The Molecular Mechanism Of CircularRNA Hsa_circ₀004872 Regulating The Development Of Gastric Cancer

Background:Gastric cancer?GC?is a common malignant tumor of the digestive system,the high mortality and morbidity have attracted much attention.To explore the molecular mechanism of the development of gastric cancer and search for effective molecular diagnostic markers is extremely important to improve the survival rate of gastric cancer patients.Circular RNA?circRNA?is named as a circular structure formed by covalent bonding at its 5'end and 3'end.The characters of richness,stability,conservation and time-space specific give it unique advantages for tumor diagnosis.The relationship between circRNA and gastric cancer is rarely involved.It maybe important for revealing the mechanism of gastric cancer and early diagnosis that study circRNA involved in the development and progression of gastric cancer,its mechanism in-depth and targeted therapy and prognosis of gastric cancer.Objective:1)To explore the expression of hsa circ 0004872 in human gastric cancer and its function in proliferation,invasion and migration;2)To elucidate the molecular mechanism by which hsa circ 0004872 plays a regulatory role in gastric cancer3)To explore the upstream regulation mechanism of hsa circ 0004872 expression disorder in gastric cancerMethods:1.The expression of hsa circ₀004872 in clinical tissue samples and its stability in cells was detected:we detected the expression level of hsa circ₀004872 in gastric cancer and the corresponding non-cancerous tissues and analyzed the trend of its expression in gastric cancer statistically.RNA was treated with RNaseR and l actinomycim D respectively and then we detected the expression of hsa circ 0004872 and linear RNA,analyzed if it is more stable than the linear RNA.2.The biological function of hsa circ 0004872 in GC cells was detected:the overexpression vector or siRNA ofhsa circ 0004872 was transfected into GC cell lines and the effect on the proliferation,invasion and metastasis of GC cells was detected by EdU.?CCK8S scratch assays and Transwell?with or without matrigel?�3.Whether hsa circ 0004872 can act as a"miRNA sponge"in GC was detected:the localization ofhsa circ 0004872 in GC cells was detected by using fluorescence in situ hybridization?FISH?,and whether it can act the"miRNA sponge"was by using RNA-binding protein immunolocalization assay?RIP?,biological softwares predicted the miRNAs which were possible binding to the circRNA,and determined the miRNA by qRT-PCR FISH experiment and the dual luciferase activity assay detected whether there was a direct interaction between the circRNA and the miRNA.The expression of hsa circ 0004872 and miRNA in GC tissues and adjacent tissues was detected by qRT-PCR and analyzed the correlation between hsa circ 0004872 and miRNA expression in GC tissues.4.The biological functions and mechanism of miRNA regulated by hsa circ 0004872 were detected:mimics or inhibitor of miRNA was transfected into GC cells,and the effects on proliferation,invasion and metastasis of GC cells were detected by EdU,CCK8,scratch assays and Transwell?with or without matrigel?.The miRNA mimics or inhibitor were transfected into GC cells,and their regulation of target proteins Smad4 and p21 were detected by qRT-PCR,Western Blot and dual luciferase activity assay.5.The biological functions of hsa_circ₀004872 were detected whether by regulating the above miRNA:The overexpression plasmids and miR-224-5p mimics were co-transfected in GC cells,and their effects on the proliferation,invasion and metastasis of GC cells were detected by EdU,CCK8,scratch assays and Transwell?with or without matrigel?.The expression of Smad4 and p21 which was the downstream protein of the miRNA was detected by Western Blot.6.Animal experiments were conducted to verify the biological function of hsa circ 0004872:nude mice xenograft model and nude mice tail vein injection experiment was used to detect whether hsa_circ₀004872 had the ability to affect the proliferation,invasion and metastasis of GC cells in vivo.7.The molecular mechanism of hsa_circ₀004872 maladjustment in GC cells was investigated:ADAR1 overexpressed plasmid or siRNA was transfected in GC cells,and the expression of hsa_circ₀004872 was detected by qRT-PCR and Western Blot.The expression of ADAR1 in GC tissues and adjacent tissues was analyzed by using NCBI GEO databases.Survival curves were analyzed by Kaplan-Meier Plotter databases.QRT-PCR,ChIP assay and dual luciferase activity assay was used to test whether transcription factors could directly regulate the expression of ADAR1.The expression of transcription factors in GC tissues and adjacent tissues was analyzed by using NCBI GEO databases and analyzed the correlation between the expression of the ADAR1 and transcription factors in GC tissues.Results:1.The expression of hsa_circ₀004872 in GC tissues and the stability analysis1)Database analysis showed that hsa_circ₀004872 was a circRNA formed by reverse splicing of MAPK1 exons 2,3 and 4.2)QRT-PCR results showed that hsa_circ₀004872 was down-regulated in 85.7%of GC tissues,and the expression was significantly down-regulated in GC according to statistical analysis?p<0.0001?.3)Stability analysis of hsa circ 0004872:RNaseR digestion assay and actinomycin D assay confirmed that hsa circ 0004872 had higher stability than the