| Objective: Gastric cancer is the sixth most common malignancy and the third leading cause of cancer-related death in China.Most gastric cancer patients go to hospital at advanced stage due to the nonspecific clinical symptoms.Gastric cancer patients have a poor prognosis,with a long-term survival rate of less than 30 percent,due to high recurrence and metastasis rates.The achievement of early diagnosis and individual treatment is the most effective treatment strategy for gastric cancer patients.Biomarkers in blood,tissue and other body fluids that indicate disease-specific properties can be used in cancer diagnosis and assessment of drug effects.Some biomarkers,such as human epidermal growth factor receptor 2(HER2),programmed cell death ligand 1(PD-L1),CA199,CA724,CEA and so on,have been widely used in clinical practice to improve the early diagnosis and find the patients who are possibly sensitive to immunotherapy and targeted therapy and further increase the treating effect.However,based on the limited number of biomarkers,less than 20% of patients are sensitive to immunotherapy and targeted therapy,thus more tumor biomarkers need to be found.With the development of high-throughput sequencing technology and bioinformatics,a large number of non-coding RNAs(nc RNAs)have been found.It has been found that nc RNAs,previously thought to have no function,actually play an important regulatory role in the occurrence and development of diseases.Circular RNAs(Circ RNAs)are a kind of non-coding RNA,which has been confirmed to exist widely in organisms.They perform a variety of biological functions,such as signal transduction and protein translation,and can participate in the regulation of pathological processes of disease through a variety of mechanisms.Their abnormal expression is strongly associated with the occurrence and development of malignancies such as stomach,liver and lung cancers.Circ RNA expression in eukaryotic cells is diverse,conserved,stable and specific,making circRNA a potential diagnostic marker and therapeutic target for oncology therapies.The circular RNAhsa＿circ＿0064554 is encoded by SATB homeobox protein 1(SATB1)and about 2042 nucleotides long.SATB1 is a nuclear matrix-associated protein that exhibits cell specificity and has been reported to be significantly highly expressed in gastric,colon,breast,bladder,and other tumor tissues,facilitating the occurrence and development of tumors.Hsa＿circ＿0064554 has multiple RNA binding protein(RBP)binding sites and miRNA response element(MRE),It may play an important physiological role by binding RBP or miRNA.The expression of hsa＿circ＿0064554 in gastric cancer and its mechanism of action remain unclear.There are no related studies of this RNA.Through bioinformatics analysis and experimental validation in tumor cells and tissues,we found that hsa＿circ＿0064554 is differently expressed in gastric cancer and its expression level is correlated with various pathological features.The aim of this study is to experimentally explore and validate the role and regulatory mechanisms of hsa＿circ＿0064554 in the occurrence and development of gastric cancer,and to further analyze whether hsa＿circ＿0064554 can be used in the early diagnosis and targeted treatment of gastric cancer.Our study may provide an experimental basis for a subsequent in-depth study.It is also hoped that this study will provide some new evidence for the exploration of the molecular mechanisms of gastric cancer,facilitate the translation of experimental studies into clinical applications,and enable accurate treatment and early diagnosis of gastric cancer at an early stage.Methods: First,circRNA-microarrays were downloaded from the GEO database and the data was processed and analyzed using bioinformatics methods.Circ RNAs that are differentially expressed in gastric cancer and encoded by oncogenes or antioncogenes were screened.Then,the expression of circRNAs was validated in cells and tissues.Gastric cancer cell lines MGC-803,HGC-27,AGS,and normal gastric epithelial cell line GSE-1were used in the PCR experiments.Based on the secondary structure of the circRNAs searched in the CSCD database,we analyze their possible regulatory mechanisms.Considering that the circular structure makes circRNAs difficult to digest by RNase,RNase R assay was performed to verify the circular structure of hsa＿circ＿0064554,with the associated m RNA as a control.The hsa＿circ＿0064554 plasmid was transfected into the gastric cancer cell line MGC-803,where hsa＿circ＿0064554 was most significantly downregulated,allowing the construction of hsa＿circ＿0064554-over-expressed gastric cancer cell line.CCK-8 assay and Transwell assay were performed to verify the biological function of hsa＿circ＿0064554.A clinicopathological analysis was subsequently performed.Previously,we found that down-regulated hsa＿circ＿0064554 could potentially regulate the biological behavior of gastric cancer.In the following,we analyze the functional mechanism of hsa＿circ＿0064554 through bioinformatic analysis and in vitro experiments.miRNAs which had potential binding sites with hsa＿circ＿0064554 were screened by searching the Circ Interactome and Starbase databases.The expression