Exclusive heat-not-burn cigarette smoking alters the profile of circulating microRNAs

Background Traditional combustion cigarette (TCC) smoking is an established risk factor for several types of cancer and cardiovascular diseases. Circulating microRNAs (miRNAs) represent key molecules mediating pathogenetic mechanisms, and potential biomarkers for personalized risk assessment. TCC smoking globally changes the profile of circulating miRNAs. The use of heat-not-burn cigarettes (HNBCs) as alternative smoking devices is rising exponentially worldwide, and the circulating miRNA profiles in chronic HNBC smokers are unknown. We aimed at defining the circulating miRNA profile of chronic exclusive HNBC smokers, and identifying potentially pathogenetic signatures. Methods Serum samples were obtained from 60 healthy young subjects, stratified in chronic HNBC smokers, TCC smokers, and Non Smokers (20 subjects each). Three pooled samples per group were used for small RNA sequencing, and the fourth sub-group constituted the validation set. Findings Differential expression analysis revealed 108 differentially expressed miRNAs; 72 exclusively in TCC, 10 exclusively in HNBC, and 26 in both smoker groups. KEGG pathway analysis on target genes of the commonly modulated miRNAs returned cancer and cardiovascular disease associated pathways. Stringent abundance and fold-change criteria nailed down our functional bioinformatic analyses to a network where miR-25-3p and miR-221-3p are main hubs. Interpretation Our results define for the first time the miRNA profile in the serum of exclusive chronic HNBC smokers and suggest a significant impact of HNBCs on circulating miRNAs. Funding Sapienza University of Rome grant 2019, # RG11916B8621D7FF to GBZ and grant 2021 # RM12117A5D7688BC to RC. IC is supported by grants # RM12117A805ED2FD and # RG11916B85CDBF76 from Sapienza University of Rome, and by grant # RG1221816BC8E766 from the Italian Health Ministry. FP is supported by Grant # A0375-2020-36621 from Regione Lazio (POR-FESR 2014-2021). ### Competing Interest Statement Giuseppe Biondi-Zoccai has consulted for Amarin, Balmed, Cardionovum, Crannmedical, Endocore Lab, Eukon, Guidotti, Innovheart, Meditrial, Microport, Opsens Medical, Terumo, and Translumina, outside the present work. All other authors report no conflict of interest. ### Funding Statement Sapienza University of Rome grant 2019, # RG11916B8621D7FF to GBZ and grant 2021 # RM12117A5D7688BC to RC. IC is supported by grants # RM12117A805ED2FD and # RG11916B85CDBF76 from Sapienza University of Rome, and by grant # RG1221816BC8E766 from the Italian Health Ministry. FP is supported by Grant # A0375-2020-36621 from Regione Lazio (POR-FESR 2014-2021). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics Committee of Policlinico Umberto I - Sapienza University of Rome gave approval for this work (protocol number 813/14). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are either contained in the manuscript or available at GEO.