Mechanism Of Persistent Infection And Promotion Of Tumor Progression Of Fusobacterium Nucleatum Mediated By MiR-31

Fusobacterium nucleatum（F.nucleatum）is a Gram-negative,obligate anaerobic bacterium located in human oral cavity.It plays an integral role in oral biofilm,and integrates other bacteria by secreting various adhesins.F.nucleatum could also be considered as a pathogen to cause a variety of infectious diseases in the oropharynx or the other parts,including pericarditis,brain abscess,appendicitis,osteomyelitis,pelvic inflammatory disease.Recent studies have shown that F.nucleatum enriched in colorectal cancer（CRC）tissues,affecting multiple stages of cancer progression,including tumor cell proliferation promotion,tumor immune escape,recurrence,and chemoresistance.F.nucleatum can act as a potential biomarker for the diagnosis and prognosis evaluation of CRC,and is also a potential target for the treatment of CRC.Among all malignant tumors,the incidence of CRC ranked third,and the mortality rate ranked second worldwide.Further study of Fusobacterium nucleatum is of great significance for understanding the molecular mechanism of the occurrence,metastasis and recurrence of CRC.There are various types and large numbers of microorganisms in the human intestinal tract,forming a complex environment.And the living conditions of F.nucleatum are harsh.Therefore,as an invasive bacteria,it is difficult for F.nucleatum to colonize in human intestinal tract.Autophagy can maintain cell homeostasis and plays a vital role in the body’s elimination of infectious pathogens.It is one of the important means of combating pathogenic microbial infections in the body’s innate immune response.Inhibition of autophagy will cause immune response disorder and cannot clear the infected pathogenic microorganisms,resulting in its persistent infection.After migrating into the human intestine,F.nucleatum can evade the clearance of the immune system through its unique mechanism,which is closely related to autophagy inhibition.Microbial infection is an important factor in tumor development.The persistent intracellular infection of F.nucleatum can active the tumor signaling pathway and promote the transformation of intestinal epithelial cells into tumor cells.The study of intracellular immune escape and tumor-promoting mechanisms of F.nucleatum can help us better understand the interaction between F.nucleatum and colorectal cancer,so as to achieve the purpose of precise prevention and treatment of CRC.In the early stage of this study,microarray screening of microRNA expression profiles revealed that microRNA-31（miR-31）and F.nucleatum play an important role in the persistent infection of colorectal epithelial cells and the promotion of tumorigenesis.On this basis,we intend to further study the molecular mechanism of miR-31 mediating the persistent infection of F.nucleatum in colorectal epithelial cells and promotion of tumor progression in cell infection model,animal infection model and tumorigenic model.【Objectives】1.To clarify the roles of miR-31 in persistent infection and promotion tumor progression of F.nucleatum.2.To clarify the mechanisms of autophagic flow inhibition via miR-31 after F.nucleatum infection.3.To clarify the mechanisms that miR-31 can promote colorectal cancer progression after F.nucleatum infection.【Methods】1.The effects of miR-31 in persistent infection and tumor progression of F.nucleatum.（1）qRT-PCR was conducted to analyse the abundance of F.nucleatum 16s RNA and miR-31 colorectal cancer clinical samples.The relationship between F.nucleatum,miR-31and colorectal cancer was analysed.（2）A F.nucleatum infection cell model was established with NCM460,and the intracellular colonization of F.nucleatum after infection was observed by transmission electron microscope.The growth curve of F.nucleatum was drawn to evaluate the function of miR-31 in cell proliferation.Cell migration and invasion experiments were conducted to demonstrate the effect of F.nucleatum infection on NCM460 cell migration and invasion.（3）A F.nucleatum infection mice model was established with C57BL/6,and the expression of cytokines in serum was evaluated by Lumnix while the HE staining of colorectal tissue was conducted to evaluate pathological damage.