The Biological Effects And Mechanisms Of Circular RNA Circ1477 In Gastric Cancer And Its Clinical Value

Objective: Gastric cancer(GC)is a serious disease which greatly threatens the public health of our country.Thus,it is urgent to explore non-invasive and effective diagnostic biomarkers and seek potential therapeutic targets for GC.Circular RNAs(circ RNAs)are becoming new promising biomarkers and therapeutic targets for cancer due to their unique properties,biological effects and mechanisms involved in tumor progression.This study is aimed to identify a new circ RNA biomarker for GC and establish corresponding examination methods,and then elucidate its expression changes,properties,biological effects and mechanisms during GC progression in order to provide new therapeutic targets for GC.Methods: Bioinformatics analysis was used to predict potential GC related circ RNAs.RT-q PCR analysis was established to detect such candidate circ RNAs levels in collected samples including paired GC tissues and adjacent normal tissues,paired plasma samples and plasma exosome samples from GC patients and healthy controls.Their clinical values were evaluated using ROC analysis,survival analysis and correlation analysis between expression levels and clinic pathological features.The most valuable circ RNA was then selected for further study.Its biological characteristics including stability,half-life time and subcellular localization were determined by RNase R assay,actinomycin D assay and RNA FISH.Its flanking Alu elements and RNA binding proteins(RBPs)were predicted by bioinformatics analysis.The regulatory effects of Alu elements and RBPs on circ RNA biosynthesis were verified by using circ RNA wild-type and mutant expression plasmids combined with RNA interference(RNAi)technology.Specific si RNAs and overexpression vectors were used to knock down and overexpress this circ RNA in GC cells.Cell functional experiments including colony formation assay,CCK8 assay,Transwell migration and invasion assays were conducted to detect biological effects of this circ RNA on GC progression in vitro.Subcutaneous xenograft tumor assay and intraperitoneal metastasis assay of nude mice were performed to uncover its effects on GC progression in vivo.To explore the role of this circ RNA as miRNA sponges,the target miRNA and downstream m RNA were predicted with bioinformatics.RIP assay combined with dual-luciferase reporter gene assay and functional rescue experiments in GC cells were conducted to verify that target miRNA could bind to and mediate this circ RNA effects on GC progression.Cell functional experiments were used to identify the role of target miRNA in GC cells.The downstream gene of target miRNA was predicted with bioinformatics.RT-q PCR analysis combined with Western blot,immunohistochemistry,dual-luciferase reporter gene assay and functional rescue experiments in GC cells were performed to confirm that the downstream gene of target miRNA could mediate effects of circ RNA on GC progression.To explore the regulatory effects of this circ RNA on its parental gene,the expression level and biological functions of parental gene were determined by RT-q PCR analysis,Western blot,gain-and loss-of-function experiments in GC tissues or cells.Western blot and immunohistochemistry were conducted to detect the regulatory effects of this circ RNA on parental gene in vitro and in vivo.Bioinformatics analysis was applied to predict the RBP interacting with this circ RNA.RIP assay combined with Western blot,immunohistochemistry and dual-luciferase reporter gene assay were used to verify that the RBP could mediate the regulatory effects of circ RNA on its parental gene.Results: The circ1477 and circ2059 were predicted and selected to be potential GC related circ RNAs and corresponding RT-q PCR methods were established.They were all significantly downregulated in GC tissues and upregulated in GC plasma samples while only circ1477 was significantly upregulated in GC plasma exosomes samples compared to that of healthy controls.ROC analysis showed that circ1477 could serve as a good diagnostic biomarker for GC,which presented great correlationships with tumor size and lymphatic metastasis in tissues,with tumor differentiation in plasma and with tumor distant metastasis in plasma exosome samples.Then,circ1477 was selected for further study.Circ1477 was mainly located in cytoplasm of cells and showed resistance to RNase R digestion with high stability.The flanking Alu Sx3 and Alu Jb elements promoted circ1477 circularization while EIF4A3 did not affect its biogenesis markedly.Knockdown of circ1477 promoted GC cell proliferation,migration and invasion while its overexpression inhibited the growth and metastasis of GC in vitro and in vivo.Circ1477 could competitively bind with miR-190 b or miR-190a-5p to inhibit GC progression.Both of miR-190 b and miR-190a-5p were upregulated in GC tissues and cells and exhibited promotive effects on GC cell proliferation,migration and invasion.TP53INP1 and DAG1 were the predicted common downstream targets of miR-190 b and miR-190a-5p,and their expression levels could be positively regulated by circ1477.TP53INP1 was further demonstrated to be the real target gene of miR-190 b and miR-190a-5p and mediate the suppressive effects of circ1477 in GC progression in vitro and in vivo.GPBP1L1 was parental gene of circ1477 and significantly upregulated in GC tissues and cells.Knockdown of GPBP1L1 inhibited GC cell proliferation,migration and invasion while its overexpression presented opposite effects.Circ1477 downregulated GPBP1L1 at protein level in vitro and in vivo but not at transcriptional level.Circ1477 interacted with HuR and downregulated HuR protein level in vitro and in vivo through enhancing its degradation.HuR upregulated the expression of GPBP1L1 at post-transcriptional level.Conclusion: The circ1477 is a GC related circ RNA which inhibits GC growth and metastasis through regulating miR-190b/miR-190a-5p-TP53INP1 axis or inhibiting HuR-GPBP1L1 axis,and serves as a promising diagnostic biomarker and therapeutic target for GC.