| BackgroundPatients with Syphilis serofast refers to part of patients remain low titer of rapid plasma region test after regular treatment. during the current clinical work, they may be given standard benzathine penicillin treatment in accordance with the level of RPR titer. However, we still lack the efficacy research of benzathine penicillin therapy in patients with serofast. Now the pathogenesis of serofast was not clear, it may closely related with the immune suppression. MicroRNAs is a length of about 21 bp non-coding negative regulation of RNA molecules, involved in many important physiological and pathological pathways, including inflammation, tissue repair, apoptosis and so on. Pathogens can use microRNA gene transcription regulation to replicate and escape from the host immune system. The current research has found that microRNA plays an important role in the processin mycobacterium tuberculosis, helicobacter pylori and other infectious disease, but studies of treponema pallidum is still blank. At first, from the clinical point of view, we explore the curative effect of benzathine penicillin for the patients with syphilis serofast. Secondly, from the perspective of functional genomics, use the Illumina sequencing technologies to screen and identified specific microRNA expressionin in patients with secondary syphilis and serofast Peripheral blood mononuclear cells(Peripheral blood mononuclear cell, PBMC) and through molecular biology technology research with the target genes and between control network and mechanism of action, and discussed the mechanism of syphilis serofast, provide valuable ideas to the final solution for syphilis serofast.ObjectsDemographic, clinical, and laboratory data of 697 late latent syphilis patients and 971 latent syphilis of unknown duration were retrospectively analyzed. All syphilitic cases were outpatients who visited the STD clinic of Peking Union Medical College Hospital, China, from January 2001 to January 2012. Clinical data included stage of diagnosis, symptoms present at the time of diagnosis, treatment information, follow-up clinical symptoms and lab testing. We choosed and analyzed the records of 70 patients with syphilis serofast retrospectively and the patients were divided into treatment by benzathine penicillin therapy 2.4 million units group (n= 35) and without any treatment as control group (n= 35). The mean age was 30 years old, there was no significant difference in the sex and age.Collected 28 cases of secondary syphilis.27 cases of patients serofast and 40 healthy volunteers during June 2013-2014 in our hospital. there was no significant difference in the sex and age. All patients met the criteria for the diagnosis of syphilis in the "People’s Republic of China infectious disease prevention and control". And except during pregnancy or breast-feeding women, HIV antibody positive, nerve syphilis and tuberculosis, autoimmune diseases, cancer, chronic liver disease, kidney disease and other systemic diseases.Methods1. Retrospective analysis of our sexually transmitted disease center in 2001-2013, with complete follow-up records of 70 patients with syphilis serofast, it can be divided into with treatment by benzathine penicillin therapy 2.4 million units group (n= 35) and without any treatment as control group (n= 35). After retreatment for 12 months, patients were reviewed by RPR titer to view results. The treatment is valid by means of the RPR drop 2 degree or higher, and the RPR fall< 2 degree of decision results as invalid.2. Select five cases of secondary syphilis and syphilis serofast and healthy volunteers as a control group during June 2013-2014 June period in our hospital dermatology center. We use Illumina HiSeq sequencing method for PBMC samples of 15 patients to study microRNA expression profiles and analysis the differentially genes, Gene Ontology (GO) and KEGG function. At the same time, we expand more samples, collected 23 cases of patients with secondary syphilis,22 cases of syphilis serofast and 35 healthy volunteers PBMC samples to confirm the expression of microRNA by real-time fluorescence quantitative PCR (Real time-PCR, RT-PCR) validation.Results1. In the benzathine penicillin treatment group,26 patients was effective, but 9 patients were still serofast. the effective rate was 74.2%; In the no treatment control group,28 patients were effective,7 patients were still invalid, the effective rate was 80%, the difference was not statistically significant (P> 0.05).2. All 15 samples were success to go through the Illumina HiSeq sequencing. Among the Secondary/serofast syphilis and normal control group, microRNAs expression level between the two groups(1) base on the Ratio>1 and P<0.05, identified 146 differentially differentially expressed microRNAs between the secondary syphilis and normal control group, increase 54 kinds of microRNAs and decrease of 92; 111 differentially differentially expressed microRNAs between serofast group and normal control group, increase 61 kinds of microRNAs and decrease of 50; 142 differentially differentially expressed microRNAs between the secondary syphilis and serofast group, increase 88 kinds of microRNAs and decrease of 54; (2) based on the Ratio≥2 和 P≤0.05, identified 58 differentially differentially expressed microRNAs between the secondary syphilis and normal control group, increase 33 kinds of microRNAs and decrease of 25; 53 differentially differentially expressed microRNAs between serofast group and normal control group, increase 40 kinds of microRNAs and decrease of 13; 49 differentially differentially expressed microRNAs between the secondary syphilis and serofast group, increase 32 kinds of microRNAs and decrease of 17. At the same time we predict 657 kinds of new microRNAs.3. Enlarge the samples,35 cases of normal collection,18 patients with secondary syphilis and 17 patients with syphilis serofast peripheral blood mononuclear cells, and the results show significant difference in 13 mircroRNAs, miR-299-3p, miR-451-a, miR-548-3j-3p, miR-654-5p, miR-3135b, miR-338-5p, Novel-5, Novel-62, Novel-73, Novel-124, Novel-128, Novel-163 and Novel-244 by Real time PCR analysis, we found expression of Novel-128 and Novel-224 in the secondary syphilis and syphilis serofast in PBMC were lower, Novel-163 in the secondary syphilis is decreased, expression of Novel-5 was higher, miR-338-5p only elevated in patients with syphilis serofast.4. Based on the GO and KEGG enrichment analysis results show that difference expression of target genes regulated by microRNA between the secondary syphilis and serofast patients, involved in metabolism (amylaceum, protein modification), the growth (positive and negative regulation of cell proliferation and proliferation, cell cycle), differentiation, cell apoptosis), various functions such as signal transduction; At the same time, these target genes involved in pathways through:metabolic pathways (cholesterol biosynthesis, glycogen synthesis pathway, fatty acid synthesis, purines and pyrimidines), T cell receptor signaling pathway, cell growth, cell cycle, connection way (adhesion connection and close connection), the cell cycle, cell adhesion, etc.5. Novel-224 and miR-338-5p were positively correlated, Novel-5 and Novel-128, miR-338-5p and Novel-163 were also exists function correlation.Conclusion1. We use benzathine penicillin to retreat the late latent syphilis and unknown duration latent syphilis serofast,26 of 35 cases were effective, but the control group also have 28 cases were effective (P>0.05), so the effectiveness of benzathine penicillin remains to be reach, the results also showed that the RPR of patients with syphilis can gradually decrease over time.2. The microRNA expression profiles of patients with syphilis serofast in peripheral blood mononuclear cells were specific, miR-338-5p, Novel-163, Novel-128, Novel-244 and Novel-5 differentially expressed in patients with syphilis serofast and related to each other, it can be used as the evaluation index of the disease.3. miR-338-5p, Novel-5, Novel-163, Novel-128, Novel-244 and its target genes related to biological functions of secondary syphilis, such as T cell receptor signaling pathway, metabolism.
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