| Objective: Gastric cancer(GC)is the fifth leading cancer worldwide and the fourth leading cause of cancer death,with high morbidity and mortality.GC patients in our country account for nearly half of the global GC patients.The main reason is that most of the GC patients in my country are in the advanced stage,with a high risk of recurrence and metastasis,and a poor prognosis.Therefore,in-depth research on the specific pathways and key biomarkers of GC invasion and metastasis,and the discovery of potential therapeutic targets for GC are of great significance for the diagnosis and treatment of GC,and provide experimental basis and new ideas for the optimization of clinical decision-making.In recent years,it has been discovered that circular RNAs(circRNAs)are special in the field of non-coding RNAs,with a large number of covalently closed-loop structures in the cytoplasm of eukaryotes.It is mainly formed by back splicing of exons of precursor mRNA(pre-mRNA),which is not easily degraded by exonuclease and is more stable than linear RNAs.More importantly,circRNAs can regulate the transcriptional or posttranscriptional level.Many circRNAs have been shown to be stably differentially expressed in some cancers,indicating that circRNAs have great potential as molecular markers of cancer.At present,it is believed that circRNAs mainly affect the occurrence and development of diseases(including tumors)through four mechanisms.Among them,the most studied is to use its own abundant micro RNA(micro RNA,mi RNA)binding sites to act as a mi RNA sponge to regulate the expression of mi RNA downstream target genes.This regulatory mechanism is also called the competing endogenous RNA mechanism(competing endogenous RNA,ceRNA).It is well known that mi RNAs can bind mRNAs to cause silencing of downstream target genes,while ceRNAs can regulate the expression patterns of downstream target genes by competitively binding mi RNAs.The most common ceRNAs are long noncoding RNAs(lnc RNAs)and circRNAs,which can reverse gene silencing caused by mi RNAs by binding to mi RNA response elements(MREs).Hsa＿circ＿0001190 is an unreported circRNA located on chromosome 21 with 1568 bases.Its parent gene is called dual specificity tyrosine-phosphorylation-regulation kinase1A(DYRK1A),which affects multiple signaling pathways including Erk and Akt,and is closely associated with tumorigenesis.However,the potential role and mechanism of hsa＿circ＿0001190 in GC are not yet known,and further research is needed.Therefore,we first used a high-throughput Gene Expression Omnibus(GEO)for bioinformatics analysis,and confirmed that the expression of hsa＿circ＿0001190 was significantly different in GC tissues and adjacent tissues.The purpose of this study was to clarify the role of hsa＿circ＿0001190 in GC through in vitro and in vivo experiments,to explore the correlation between hsa＿circ＿0001190 and the clinicopathological characteristics of GC,to study the role and internal mechanism of hsa＿circ＿0001190 in GC,and to provide experiments for the mechanism of GC invasion and metastasis.It also provides new possibilities for the translation of basic research into clinical decisionmaking.Methods: First,we used a variety of bioinformatics analysis methods to jointly analyze and screen out the significantly differentially expressed circRNAs in the GEO database,and perform structure visualization,function prediction,and pathway enrichment analysis.To further verify the effect of candidate circRNAs on GC,we selected 37 pairs of tissue specimens from patients with pathologically confirmed gastric adenocarcinoma,and extracted RNA from cancer and adjacent tissues for RT-qPCR detection.In addition to verifying the expression of hsa＿circ＿0001190 in fresh tissue samples,we also selected paraffin sections of 120 GC patients who underwent radical surgery,performed in situ hybridization experiments,and conducted semi-quantitative analysis of hsa＿circ＿0001190 to explore its relationship with the clinicopathological characteristics of GC patients.Multivariate Cox regression analysis and Kaplan-Meier survival analysis were used to observe whether it was significantly related to the survival and prognosis of GC patients.The expression levels in 5 GC cell lines were detected by RT-qPCR experiments,and two GC cell lines,MGC-803 and HGC-27,were selected for subsequent experimental analysis.The lentiviral vector was used to construct a cell line that stably overexpressed and silenced hsa＿circ＿0001190,and the expression efficiency was verified by RT-qPCR experiments.By designing in vivo and in vitro experiments,including wounding healing experiment,Transwell experiment,clone formation experiment,CCK-8 proliferation experiment and nude mouse subcutaneous tumorigenesis experiment,the