环状样RNA竞争内源性msRNAs调控变异链球菌胞外多糖 代谢和致龋性的作用及其机制研究

Exopolysaccharides play the crucial role in dental plaque cariogenic pathogenicity. Streptococcus mutans msRNA (microRNA- size small RNAs) which bind to the 5’ UTR region and results in inhibition of vicR transcription. This process could reduce the caries of dental plaque by interfering with the bacterial extracellular polysaccharide metabolism and biofilm phenotype construction. Based on the previous S. mutans noncoding RNAs research, the applicant approved for the first time: circular like RNAs exist in S. mutans which has the specific binding sites to msRNAs. However, the biological characteristics and effects of bacterial circular like RNAs as well as the relationship with msRNAs have been poorly understood. Therefore, the applicant hereby put forward the new hypothesis: the circular like RNAs could compete with endogenous msRNA which regulates the polysaccharides metabolism and dental plaque cariogenic pathogenicity. In order to verify the hypothesis, second-generation sequencing, structural analysis and co-immunoprecipitation techniques would be applied in this project. The goal of this study was to define: the biological characteristics of S. mutans circular like RNAs; the mechanism of circular like RNAs competing with endogenous msRNAs; compete with endogenous msRNAs; the circular like RNAs compete with endogenous msRNAs which regulates the polysaccharides metabolism and dental plaque cariogenic pathogenicity. This project would provide a new perspective for the post-transcriptional control of S. mutans non-coding RNAs. Furthermore, new anti-caries measures would be developed to achieve the effective prevention and treatment of dental caries.

胞外多糖是牙菌斑生物膜致龋的重要毒力因子。变异链球菌msRNAs抑制必需转录因子vicR转录，干预胞外多糖合成从而降低牙菌斑生物膜致龋性。申请人新发现和筛选到与变异链球菌msRNAs有特异性结合位点的环状样RNA。然而，细菌中环状样RNA的生物学功能及其与msRNAs的相互关系、效应、机制等均未见报道。据此，申请人提出环状样RNA可竞争内源性msRNAs从而调控变异链球菌胞外多糖代谢和致龋性的科学假说。本项目拟通过序列测定、结构分析、环状样RNA干扰与过表达、免疫共沉淀等技术：①探讨环状样RNA生物学功能及其竞争内源性msRNAs效应和机制；②阐明环状样RNA竞争内源性msRNAs调控胞外多糖代谢的分子机制；③解析环状样RNA在变异链球菌中调控致龋性强弱的效应。环状样RNA研究为变异链球菌非编码RNA调控提供新视角，为实现龋病的有效防治及新的防龋措施转化提供理论依据。

龋病属于微生物生态失衡性疾病，它的发生与牙菌斑生物膜沉积密切相关。变异链球菌是主要致龋菌，龋病的发生发展与细菌胞外多糖（葡聚糖）合成密切相关。变异链球菌msRNAs抑制必需转录因子vicR基因转录，干预胞外多糖合成从而降低牙菌斑生物膜致龋性。本项目在变异链球菌转录后调控层面，新发现和筛选到与变异链球菌msRNAs有特异性结合位点的环状样RNA，发现环状样RNA可差异性调控变异链球菌胞外多糖代谢，借助最主要的糖代谢通路vicRKX及其下游的rnc基因为影响因素，探讨变异链球菌环状样RNA生物学功能及其调控胞外多糖代谢的分子机制，研究发现：①环状样RNA分子circ4/6/7/8在添加蔗糖的培养条件下表达显著差异；②借助RNA近端连接技术验证vicR 5’UTR与msRNA1657的结合，免疫共沉淀技术验证ASvicR与msRNA1657的特异性结合；③变异链球菌环状样RNA circ 6/7对生物膜表型呈现差异性的调控效应；④过表达ASvicR显著抑制大鼠龋病发生。本课题较好地完成了研究任务，研究内容为变异链球菌非编码RNA调控提供新视角，为实现龋病的有效防治及新的防龋措施转化提供理论依据。