Background. Traditional combustion cigarette (TCC) smoking still is a major risk factor for several types of cancer and cardiovascular diseases. Circulating microRNAs represent key molecules mediating pathogenetic mechanisms, and potential biomarkers for personalized risk assessment. Several transcriptomics studies showed that TCC smoke exposure globally changes the profile of circulating miRNAs, both in the presence or absence of co-morbidities. The use of heat-not-burn cigarettes (HNBCs) as novel smoking devices is rising exponentially worldwide, with still unknown long-term effects on health. Comprehensive data on the circulating miRNA profile in chronic HNBC smokers is still lacking. Purpose. To define for the first time the profile of circulating miRNAs in serum samples of chronic exclusive HNBC smokers, and to identify potentially pathogenetic circulating miRNAs signatures. Methods. Serum samples were obtained from subjects enrolled in the SUR-VAPES CHRONIC trial, including 3 groups of 20 healthy young subjects, stratified as chronic HNBC smokers (mean exclusive use=1.5 years), TCC smokers, and non-smokers (NS). 4 sub-groups of 5 subjects were randomly created (tested for non-statistical differences in available anthropometric and clinical parameters). 3 sub-groups were used to create 3 pooled samples per group for small RNA sequencing; the fourth sub-group constituted the validation set. Data were analyzed using DEseq2. Results. The principal component analysis showed a neat separation between NSs and both smoker groups, with a less pronounced difference between TCC and HNBC smokers sera. Differential expression analysis revealed 101 miRNAs significantly modulated between groups (Adj p<0.05). We found a global downregulation of circulating miRNAs with only 20 miRNAs upregulated in both groups of smokers. Head-to-head comparison revealed that 65 miRNAs were exclusively modulated in TCC, 26 miRNAs were commonly modulated in both smoker groups, and 10 miRNAs were exclusively modulated in HNBC samples. Most miRNAs of the HNBC group displayed an intermediate level between NS and TCC groups. KEEG pathway analysis on target genes of significantly modulated miRNAs with high counts (cutoff>500 norm DESeq counts) revealed a relevant number of cancer-associated pathways, followed by signaling and cardiovascular disease pathways. Although TCC smokers presented a significantly higher number of dysregulated miRNAs in their sera, the target and pathway analyses returned overlapping terms for both commonly deregulated miRNAs and those deregulated only in one of the smoker groups. Conclusions. Our results define for the first time a global miRNA profile in the serum of exclusive chronic HNBC smokers. Despite the globally lower extent of the differences observed, data suggest a significant impact of HNBCs on circulating miRNAs with overlapping of predicted target pathways compared to the miRnome of TCC smokers.

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 profile of chronic HNBC smokers is 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 nonsmokers (20 subjects each). Three pooled samples per group were used for small RNA sequencing, and the fourth subgroup constituted the validation set. Results: 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. Conclusion: 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.