effects of hsa＿circ＿0001190 on the invasion,migration and proliferation of GC cells in vitro and in vivo were observed.We plan to perform high-throughput sequencing on GC cells with high expression of hsa＿circ＿0001190 to explore the specific pathways that hsa＿circ＿0001190 affects the malignant biological behavior of GC cells,and to identify key mi RNA molecules and downstream target genes in the ceRNA mechanism.Using multiple databases such as ENCORI,circ Bank,mi RDB,and Target Scan Human for joint prediction and analysis,combined with high-throughput sequencing results,mi RNAs and downstream target genes that may bind to hsa＿circ＿0001190 were screened out.Previous studies have confirmed that candidate target genes,Phosphatase And Tensin Homolog(PTEN),can inhibit the expression of Vascular Endothelial Growth Factor A(VEGFA)through the Akt pathway,thereby inhibiting tumor angiogenesis.Therefore,we reasonably speculate that hsa＿circ＿0001190 competes to bind to this mi RNA and prevents the inhibition of downstream target genes,thereby inhibiting tumor angiogenesis and ultimately exerting a tumor suppressor effect.The binding sites of circRNA and mi RNA were clarified by using dual-luciferase reporter gene assay.Western Blot assay detected the expression of all downstream target genes in GC cells overexpressing and silencing hsa＿circ＿0001190.To further investigate whether hsa-mi R-23a-3p is involved in the regulatory pathway of hsa＿circ＿0001190,the mimics of hsa-mi R-23a-3p was further transfected into MGC-803 and HGC-27 cells stably overexpressing hsa＿circ＿0001190,and then we observed the functional and protein level changes of GC cells.Finally,we detected whether the tube-forming ability of endothelial cells was significantly changed in the co-culture system of GC cells and human umbilical vein endothelial cells(HUVEC).And the Rescue experiment was used to verify whether hsa＿circ＿0001190 could affect the expression of downstream proteins through hsa-mi R-23a-3p,thereby changing angiogenesis of GC,and ultimately affecting the malignant biological behavior of GC cells.Results: 1.The results of bioinformatics analysis showed that in the combined analysis of microarray data of 36 GC patients in 5 GEO datasets,a total of 39 circRNAs were identified that were significantly differentially expressed.The expression of hsa＿circ＿0001190 and hsa＿circ＿36287 was significantly lower in GC tissues,and significantly higher in adjacent tissues(P<0.05).The secondary structures of two candidate circRNAs were retrieved from the Cancer-Specific Circ RNA Database(CSCD)and found that both contained a large number of mi RNA binding sites.2.Further RT-qPCR analysis of 37 pairs of GC patients’ cancer and adjacent tissues showed that the expression levels of hsa＿circ＿0001190 and hsa＿circ＿0036287 in adjacent tissues were significantly higher than those in tumor tissues(hsa＿circ＿0001190: P<0.001;hsa＿circ＿0036287: P=0.014).3.The expression levels of hsa＿circ＿0001190 and hsa＿circ＿0036287 in human gastric cancer cell lines SGC-7901,MGC-803,HGC-27,MKN-45 and AGS were detected by RT-qPCR.The experimental results showed that the expression of both of them was the highest in the control group GES-1,and the expression of MGC-803 and AGS was the lowest.hsa＿circ＿0036287 was excluded due to low background expression.In conclusion,we selected hsa＿circ＿0001190 as the target,MGC-803 and HGC-27 as ideal gastric cancer cell lines,and successfully constructed cell lines that stably overexpressed and silenced hsa＿circ＿0001190 for further experimental research.4.The results of in situ hybridization showed that hsa＿circ＿0001190 was mainly expressed in the cytoplasm of cells.Correlation analysis was performed of 120 GC patients,and we found that the expression of hsa＿circ＿0001190 was significantly correlated with N stage(P=0.016)and lymph node metastasis radio(P=0.007).The N stage of patients in the low expression group was significantly later,and the lymph node metastasis radio was significantly higher.Kaplan-Meier survival analysis showed that the survival prognosis of patients with high hsa＿circ＿0001190 expression was significantly better than that of patients with low expression(5-year survival rate: 55.6% vs 19.7%,P<0.001).Multivariate Cox regression analysis showed that high expression of hsa＿circ＿0001190 was an independent predictor of better prognosis in GC patients(HR=0.367,95%CI: 0.227-0.596,P<0.001).5.The results of wounding healing and Transwell experiments showed that hsa＿circ＿0001190 could significantly inhibit the invasion and migration of GC cells in vitro.However,the clone formation and CCK-8 cell proliferation experiments showed that this circRNA had little effect on the proliferation ability of GC cells.The results of the