{"doi":"10.1101/2022.03.30.486482","title":"Substance P and adenosine signaling pathways regulate exosomal sorting of miR-21 in colonic epithelial cells","abstract":"Abstract Background & Aims MicroRNAs (miRNAs) are transported in body fluids within exosomes, and exosomal-miRNA sorting is highly selective. In colonic epithelial cells (CECs), Substance P-neurokinin-1-receptor (SP/NK1R) signaling regulates miR-21 sorting into secreted exosomes. Here, we studied the molecular mechanisms driving miR-21 sorting into colonic epithelial exosomes (CEEs) under SP-stimulation. Methods We performed studies with human colonic epithelial NCM460 cells overexpressing neurokinin-1-receptor (NCM460-NK1R) and in intestinal-epithelial-specific NK1R knockout mice. SP-regulated gene targets were validated by real-time polymerase chain reaction (RT-PCR) and immunoblotting. Small non-coding RNAs (sncRNAs) were isolated from NCM460-NK1R cells and secreted exosomes, and 3′-end adenylated and 3′-end uridylated fractions were separated. Cellular and exosomal sncRNA fractions were processed for miR-21 and miR-1307-3p expression and 3′-end adenylation to uridylation (A/U) ratios using RT-PCR. Pharmacological inhibition studies in NCM460-NK1R cells were performed in the presence of the Adenosine A2B receptor (ADORA2B) antagonist, PSB-1115, followed by RT-PCR and immunoblotting. Mass spectrometry validated in silico interacting partners of miR-21 in CECs. Results In CECs, miR-21 is predominantly polyuridylated under SP-stimulation and preferentially sorted to CEEs. SP/NK1R signaling activation upregulates extracellular adenosine (ADO) signaling via ADORA2B concomitant with reduced ADO uptake via epithelial-specific equilibrative nucleoside transporter-2 (ENT-2). Mass spectrometry and immunoblotting revealed upregulation of TUT7 in SP-stimulated CEEs. Knockdown of TUT7 decreased both TUT7 and miR-21 content in these exosomes. Pharmacological inhibition of ADORA2B in CECs resulted in decreased TUT7 and miR-21 in CEEs, regardless of SP stimulation. Conclusions SP/NK1R coupling in CECs activates ADORA2B and its downstream signaling cascade which mediates TUT7/miR-21 interaction and subsequent miR-21 polyuridylation and exosomal export. Synopsis Substance P-neurokinin-1 receptor signaling regulates terminal uridyl transferase7-mediated predominant 3’-end polyuridylation and exosomal recruitment of miR-21 in human colonic epithelial NCM460 cells overexpressing NK-1R via activation of Adenosine A 2B receptor and its downstream signaling cascade. Because Adenosine A 2B receptor signaling is involved in IBD pathogenesis, it could therefore be exploited as a pharmacological target for the therapeutic benefits in IBD.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2022,"id":307001,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9507,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":424051,"name":"Ivy Ka Man Law","orcid":"0000-0002-7430-1215","position":1,"is_corresponding":false},{"id":1001565,"name":"Amy K. Bugwadia","orcid":null,"position":2,"is_corresponding":false},{"id":733350,"name":"Jill M. Hoffman","orcid":null,"position":3,"is_corresponding":false},{"id":439511,"name":"Charalabos Pothoulakis","orcid":"0000-0002-5311-2634","position":4,"is_corresponding":false},{"id":806808,"name":"Sameena Wani","orcid":"0000-0002-3131-3603","position":0,"is_corresponding":true}],"reference_count":63,"raw_metadata":null,"created_at":"2026-07-19T00:32:52.703737Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}