| Background and objectivesGastric cancer(GC)is a widespread malignant tumor of the digestive system that directly damages and seriously affects human health.Although tumor treatment has made great advance for the past years,the invasion and metastasis of middle and advanced Gastric cancer are still the main cause of high postoperative mortality of patients.It is very important to probe the possiblepathogenesis of gastric cancer and to find new diagnostic label and possible therapeutic aims for the control of the occurrence and development of gastric cancer.CircRNAs are circular non-coding RNA molecules that are widespread found in mammals and regulate gene expression at the transcriptional or post-transcriptional levels.Researchs have discover that circRNAs can directly or indirectly effect the expression standard of downstream genes,affect the growth,apoptosis,migration and attack of malignant tumor cells,and participate in the growth,invasion and removel of tumor cells.Last several years,largelystudies have shown that circRNAs and miRNA molecules can inhibit the functional activity of miRNA through the mechanism of sponge adsorption,thus regulating miRNA at the transcriptional level.However,most of the current studies on the functional role of circRNA in the progression of gastric cancer and its mechanism still remain at the level of gene expression detection,and only a few studies have conducted in-depth discussions on the function and mechanism of circRNA in gastric cancer.First,this study applies a high-throughput sequencing technology to detect the MNNG induced the gastric epithelial cell lines and progression of malignant transformation of normal controls all transcription genome expression spectrum in cell lines,find out the differentially expressed circRNA,and probeits role in gastric carcinoma and mechanism,to excavate the possible labels of cancer of the stomach for gastric carcinoma aimed cureoffer new aims and accumulation theory and data.Methods1.Select the gastric epithelial mucosal cell GES-1-T that has undergone malignant transformation after treatment with MNNG in the early stage and the normal gastric epithelial mucosal cell line GES-1-N of the negative control for high-throughput sequencing detection of circular RNA whole transcriptome,preliminary screening The circular RNA with obvious differential expression was tested,and the circular RNA hsa?circ?006100 screened out in gastric cancer tissues and cells was tested experimentally by qRT-PCR method,and the expression change of hsa?circ?006100 was found to be consistent with the clinical disease of gastric cancer patients at different stages.The interrelationship between the characteristics of Neo-Confucianism.2.After overexpressing hsa?circ?006100 with plasmid,use CCK-8 and clone formation experiment in gastric cancer cell lines AGS and MGC-803 to detect the changes in the proliferation level of gastric cancer cells after hsa?circ?006100 overexpression,and use Hoechst staining experiment to detect the apoptosis of gastric cancer cells by hsa?circ?006100 Transwell test was used to detect the effect of hsa?circ?006100 on the migration and invasion of gastric cancer cells.3.Use multiple bioinformatics software such as Targetscan,RegRNA and miRanda to predict the miRNA that hsa?circ?006100 may be linked to,and use the dual luciferase report experiment to further analyze and verify the miRNA that is predicted to be regulated by hsa?circ?006100.The expression of miR-195 was tested by the qRT-PCR technology,and the relationship between its expression changes and the clinicopathological characteristics of gastric cancer patients at different stages was analyzed.After the hsa?circ?006100 overexpression plasmid and miR-195mimics were co-transfected into gastric cancer cell lines,CCK-8,clone formation test,Hoechst staining test and Transwell test were used to investigate whether there is an interaction between the two and the changes in gastric cancer cell function.4.Use miRanda,Pictar,Targetscan and other bioinformatics forecast software to analyze and forecast miR-195's maybe target genes,and use dual luciferase report experiment and multiple cell function related experiments to further study and analyze miR-195 For the targeting effect of GPRC5A.5.Finally,the tumor formation experiment in nude mice confirmed the effect of hsa?circ?006100 on gastric cancer in vivo.Results1.Circular RNA high-throughput gene sequencing to detect malignant transformation of gastric mucosal epithelium after MNNG induction the cell line GES-1-T and the normal gastric mucosal epithelial cell line GES-1-N without malignant transformation found that the expression of 4650 circular RNAs was significantly different,of which there was a relative increase in GES-1-T.1509 were high,and 3141 had a lower relative expression in GES-1-T cells.According to the consequence of qRT-PCR tests in gastric cancer tissues and abundant gastric cancer cell lines,hsa?circ?006100 with the most up-regulated expression