corresponding linear RNA.2.The biological functions of hsa circ 0004872Overexpression of hsa circ 0004872 in GC cells could significantly inhibit the proliferation of GC cells by EdU and CCK8 experiments,and significantly supress the invasion and metastasis of GC cells via scratch assay and Transwell.Interference the expression of hsa circ 0004872 showed the inverse results.3.hsa circ 0004872 played regulated role via acting as " molecular sponge" of miR-224-5pThe results of fluorescence in situ hybridization?FISH?indicated that hsa circ 0004872 was localized in the cytoplasm,suggesting that it may play a role as a"miRNA sponge".RNA-binding protein immunoprecipitation assay?RIP?results showed that hsa circ₀004872 could be detected by immunoprecipitated by Ago2 antibody and it could be used as a"miRNA sponge".Then we used three biological websites to predict the potential binding miRNAs.After overexpression or interference of hsa circ 0004872,the expression level of these miRNAs was detected by qRT-PCR.However,it had almost no regulatory effect on other miRNAs except for downregulating the expression of miR-224-5p.FISH results confirmed the co-localization of hsa circ 0004872 and miR-224-5p.Dual luciferase activity assay confirmed the direct interaction between hsa circ 0004872 and miR-224-5p.The expression levels of hsa circ 0004872 and miR-224-5p in clinical tissue samples were detected,and we found that the expression levels of miR-224-5p in GC tissues were significantly up-regulated compared with that in adjacent tissues,and the expression levels of hsa circ 0004872 and miR-224-5p in GC tissues were negatively correlated.The above experiments fully confirmed that hsa circ 0004872 could play a regulatory role as a"molecular sponge" of miR-224-5p.4.The biological functions and regulatory mechanism of miR-224-5pWe transfected miR-224-5p mimics into GC cells and found miR-224-5p could significantly promote the proliferation of GC cells by EdU and CCK8 experiments and miR-224-5p could significantly promote the invasion and metastasis of GC cells by scratch assay and Transwell.Western Blot and qRT-PCR showed that miR-224-5p could significantly down-regulate the expression levels of Smad4 and p21.We transfected miR-224-5p inhibitor into GC cells and got the inverse results.Dual luciferase activity assay confirmed that miR-224-5p could regulate the expression of Smad4 and p21 by targeting the 3'UTR region of Smad4 and p21 directly.5.Hsa circ 0004872/m;iR-224-5p/Smad4?p21?regulated the proliferation,invasion and metastasis of GC cells.Co-transfected hsa circ₀004872 overexpressed plasmids and miR-224-5p mimics into GC cells,EdU and Transwell showed that overexpression of miR-224-5p partially restored the inhibitory effect of hsa circ 0004872 on cell proliferation,invasion and metastasis.Western Blot results showed that increased expression of Smad4 and p21 induced by high expression of hsa circ 0004872 could be partially recovered by overexpression of miR-224-5p.6.Hsa_circ₀004872 inhibited the proliferation,invasion and metastasis of GC cells in nude miceHsa circ 0004872 stable transfected cells and control cells were injected into nude mice subcutaneous and tail vein respectively.The results of subcutaneous injection showed that the tumorigenic ability of GC cells in nude mice was decreased after the high expression of hsa circ 0004872.And tail vein injection showed that the engraftment ability of GC cells in the lung was significantly inhibited after the high expression ot hsa circ 0004872.7.The molecular mechanism of hsa circ 0004872 downregulation in GC cellsOverexpression of ADAR1 in GC cells reduced the expression of hsa circ 0004872 significantly while the expression of hsa circ 0004872 was significantly up-regulated after interfering ADAR1 in GC cells,The phenomenon suggested that ADAR1 could regulate the expression of hsa circ 0004872 in GC cells.NCBI GEO database showed that the expression of ADAR1 in GC tissues was significantly up-regulated.Kaplan-meier Plotter database showed that GC patients with high ADAR1 expression had a lower survival period.Overexpression of Smad4 in GC cells significantly decreased the expression of ADAR1,in the meantime,ChIP assay verified that Smad4 can bind in the promoter region of ADAR1,and dual luciferase activity assay showed that Smad4 can target and regulate the promoter region of ADAR1.NCBI GEO database showed that the expression of Smad4 in GC tissues was significantly down-regulated,and statistical analysis showed that there was a negative correlation between Smad4 and ADAR1 expression.Kaplan-meier Plotter database showed that GC patients with low Smad4 expression had a lower survival period.Conclusion:In GC cells,the overexpression of ADAR1 inhibited the formation of circRNA hsa circ 0004872,and hsa circ 0004872 could inhibit the development of GC by acting as a"molecular sponge"for miR-224-5p.MiR-224-5p could target the 3'UTR of Smad4 and p21 and inhibit their expression,thus miR-224-5p could promote the proliferation,invasion and metastasis of GC cells.As a transcription factor,Smad4 acted on the promoter region of ADAR1 to inhibit the expression of ADAR1,thus forming a negative feedback regulatory loop and continuously amplifying ADAR1-mediated regulatory signals.Therefore,hsa circ 0004872 could be used as a potential target for the diagnosis and treatment of GC.