 2018,about half of which occurred in China.Clinical radical resection is restricted due to the stage of gastric cancer,the location of gastric cancer and other reasons,and there are no universally applicable drugs.It is urgent to conduct in-depth research on the occurrence and development of gastric cancer,especially the process of malignant proliferation.The FOXP3 transcription factor has been reported to participate in pathological processes such as proliferation and migration of various tumors.The function of FOXP3 was closely related to its localization and phosphorylation(p-FOXP3)in cells.Our and other studies have found that FOXP3 was closely related to the progression of gastric cancer.How does FOXP3 affect the proliferation of gastric cancer? How is the expression of FOXP3 regulated? How does the FOXP3 protein in the cytoplasm translocate into the nucleus to participate in the regulation of downstream gene expression? In-depth research on these issues can deepen our understanding of the occurrence and development of gastric cancer,provide possible detection indicators and drug targets for clinical diagnosis and treatment of gastric cancer,and provide a theoretical basis for these.Materials and Methods:PCR,site-directed mutagenesis technology and homologous recombinationmediated ligation were used to construct overexpression or knockdown plasmids of each gene as well as wild-type and site-directed mutant luciferase reporter gene plasmids.Short hairpin RNA(shRNA)combined with lentivirus was used to screen gastric cancer cell lines with stable knockdown of endogenous gene expression.Quantitative real-time PCR(qRT-PCR)and Western blot(WB)were used to detect the expression of various genes.The vitality and proliferation of gastric cancer cells were detected by CCK-8 cell viability assay and EdU cell proliferation assay.The subcutaneous tumor formation experiments in nude mice was detected by nude mice subcutaneous tumor formation experiment.Luciferase reporter assay combined with chromatin immunoprecipitation(ChIP)or RNA binding protein immunoprecipitation(RIP)to detect the binding of FOXP3 and the promoters of PCNA and MKI67,the binding between miR-34a-5p and the 3'UTR of FOXP3 as well as the binding between PRKAR2A-AS1 and miR-34a-5p.Immunofluorescence(IF),nuclear protein / cytoplasmic protein separation technology combined with WB were used to analyze the expression and localization of FOXP3 and p-FOXP3 in cells.Results:The results of CCK-8 cell viability assay and EdU cell proliferation assay found that knockdown of FOXP3 increased the viability of gastric cancer cells and promoted the proliferation of gastric cancer cells.The results of subcutaneous tumor formation experiments in nude mice found that knocking down FOXP3 promoted the growth of gastric cancer in vivo.The results of qRT-PCR and WB showed that the knockdown of FOXP3 promoted the expression of proliferation-related genes PCNA and MKI67 at the messenger RNA(mRNA)and protein level.The results of luciferase reporter assay and ChIP confirmed that FOXP3 directly bound to the promoter regions of PCNA and MKI67 to inhibit their transcriptional activity.The results of CCK-8 cell viability assay,Ed U cell proliferation assay and subcutaneous tumorigenesis experiments in nude mice found that knocking down miR-34a-5p reduced the activity of gastric cancer cells,inhibited the proliferation of gastric cancer cells and inhibited the growth of gastric cancer in vivo.The trend of the corresponding experimental results after knocking down PRKAR2A-AS1 is opposite to knocking down miR-34a-5p.Luciferase reporter assay,RIP combined with WB confirmed that miR-34a-5p directly binding to the 3'UTR of FOXP3 to inhibit its expression,and PRKAR2A-AS1 antagonized the miR-34a-5p-medited translation inhibition of FOXP3 by competitively binding to miR-34a-5p.The results of IF,nuclear protein / cytoplasmic protein separation and WB confirmed that FOXP3 is more localized in the nucleus in normal gastric epithelial cells and more in the cytoplasm in gastric cancer cells.The vast majority of p-FOXP3 is located in the nucleus.The activation of PKA pathway increases the proportion of pFOXP3 without affecting the expression of FOXP3,and promotes the translocation of p-FOXP3 from the cytoplasm to the nucleus.Endogenous PRKAR2A-AS1 knockdown reduced the expression of FOXP3 and the proportion of p-FOXP3,and inhibited the translocation of p-FOXP3 from the cytoplasm to the nucleus,and this inhibition was antagonized by the activation of the PKA pathway.Conclusion:In summary,we have the following conclusion: FOXP3 binds to the promoters of PCNA and MKI67 to up-regulate their expression and thereby inhibits the proliferation of gastric cancer cells and the growth of gastric cancer in vivo.MiR-34a-5p binds to the 3'UTR of FOXP3 to inhibit its expression,and PRKAR2A-AS1 antagonize the miR-34a-5p-medited translation inhibition of FOXP3 by competitively binding to miR-34a-5p.The activation of PKA signaling pathway promotes phosphorylation of FOXP3 and promotes the translocation of p-FOXP3 from the cytoplasm to the nucleus and thereby inhibits the proliferation of gastric cancer cells.PRKAR2A-AS1 promotes the phosphorylation of FOXP3 and promotes the translocation of p-FOXP3 from the cytoplasm to the nucleus through the PKA pathway.These studies can deepen the understanding of the occurrence and development of gastric cancer,and also provide possible detection indicators and drug targets for clinical diagnosis and treatment of gastric cancer,and provide a theoretical basis for these.