was selected as the research object.And hsa?circ?006100 has a significant correlation with the differentiation standard of malignant tumors,the TNM staging of gastric cancer sicks,and lymph node metastasis.2.CCK-8 and clone formation experiments showed that compared with the negative control group,the proliferation ability of hsa?circ?006100 overexpressed gastric cancer cell line MGC-803 and AGS was enhanced,and the difference was statistically significant(P<0.05);Hoechst staining experiment showed that hsa?circ?006100 The apoptosis rate of gastric cancer cell lines MGC-803 and AGS overexpressed was reduced.The Transwell experiment showed that the migration and invasion ability of gastric cancer cell lines MGC-803 and AGS overexpressed hsa?circ?006100 were enhanced.3.Bioinformatics software combined with literature analysis to predict the combined target miRNA of hsa?circ?006100 found that miR-1236-5p,miR-195,miR-1236-3p,miR-10a-5p,miR-107 are related to the progression of gastric cancer.The qRT-PCR experiment detected the changes in the expression levels of five candidate miRNAs in gastric cancer cell line MGC-803 when the expression of hsa?circ?006100 was interfered,and the results showed that hsa?circ?006100 had the most significant effect on the changes in miR-195 expression.The dual luciferase reporter gene verified that there is a combine site between hsa?circ?006100 and miR-195,and overexpression of hsa?circ?006100 can inhibit the expression of miR-195 in gastric cancer cell lines MGC-803 and AGS.In addition,gastric cancer patients with low expression of miR-195 have the characteristics of poor cell differentiation,late TNM staging and high lymph node metastasis rate.The results of CCK-8 and clone formation experiments showed that compared with the negative control group,miR-195 mimics in gastric cancer cell lines MGC-803 and AGS could partially reverse the proliferation effect caused by overexpression of hsa?circ?006100.Hoechst staining experiment results It is shown that miR-195 mimics in gastric cancer cell lines MGC-803 and AGS can partially reverse the apoptosis-inhibiting effect caused by overexpression of hsa?circ?006100.The results of Transwell experiments show that miR-195 in gastric cancer cell lines MGC-803 and AGS The mimic can sectional reverse the migration and invasion effects caused by the overexpression of hsa?circ?006100.4.qRT-PCR experiment to detect biological information in gastric cancer cell line MGC-803 overexpressing miR-195 the expression alters of five tumor-related mRNAs WNT3A,GDPD5,CHEK1,NOTCH2,and GPRC5A predicted by the scientific software,the results show that GPRC5A is most significantly affected by miR-195.The dual luciferase reporter gene confirmed that there is a binding site between miR-195 and GPRC5A.The expression standard of GPRC5A in gastric cancer tissues was obvious higher than that of normal tissues adjacent to cancer.The expression of GPRC5A and miR-195 in gastric cancer tissues and gastric cancer cell lines is negatively related.The results of CCK-8 and clone formation experiments indicate that up-regulating the expression of miR-195 can reverse the proliferation-promoting effect caused by up-regulation of GPRC5A;Hoechst staining experiments show that up-regulating the expression of miR-195 can reverse the inhibition of apoptosis caused by up-regulation of GPRC5A Effect;Transwell experiment results show that after up-regulating the expression of miR-195,it can reverse the migration and invasion promotion effect caused by up-regulation of GPRC5A.5.In the nude mouse tumor formation test,the xenograft tumor volume of the hsa?circ?006100 silenced MGC-803 group was smaller than that of the control group.Inhibiting the expression of miR-195 reversed the xenograft tumor volume reduction.HE staining results showed that the number of tumor microvessels was reduced when hsa?circ?006100 expression was low and miR-195 was overexpression.Immunohistochemistry results showed that hsa?circ?006100 expression was silent and PCNA staining was weakened.Western blot results showed that hsa?circ?006100 expression was silent,Bcl-2,GPRC5A,EGFR,and E-cadherin.,The expression of N-cadherin was weakened,and the expression of Cleaved Caspase3 was increased.Inhibiting the expression of miR-195 partially reversed the expression changes caused by hsa?circ?006100.ConclusionHsa?circ?006100 is highly expressed in gastric cancer tissues and cells,which can significantly facilitate the proliferation,migration,invasion and EMT function of gastric cancer cells.Hsa?circ?006100 can bind to miR-195 to play the role of ceRNA,and GPRC5A is significantly up-regulated in gastric cancer tissues and can bind to miR-195.In this study,we first found that hsa?circ?006100 indirectly regulates GPRC5A by competitively binding miR-195 to affect the biological behavior of gastric cancer cells,which is expected to offer a new idea for the pathogenesis and development of gastric cancer. |
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