subcutaneous tumorigenesis experiment in nude mice showed that the tumor growth rate of the left axillary tumor injected with hsa＿circ＿0001190 overexpressing MGC-803 was significantly lower than that of the right control group(P=0.035);the tumor weight of the left axilla was significantly smaller than that of the right control group(P=0.005).6.High-throughput sequencing results showed that mRNA differential analysis indicated that overexpression of hsa＿circ＿0001190 led to the up-regulation of PTEN,NCLP1,COL3A1 and RASIP1 expressions,which were positively regulated by hsa＿circ＿0001190,suggesting that they have the potential to become downstream target genes;and mi RNA differential analysis showed that 17 mi RNAs including hsa-mi R-23a-3p and hsa-mi R-191-5p were down-regulated and negatively regulated with hsa＿circ＿0001190,suggesting that these mi RNAs may be regulated by the binding of hsa＿circ＿0001190.Further functional enrichment analysis found that compared with the hsa＿circ＿0001190 overexpression group,the NC group was significantly enriched in angiogenesis-related pathways,indicating that the low expression of hsa＿circ＿0001190may promote tumor angiogenesis.7.Multiple databases such as ENCORI,circ Bank,mi RDB and Target Scan Human jointly predicted,and combined with high-throughput sequencing results,the key mi RNA molecule—hsa-mi R-23a-3p and the key mRNA—PTEN were jointly screened.Based on this,we reasonably speculate that hsa＿circ＿0001190 can reverse the inhibitory effect of hsa-mi R-23a-3p on the target gene PTEN by competitively binding to hsa-mi R-23a-3p,thereby inhibiting tumor angiogenesis and ultimately exerting a tumor suppressor effect.8.The results of the dual luciferase reporter gene assay showed that compared with WT+mi R(+)NC,the luciferase activity of WT+mi R(+)group was significantly decreased(P=0.003).And there was no significant difference in luciferase activity between the MT+mi R(+)NC and the MT+mi R(+)group(P>0.05).9.After transfection of hsa-mi R-23a-3p mimics,Western Blot detection showed that in the two cell lines of MGC-803 and HGC-27,the expression of PTEN protein in the overexpression group was significantly lower than that in the NC group(P<0.05).10.Western blot detection showed that the expression of PTEN in MGC-803 and HGC-27 cells in the hsa＿circ＿0001190 overexpression group was higher than that in the control group(P<0.01).Akt protein expression,MGC-803 cells in the overexpression group were lower than those in the control group,and the trend was obvious,but did not show statistical significance(P=0.072);HGC-27 cells were significantly lower than those in the control group(P=0.048).VEGFA protein expression,MGC-803 and HGC-27 cells in the overexpression group were lower than those in the control group(P<0.05).11.The results of angiogenesis experiments showed that the tube-forming ability of HUVECs was significantly reduced when co-cultured with MGC-803 cells in the hsa＿circ＿0001190 overexpression group(P<0.01);when co-cultured with MGC-803 cells in hsa＿circ＿0001190 silenced group,the tube-forming ability of HUVEC was significantly increased(P<0.01).Co-culture of HUVEC with overexpressing hsa-mi R-23a-3p cells significantly increased the tube-forming ability(P<0.05).The results of the Rescue experiment showed that the tube-forming ability of HUVECs was significantly decreased after co-culture with MGC-803 in the circ(+)+mi R(+)NC group(P<0.05);while HUVEC co-cultured with circ(+)NC+mi R(+)group MGC-803,the tube-forming ability was significantly increased(P<0.01).However,the circ(+)+mi R(+)cotransfection group showed that the tube-forming ability of HUVECs in the co-culture system was significantly lower than that of the circ(+)NC+mi R(+)group(P<0.01).12.The results of the Western blot suggested that in MGC-803 cells and HGC-27 cell lines,compared with the circ(+)NC+mi R(+)NC group,the expression of PTEN in the circ(+)NC+mi R(+)group was significantly decreased(MGC-803: P=0.006;HGC-27:P<0.001);the expression of PTEN in the circ(+)+mi R(+)NC group was significantly increased(MGC-803:P=0.032;HGC-27:P=0.006).Compared with the circ(+)NC+mi R(+)group,the circ(+)+mi R(+)co-transfection group showed a significant increase in the expression of PTEN.(MGC-803: P=0.049;HGC-27: P=0.037).Conclusion: The high expression of hsa＿circ＿0001190 is significantly correlated with the better prognosis of GC patients.Through competitive binding to hsa-mi R-23a-3p,the expression level of its target gene PTEN is increased,resulting in the inhibition of Akt activation,and then the down-regulation of VEGFA protein expression.Finally,it inhibits malignant biological behavior such as angiogenesis in GC,and exerts a tumor suppressor effect. |
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