Transcriptome-wide map of m⁶A circRNAs identified in hypoxic pulmonary hypertension rat model

Abstract

32Hypoxic pulmonary hypertension (HPH) is a lethal disease. CircRNAs and m 6 A 33 circRNAs have been reported to be associated with cancer progression, but the 34 expression profiling of m 6 A circRNAs has not been identified in HPH. This study was 35 to investigate the transcriptome-wide map of m 6 A circRNAs in HPH. In this study, 36 hypoxia-induced PH rat model was established. Total RNA was extracted and purified 37 from lungs of rats, then circRNAs were detected and annotated by RNA-seq analysis. 38 m 6 A RNA Immunoprecipitation (MeRIP) was performed following rRNA depletion, 39 and RNA-seq library was constructed. CircRNA-miRNA-mRNA co-expression 40 network was also constructed. In vitro, total m 6 A was measured. m 6 A circXpo6 and 41 m 6 A circTmtc3 were detected in pulmonary artery smooth muscle cells (PASMCs) and 42 pulmonary artery endothelial cells (PAECs) exposed to 21% O 2 and 1% O 2 for 48 h, 43 respectively. m 6 A abundance in 166 circRNAs was significantly upregulated and m 6 A 44 abundance in 191 circRNAs was significantly downregulated in lungs of HPH rats. 45 m 6 A abundance in circRNAs was significantly reduced in hypoxia in vitro. m 6 A 46 circRNAs were mainly derived from single exons of protein-coding genes. m 6 A 47 influenced the circRNA-miRNA-mRNA co-expression network in hypoxia. m 6 A 48 circXpo6 and m 6 A circTmtc3 were downregulated in hypoxia. In general, our study 49 firstly identified the transcriptome-wide map of m 6 A circRNAs in HPH. m 6 A level in 50 circRNAs was decreased in lungs of HPH rats and in PASMCs and PAECs exposed to 51 hypoxia. Downregulated or upregulated m 6 A level influenced circRNA-miRNA-52 mRNA co-expression network in HPH. Moreover, we firstly identified two 53 downregulated m 6 A circRNAs in HPH: circXpo6 and circTmtc3. We suggested that 54 m 6 A circRNAs may be used as a potential diagnostic marker or therapy target in the 55 future.56 57 58 59 60 3 61 62 Author summary 63 HPH is a disease with great morbidity and mortality. It is often caused by chronic 64 hypoxic lung diseases, such as chronic obstructive pulmonary disease and interstitial 65 lung diseases. It lacks effective therapy methods so far. CircRNAs are a type of non-66 coding RNAs and can be used as biomarkers because they are differentially enriched in 67 specific cell types or tissues and not easily degraded. m 6 A is identified as the most 68 universal modification on non-coding RNAs in eukaryotes. CircRNAs can be modified 69 by m 6 A. m 6 A circRNAs in HPH is not well understood yet. Here we identify the 70 transcriptome-wide map of m 6 A circRNAs in HPH. We elucidate that m 6 A level in 71circRNAs is decreased in lungs of HPH rats and in PASMCs and PAECs exposed to 72 hypoxia. We find that downregulated or upregulated m 6 A level influences circRNA-73 miRNA-mRNA co-expression network in HPH. Moreover, we are the first to identify 74 two downregulated m 6 A circRNAs in HPH: circXpo6 and circTmtc3. We suggest that 75 m 6 A circRNAs may be used as a potential diagnostic marker or thera...

Role of m6A RNA methylation in cardiovascular disease (Review)

“…RIP-PCR confirmed that the expression of circXpo6 and circTmtc3 were significantly decreased in both hypoxia-induced pulmo-nary artery smooth muscle cells and endothelial cells ( 110 ). Collectively, the study ( 110 ) first revealed that m 6 A affected the stability of circRNAs, subsequently influencing the circRNA-miRNA-mRNA network, leading to the activation of the Wnt and FoxO signaling pathways and ultimately promoting HPH. The results of that study ( 110 ) suggested that m 6 A-modified circRNAs were associated with pulmonary hypertension.…”

“…GO and Kyoto Encyclopedia of Genes and Genomes pathway analysis identified different host genes of circRNAs with a hyper- and hypomethylated m 6 A level. m 6 A-modified cicRNAs tended to decrease in hypoxia ( 110 ).…”

“…A circRNA-miRNA-mRNA network was previously constructed to explore the regulation of gene expression in HPH. Key mRNAs associated with the Wnt ( 112 ) and FoxO signaling pathways ( 113 ), and miRNAs reported to be involved in HPH were filtered, and the most enriched novel circRNA Xpo6 and Tmtc3 were then identified ( 110 ). RIP-PCR confirmed that the expression of circXpo6 and circTmtc3 were significantly decreased in both hypoxia-induced pulmo-nary artery smooth muscle cells and endothelial cells ( 110 ).…”

“…Key mRNAs associated with the Wnt ( 112 ) and FoxO signaling pathways ( 113 ), and miRNAs reported to be involved in HPH were filtered, and the most enriched novel circRNA Xpo6 and Tmtc3 were then identified ( 110 ). RIP-PCR confirmed that the expression of circXpo6 and circTmtc3 were significantly decreased in both hypoxia-induced pulmo-nary artery smooth muscle cells and endothelial cells ( 110 ). Collectively, the study ( 110 ) first revealed that m 6 A affected the stability of circRNAs, subsequently influencing the circRNA-miRNA-mRNA network, leading to the activation of the Wnt and FoxO signaling pathways and ultimately promoting HPH.…”

“…Collectively, the study ( 110 ) first revealed that m 6 A affected the stability of circRNAs, subsequently influencing the circRNA-miRNA-mRNA network, leading to the activation of the Wnt and FoxO signaling pathways and ultimately promoting HPH. The results of that study ( 110 ) suggested that m 6 A-modified circRNAs were associated with pulmonary hypertension. However, the research was just an expression file of circRNAs in lung tissues from HPH and control mice.…”