levels of miRNAs in gastric cancer were verified.RT-qPCR was performed on the hsa＿circ＿0064554-overexpressed cell line to identify dynamic changes in miRNAs.Finally,we find that miR-23a-3p may be a downstream effector of hsa＿circ＿0064554.The binding sites of hsa＿circ＿0064554 and miR-23a-3p were verified using dual luciferase reporter assays.The expression of miR-23a-3p in gastric cancer was validated using the Starbase database.Gene microarrays containing miR-23a-3p expression data,clinicopathology data and survival data from GC patients were downloaded from the GEO database.Its associations with clinicopathological factors and survival were identified.Having clarified the targeted binding relationship between hsa＿circ＿0064554 and miR-23a-3p,we further explore the regulatory mechanism of the hsa＿circ＿0064554/ miR-23a-3p axis on the biological behavior of gastric cancer and investigate potential downstream target genes and pathways.First,miR-23a-3p mimics were transferred into the hsa＿circ＿0064554-overexpressed gastric cancer cell line.Rescue experiments were subsequently performed to investigate changes in metastasis,proliferation,and apoptosis of tumor cells,as well as changes in the downstream apoptotic pathway and the P38-MAPK pathway.Finally,it can be found that hsa＿circ＿0064554 can regulate the biological behavior of gastric cancer by binding to miR-23a-3p and further mediating downstream genes and pathways.Results: 1.Hsa＿circ＿0064554,encoded by SATB1,was down-regulated in gastric cancer(P<0.001).Hsa＿circ＿0064554 had circular structure and was tolerant to digestion from RNase R.2.RT-qPCR results demonstrated that hsa＿circ＿0064554 was significantly associated with tumor size(P=0.027),metastatic lymphatic nodes(P=0.006),positive CA199(P=0.038).Other clinicopathological factors,such as age,sex,tumor location,lymph invasion,nervous invasion,histological grade,WHO classification,Lauren type,pathological stage,CEA and HER2,were not associated with the expression level of HSA＿circ＿0064554.3.Improving the expression level of hsa＿circ＿0064554 in gastric cancer significantly suppresses tumor cell proliferation and migration.Thus,hsa＿circ＿0064554,acting as an inhibitory factor,negatively regulates the biological behavior of gastric cancer.4.Compared with normal cells,miR-23a-3p was upregulated in gastric cancer cells(P<0.01).According to the Starbase database,the expression of miR-23a-3p is higher in gastric cancer tissue than in adjacent tissue.Based on GSE62254,GC patients with higher expression of miR-23a-3p were younger(P=0.030),more advanced N stage(P<0.001)and pathological TNM stage(P<0.001),less located in antrum sites(P=0.039)than patients with lower expression of miR-23a-3p.Patients with lower expression showed longer survival(1-year OS: 81.6% vs 86.9%;3-year OS: 57.7% vs 62.6%;5-year OS: 48.6% vs57.1%).However,the Kaplan-Meier survival analysis detected a non-significant association between expression of miR-23a-3p and survival.5.MiR-23a-3p had binding sites with hsa＿circ＿0064554,their expressions showed negative relationship in GC cells(P<0.001).According to dual luciferase reporter gene assay,compared with circ-WT + miR-NC group,the luciferase activity of circ-WT + miR-23a-3p mimic group was significantly decreased.It can be concluded that hsa＿circ＿0064554 can bind to miR-23a-3p as a miRNA sponge,and inhibit its expression.6.Next,Rescue assays were performed to validate the change in malignant behaviors of tumor cells and expression of downstream genes and pathways.Increasing hsa＿circ＿0064554 inhibits the proliferation and invasion of gastric cancer cells and promotes apoptosis.When miR-23a-3p expression is restored in cells with high hsa＿circ＿0064554 expression,tumor cells can be activated partly.7.Bioinformatic analysis and RT-qPCR validation were performed to screen the downstream target genes for hsa＿circ＿0064554/miR-23a-3p axis.Results showed that expressions of PTEN and PDCD4 can be improved by hsa＿circ＿0064554,but are again suppressed when the expression for miR-23a-3p is recovered.Thus,PTEN and PDCD4 are likely targeted genes for the hsa＿circ＿0064554/ miR-23a-3p axis.8.With the improvement of hsa＿circ＿0064554,the phosphorylated P38,NLRP3,caspase 1,and cleaved caspase 1 are increased,and the P38-MAPK signaling pathway and cell pyroptosis are significantly activated.However,upon restoration of miR-23a-3p,the P38 signaling pathway and cell pyroptosis are again inhibited.As a result,hsa＿circ＿0064554/ miR-23a-3p modulates the expression of PTEN and PDCD4,further mediating the P38-MAPK signaling pathway and cell pyroptosis to achieve its inhibitory function in gastric cancer.Conclusion: Hsa＿circ＿0064554 is down-regulated in gastric cancer,and its expression is significantly correlated with tumor size,positive lymph nodes,and CA199.Hsa＿circ＿0064554 binds to miR-23a-3p as a miRNA sponge and then inhibits the expression of PTEN and PDCD4,further activating the P38-MAPK signaling pathway and NLRP3-CASP1 pyroptosis-related pathway.In this way,hsa＿circ＿0064554 negatively modulates the biological behavior of gastric cancer and is a potential tumor marker and therapeutic target. |
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