Abstract
Background & AimsTo clarify the biological roles, circularization process and secretion pathway of circRHOBTB3 in colorectal cancer (CRC) progression.MethodsWe performed a comprehensive analysis of circRNA levels in serum exosomes from multiple types of cancer patients in public databases and verified the higher level of circRHOBTB3 in CRC sera versus healthy donors by RT-qPCR. Then, the function of circRHOBTB3 in CRC was investigated in vitro and in vivo. RNA-seq and RNA pull-down assays together with mass spectrometry identified the downstream signals and the binding proteins of circRHOBTB3. Finally, Antisense oligonucleotides (ASOs) were designed to target circularization and secretion elements of circRHOBTB3 for CRC therapy.ResultscircRHOBTB3 levels were increased in the sera but was downregulated in tissue samples in CRC, and the downregulation was associated with poor prognosis. Furthermore, circRHOBTB3 acts a tumor-suppressive circRNA by repressing metabolic pathways, intracellular ROS production in CRC. Several key elements were discovered to regulate circRHOBTB3 circularization and exosomal secretion. Moreover, SNF8 was identified that sorts circRHOBTB3 into exosomes. Interestingly, we found that CRC cells could actively secrete more circRHOBTB3 than normal cells. According to the sequence of regulatory elements for circularization and exosomal secretion, we designed and synthesized ASOs, which increased circRHOBTB3 expression and blocked circRHOBTB3 exosomal secretion. More importantly, ASOs could inhibit CRC growth and metastasis in vitro and in vivo.ConclusionscircRHOBTB3 plays a tumor-suppressive role in CRC and has to be excreted out of cells to sustain cancer cell fitness. ASOs targeting regulatory elements for circularization and exosomal secretion will become a novel antitumor strategy.
Highlights
Circular RNAs comprise a large class of noncoding RNAs without a 5′ terminal cap and a 3′ terminal poly (A) tail that are produced by RNA polymerase II transcription through back-back splicing to form covalently closed RNA circles [1]
Results circRHOBTB3 is highly expressed in exosomes but downregulated in tumor tissues First, we reanalyzed the data of 4 GEO datasets (GSE100063, GSE100206, GSE100207, GSE100232), including total RNA-seq data of serum exosomes from 12 colorectal cancer (CRC) patients, 21 hepatocellular carcinoma (HCC) patients, 32 pancreatic adenocarcinoma (PAAD) patients, and 14 healthy donors, in the same sequencing platform (Fig. S1A)
The results showed a higher circRHOBTB3 level in the serum of CRC patients than in that of healthy donors, which was consistent with the database (Fig. 1G)
Summary
Circular RNAs (circRNAs) comprise a large class of noncoding RNAs without a 5′ terminal cap and a 3′ terminal poly (A) tail that are produced by RNA polymerase II transcription through back-back splicing to form covalently closed RNA circles [1]. The process of circRNA splicing is regulated by multiple factors, including the transcriptional elongation rate, multiple cis-trans regulatory sites, splicing complexes, and cis- or trans-acting elements [2]. Recent advances implicate these novel circRNAs in various biological processes, such as cell proliferation, adhesion, apoptosis and survival [3]. An increasing number of studies have reported aberrant circRNAs in breast, lung and colorectal cancer [4], the biological function, molecular mechanism and circularization patterns of circRNAs in tumor cells are still unclear. Endosomal sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways have been reported to regulate exosomes to sort cargos. To clarify the biological roles, circularization process and secretion pathway of circRHOBTB3 in colorectal cancer (CRC) progression
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