（4）A F.nucleatum infected mice model was established with APC^min/+.The mice were treated with F.nucleatum or miR-31 inhibitor adenovirus respectively after fading with AOM.The number,volume and survival rate were conducted to evaluate the effect of promoting CRC.2.The mechanismof inhibition of autophagic flow via miR-31 after F.nucleatum infection.（1）The level of autophagy was evaluated with laser confocal microscopy and WB.The amount of MAP1LC3B-II and SQSTM 1 was used to evaluate the autophagic flux.（2）The target genes of miR-31 were speculated via bioinformatics.The dual luciferase reporter assay was conducted to evaluate whether miR-31 can inhibit the expression of STX12.（3）The number of autophagy spots in cells was observed to determine autophagosome and lysozyme.Alexa Flour⁵⁵⁵ labeled lysosomal marker LAMP1,GFP labeled autophagosome marker LC3,and the fusion of intracellular autophagosomes and lysosomes were observed after knocking out STX12.（4）F.nucleatum infected mice model was established with C57BL/6.The mice were treated with F.nucleatum or miR-31 inhibitor adenovirus respectively.The pathological analysis of colorectal tissues and qPT-PCR were conducted to evaluate the role of miR-31in F.nucleatum infection.3.The mechanisms of tumor progression via RAS pathway after F.nucleatum infection.（1）RASA1 can be specifically regulated by miR-31 though the dual luciferase reporter assay.（2）The amount of RASA1 in tumor tissues and normal tissues was detected with WB.（3）The effect of miR-31 on cell proliferation after F.nucleatum infection was evaluated with CCK-8 assay.（4）The form of E-cadherin,F-actin and Tublin was observed with Laser confocal microscope after treating with miR-31 inhibitor and F.nucleatum.【Results】1.F.nucleatum can proliferate in host and induce a strong inflammatory response.（1）25 clinical specimens were analysed with gene microarray and 49 upregulated miRNAs were screened out.The abundance of F.nucleatum and miR-31 was elevated in tumor tissues.（2）miR-31 promotes intracellular colonization of F.nucleatum.（3）F.nucleatum infection can secrete a lot of pro-inflammatory cytokines and cause serious pathological damage in mouse colorectal tissues.（4）F.nucleatum infection can promote the formation of tumors in colorectal tissues in mice.2.F.nucleatum inhibits autophagic flow by upregulating the expression of miR-31.（1）F.nucleatum infection can increase the formation of autophagosomes in NCM460cells and the expression of MAP1LC3B-II and SQSTM1.Laser confocal observation showed that F.nucleatum infection can inhibit autophagic flow in cells via miR-31.（2）The target genes of miR-31 were speculated via bioinformatics.STX12 can be specifically regulated by miR-31 though the dual luciferase reporter assay.（3）Inhibition of STX12 can promote the intracellular colonization of F.nucleatum.（4）miR-31 can promote the colonization of F.nucleatum in the intestine of mice.3.F.nucleatum promotes colorectal cancer progression by miR-31-mediated RAS pathway activation.（1）miR-31 can inhibit the expression of RASA1 to activate the RAS pathway.The amount of RASA1 in tumor tissues is fewer than that in normal tissues.（2）miR-31 and F.nucleatum infection can significantly increase the proliferation of NCM460 and SW480.（3）miR-31 and F.nucleatum infection can promote the transformation of NCM460cells into mesenchymal cells,and the expression of cytoskeletal protein E-cadherin is significantly decreased;Tubulin ruptures to form a mass-like structure;F-actin structure is disordered.（4）F.nucleatum infection can improve the ability of migration and invasion of NCM460 cells.【Conclusion】（1）F.nucleatum can upregulate the expression of miR-31 in cells,mediating its persistent infection in colorectal epithelial cells and promoting tumor progr ession.（2）miR-31 inhibits autophagic flow by inhibiting the fusion of autophagosomes and lysosomes by STX12.（3）miR-31 activates RAS signaling pathway by inhibiting the expression of RASA1 to promote tumor progression.【Significance】In this study,we focused on miR-31 to explore the interaction pattern of"F.nucleatum infection,autophagy and tumor".It demonstrated that F.nucleatum infection can inhibit autophagic flow and promote cancer progression via miR-31.This study provides a theoretical basis for the clinical diagnosis,prognosis evaluation and precise prevention of colorectal cancer,and also a new idea for the study of other intracellular pathogenic microorganisms.