This is the first report of differently expressed miRNAs derived from plasma exosomes in serofast syphilis. Syphilis is a classic chronic and contagious disease with a long history. However, the diagnosis and prognosis of serofast syphilis are challenging issues in clinical settings. Therefore, we focus on exosomal miRNA derived from plasma, which may be a new and potential method for effective diagnosis.
Exosomes are biological nanovesicles composed of a lipid bilayer and known for cell-to-cell communication. Exosomes have been declared to be present in almost all bodily fluids,such as blood, milk, urine, saliva, and cerebrospinal fluid. They are capable to deliver bioactive substances such as protein, lipids, and various RNAs under normal and pathological conditions. Liquid biopsy represents a great advance in the biomarker field because it is less invasive, less time-consuming, and safer compared to classical biopsies. Among potential biomarkers detectable in liquid biopsies, microRNAs (miRNAs) are gaining more and more attention, since they are easily detectable, quite stable in biological fluids, and show high sensitivity. Compared to mRNAs, detection of miRNAs in peripheral blood is more accurate and reliable than in other body fluids(28). Consequently, miRNAs are considered to be novel biomarkers for various types of diseases, and like the messenger molecules to involute in convoluted cell biological changes and immune responses to pathogens. (29; 30; 31). This study aimed to explore a set of exosomal miRNAs in peripheral blood, and we found that miR-197-3p, miR- 1908-3p, miR-1273g-3p, miR-4485-5p were significantly upregulated in seofast patients.
Prior to our study, also for the exosomal microRNA, a study has been carried out on neurosyphilis(32). Exosomal miRNAs are derived from serum and cerebrospinal fluid, and the results show that miR-590-5p, miR-570-3p, and miR-570-5p are significantly upregulated in patients with neurosyphilis in both cerebrospinal fluid and serum. The other kinds of research(33; 34) focus on miRNAs from peripheral blood mononuclear cells that serve as novel biomarkers for syphilis diagnosis, especially for serofast diagnosis. MiR-223-3p and miR-195-5p from peripheral blood mononuclear cells varied from patients with primary syphilis or serofast syphilis to healthy control, despite the difficulty of distinguishing latent syphilis from the serofast state(35). In addition, known and novel predicted (np)-miRNAs in peripheral blood mononuclear cells in syphilitic patients with serofast status were identified and verified, such as miR-338-5p, np-miR-163, np-miR-128, np-miR-244, and np-miR-5(36). It is obvious that exosomal miRNAs from serum and cerebrospinal fluid in neurosyphilis and miRNAs from peripheral blood mononuclear cells in serofast are distinctive from exosomal microRNA from plasma in serofast, and it is hard to find the same differentially expressed miRNAs from each other. This may be explained by the difference in the selection of subjects. Xiao-Yong Man’s study exosomal miRNAs in the serum and CSF in patients with and without neurosyphilis, while neurosyphilis is excluded in our study. Furthermore, patients with individual differences contribute to the different results. This is worth further research.
Previous studies indicated that the serofast was possibly associated with latent infection (37), such as the special molecular subtypes of TP repeat gene found in serofast(38), which was admittedly associated with the immune escape of pathogens and chronic infection of syphilis (39; 40). The suppression and disorders of the host immune function have been observed in serofast patients (39; 40). Thus, we are interested in the role which DEmiRNAs play in the pathogenesis of serofast. Our study reveals that a number of miRNAs regulated the host immune response, and as one of the highest differentially expressed miRNAs in the circulating exosomes, miR-197-3p may play a crucial regulatory role in the host immune system in serofast syphilis. To uncover the potential roles of specific miRNA in the occurrence of serofast, we analyzed the relationship between the differentially expressed miRNAs and their adjacent protein-coding genes. Several studies have revealed the potential function of miR-197-3p, for example, Huang Q, et al. identified that hsa-miR-197-3p regulates the VDAC1/AKT/β-catenin signaling axis to repress the proliferation of prostate cancer (41). Li Qin et al. investigated that miR-197-3p reduces epithelial-mesenchymal transition by targeting ABCA7 in ovarian cancer cells(42). Li Y, et al. confirmed that miR-197-3p targets IGF1R and BCL2 and regulates endothelial cell proliferation and migration in Kawasaki disease(43). Tavukcuoglu Z, et al. revealed that familial Mediterranean fever-related miR-197-3p targets the IL1R1 gene and modulates inflammation in monocytes and synovial fibroblasts(44). All of the facts indicate that hsa-miR-197-3p may be an important candidate molecule for the clarification of the pathogenesis of serofast. Further investigation of the differential exosomal miRNAs to identify their role in the development of the serofast state would be deserved.
Presently, it is still an inconsistent definition of the serofast, and the main difference is about the fellow-up time. 6 and 12 months after treatment for primary and secondary syphilis and 24 months for early latent syphilis for fellow-up is recommended in USA(45). In UK, recommended clinical and serological (RPR or VDRL) follow-up is at three, six and 12 months, then if indicated, six monthly until VDRL/RPR negative or serofast(46). Nevertheless, Chinese diagnostic criteria for syphilis suggested that patients were considered to be in a serofast state after 12 months of recommended therapy for primary syphilis, 2 years for secondary syphilis and 3 years for late stage of syphilis if their nontreponemal test remained positive and the titres neither increased nor decreased by at least four-fold (two dilutions)(47). For practical reasons, the collection of this study was based on the Chinese diagnostic criteria for syphilis, and the fellow-up time of serofast patients were at least 12 months.
Finding from our study showed a less variation between serofast with healthy control. For this reason, we did not further analyze the differentiated miRNAs between serofast and healthy control. Comparison with serofast and serologically cured syphilis patients may be more significant for their various condition after treatment. According to the microarray, one sample from serofast was odd and then excluded when we analyzed the differentially expressed miRNAs between the serologically cured groups and serofast state groups by the analysis of clustering heatmap (Fig. 2).
The detection of circulating exosomal miRNAs is a noninvasive method for disease diagnosis, especially the lipid bilayer protecting the content of exosome itself. Our findings showed that plasma exosome-derived miR-197-3p, miR- 1908-3p, miR-1273g-3p, miR-4485-5p are potential biomarkers for the diagnosis of serofast. ROC curve analyses showed that miR- 1908-3p alone had a higher diagnostic accuracy, sensitivity, and specificity than other miRNAs in distinguishing healthy controls from patients with seofast syphilis, with an AUC value of 0.901, sensitivity of 92.3%, and specificity of 91.7%. When combined, these four miRNAs showed a stronger capacity to differentiate between patients with serofast syphilis from serological cured patients or healthy people, presenting an AUC value of 0.904–0.949, sensitivity of 92.3%-100%, and specificity of 83.3%- 91.7%. This result is important in terms of serofast syphilis diagnosis because of the vague definition of serofast and the long time for fellow-up(45) (47).
In summary, our data demonstrated that plasma exosomal miR-197-3p, miR- 1908-3p, miR-1273g-3p, miR-4485-5p were differentially expressed in syphilis patients with serofast state. MiR- 1908-3p could be useful for distinguishing healthy controls from patients with seofast syphilis. Combined four miRNAs have the potential for use as biomarkers for discriminating patients with serofast state from healthy controls, and patients with serological cure. The major limitation of this study was that the number of participants was too small to verify the diagnostic value of these candidate miRNAs. Therefore, further studies and clinical trials are necessary for developing new diagnostic methods.