Circular RNA Expression Profile And M6A Modification Analysis In Poorly Differentiated Adenocarcinoma Of The Stomach

Poorly Differentiated Gastric Adenocarcinoma?PDGA?is a type of gastric cancer?GC?with poor prognosis.Circular RNA?circ RNA?is a type of single-stranded RNA.It is a covalently closed continuous loop formed by the back-splicing mechanism and is abundantly presented in eukaryotic transcriptomes.Circ RNA can not only affect the post-transcriptional gene expression regulation,but also participate in the process of tumorigenesis and development.To the best of our knowledge,there're limited studies of the m6A?N6-methyladenosine?modification of circ RNA in cancer.Therefore,we aim to reveal the differential expression profile of circ RNA in PDGA,at the same time,identify the m6 A modification characteristics of differentially expressed circular RNAs?DECs?.Eventually provide some new horizons for the diagnosis and treatment of PDGA.Materials and methodsThree cases of PDGA tissues and their paired normal adjacent tissues were selected for analysis of DECs by microarray analysis.Among the DECs,by using the RT-q PCR technology,5 circ RNAs were selected to verify the reliability of the data from DECs profile in 41 cases of gastric cancer and paired normal adjacent tissues,as well as in 3GC cell lines and their normal control.Moreover,the receiver operating curve?ROC?was used to evaluate the diagnostic efficacy of these 5 DECs in gastric cancer and PDGA.In addition,GO?Gene ontology?enrichment analysis and KEGG?Kyoto Encyclopedia of Genes and Genomes?pathway analysis were both performed for all DECs-derived genes in the expression profile.Finally,the top 10 high & low-level DECs in the expression profile are predicted by the sequence-based RNA adenosine methylation site predictor?SRAMP?website for their possible m6 A modification sites.Then the very high & high-confidence DECs are selected for the detection of their m6 A modification levels as well as their expression levels by using Me RIP?Methylated RNA Immunoprecipitation?and RT-q PCR methods in 5 pairs of PDGA and normal adjacent tissues.The correlation between the m6 A modification levels and the expression levels was evaluated by Pearson correlation analysis.Result1.Circular RNA microarray analysis identified 30 high-expressing and 35low-expressing DECs in PDGA.2.The 5 selected DECs from the PDGA expression profile have been verified in gastric cancer tissues and cell lines,the results were generally consistent with the profile.However,compared with the normal gastric mucosal epithelial cells,hsa_circ₀102434 was low expressed in gastric cancer cell lines,which is contrary to the results of the expression profile.3.The receiver operating characteristic curve showed that hsa_circ₀077837 has the largest area under the curve,which is the best candidate for diagnosis of gastric cancer and PDGA.4.The results of GO enrichment analysis and KEGG pathway analysis suggest that DEGs may play an important role in the tumorigenesis and progression of PDGA.5.Of the 14 DECs predicted by the SRAMP website,all of them were identified with m6 A modification sites by Me RIP and RT-q PCR.Most of the DECs' m6 A modification levels were consistent with their expression levels in PDGA tissues.Pearson correlation analysis showed that 6 DECs' m6 A modification levels were significantly correlated with their own expression level in PDGA.ConclusionThe study revealed a series of DECs in PDGA,of which hsa_circ₀077837 could be used as a promising clinical diagnostic marker for GC and PDGA.Moreover,the alterations in m6 A modification levels of DECs in PDGA provided new insight into the molecular mechanism of PDGA tumorigenesis and progression.