Circulating microRNAs (miRNAs) are receiving attention as potential biomarkers of various diseases, including cancers, chronic obstructive pulmonary disease, and cardiovascular disease. However, it is unknown whether the levels of circulating miRNAs in a healthy subject might vary with external factors in daily life. In this study, we investigated whether cigarette smoking, a habit that has spread throughout the world and is a risk factor for various diseases, affects plasma miRNA profiles. We determined the profiles of 11 smokers and 7 non-smokers by TaqMan MicroRNA array analysis. A larger number of miRNAs were detected in smokers than in non-smokers, and the plasma levels of two thirds of the detected miRNAs (43 miRNAs) were significantly higher in smokers than in non-smokers. A principal component analysis of the plasma miRNA profiles clearly separated smokers and non-smokers. Twenty-four of the miRNAs were previously reported to be potential biomarkers of disease, suggesting the possibility that smoking status might interfere with the diagnosis of disease. Interestingly, we found that quitting smoking altered the plasma miRNA profiles to resemble those of non-smokers. These results suggested that the differences in the plasma miRNA profiles between smokers and non-smokers could be attributed to cigarette smoking. In addition, we found that an acute exposure of ex-smokers to cigarette smoke (smoking one cigarette) did not cause a dramatic change in the plasma miRNA profile. In conclusion, we found that repeated cigarette smoking substantially alters the plasma miRNA profile, interfering with the diagnosis of disease or signaling potential smoking-related diseases.

MicroRNAs (miRNAs) post-transcriptionally regulate mRNA expression, controlling global cell function. Altered expression or function of miRNAs causes various diseases. Chemically induced changes in miRNA expression in human tissues are not fully understood. We investigated the changes in miRNA expression by rifampicin, which modulates the expression of various genes related to drug metabolism and pharmacokinetics, in human hepatocytes, and evaluated the relationship with the gene expression changes. We found that 23 miRNAs were increased (>2-fold) and 17 miRNAs were decreased (<0.5-fold) among 150 detected miRNAs, whereas 60 genes were increased and 105 genes were decreased among 22,673 detected genes upon treatment with 10 µM rifampicin. Changes in 17 intragenic miRNAs out of 40 altered miRNAs did not occur in parallel with alterations in their host genes. We searched for the target mRNAs of the miRNAs altered by rifampicin and found that the changes in expression of 16 mRNA/miRNA pairs were inversely associated. Thus, some mRNA expression altered by rifampicin may result from miRNA regulation. In conclusion, we found that rifampicin altered miRNA expression in human hepatocytes. We obtained new insight on the mechanism of the miRNA expression changes and the complicated relationship with gene transcripts.

The profiling of the selected miRNAs can be useful to distinguish different types of liver diseases.

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SDS software v.2.4. The expression levels were evaluated using the comparative cycle 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 Statistical analysisThe data are expressed as the means ± SD. The comparisons of two groups were made using Mann-Whitney's U-test. A value of P < 0.05 was considered statistically significant. Results ALT, AST, and histopathology of the liverAfter the administration of APAP and MP, the plasma ALT and AST levels were significantly elevated (Fig. 1A). The histopathological analysis revealed that APAP caused hepatocellular necrosis at the pericentral regions, whereas MP caused hepatocellular necrosis at the periportal region (Fig. 1B). These two models were used for the subsequent studies. miRNA expression at the pericentral and periportal regions of the liverThe miRNA expression at pericentral and periportal regions of the liver was determined by TaqMan microRNA array analysis. The numbers of the detected miRNAs and the miRNAs whose expression exceeded the cutoff (Ct < 32) are shown in Table 1. No large difference was observed in these numbers between the pericentral and periportal regions within a group or between the treated and control groups. Among the miRNAs with Ct values less than 32 in all of the groups, 125 miRNAs were common to all of the groups; therefore, these 125 miRNAs were used for the subsequent analyses. Fig. 2 shows the heat maps of the expression of the 125 miRNAs in each sample; note that the miRNAs are ordered based on descending expression level in the pericentral region of the control rats. The top three miRNAs that 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 7 exhibited high expression in both the pericentral and periportal regions in all of the samples are miR-709, miR-122, and miR-720. The comparison of the miRNA levels between the control rats and fasted-control rats revealed that 36 miRNAs presented higher levels and eight miRNAs exhibited lower levels at the pericentral region of the fasted-control rats (Tables 1 and 2). At the periportal region, 52 miRNAs and two miRNAs presented higher and lower levels, respectively, in the fasted-control rats compared with the control rats. Among these miRNAs, 17 miRNAs, which are shown in bold, were found in both the pericentral and periportal regions. Thus, the results demonstrate that fasting affects the expression of some miRNAs in the liver. The comparison of the miRNA levels between the pericentral and periportal regions in control rats showed that 27 miRNAs exhibited higher expression levels in the pericentral region and that 22 miRNAs presented higher expression levels in the periportal region compared with t...