Fusobacterium Nucleatum Promotes Epithelial-mesenchymal Transition In The Infected Oral Epithelial Cells Via LncRNA MIR4435-2HG/miR-296-5p/Akt2/SNAI1 Signal Pathway

Objective: Extensive epidemiological studies have found that approximately 20% of malignancies are associated with chronic infections.As a potential carcinogenic factor,the interaction between bacterial infection and host cells has become a focus of research on tumor molecular mechanisms.Epithelial-mesenchymal transition(EMT)refers to the cellular process that consists in the conversion of epithelial cell phenotype to a mesenchymal phenotype.Phenotypically,a disassembly of cell-cell junction,actin cytoskeleton reorganization and enhanced migration and invasion ability were observed in cells undergoing EMT.EMT plays a vital role in the progression of carcinogenesis.Bacterial infection promoting tumorigenesis probably via EMT has attracted extensive attention.The association between oral bacteria and tumor,especially oral squamous cell carcinoma(OSCC)is gaining attention and has become a research hotspot in tumor-related mechanisms investigation.Studies have found that Fusobacterium nucleatum is more enriched in OSCC tissues compared with normal oral tissues,suggesting that F.nucleatum may be involved in the carcinogenesis progression.F.nucleatum is an important putative periodontal pathogen that participates in the inflammatory disorders correlated with periodontal diseases.In addition,F.nucleatum infection has been found to be associated with a variety of extra-oral diseases,such as pre-term birth,respiratory tract infections and colorectal cancers.At present,a majority of researches focus on the association between F.nucleatum infection and CRC,and demonstrate that F.nucleatum promotes tumor development by inducing inflammation and immune response in the CRC microenvironment.The well-known adhesin Fad A from F.nucleatum can activate ?-catenin signaling by binding to E-cadherin and then stimulate cell proliferation.However,the regulatory role of F.nucleatum in occurrence of EMT or malignant transformation in oral epithelial cells and the underlying mechanisms remain largely unknown.Therefore,based on the above research background,the present study aims to verify the abundance of F.nucleatum in OSCC tissues and normal tissues and explore the effects of F.nucleatum infection on EMT and the probable mechanisms involved by establishing a model of F.nucleatum infection of oral epithelial cells.Methods: 1.We collected the OSCC tissues from the patients undergoing tumor resecting and the normal control tissues from the patients undergoing impacted tooth extraction.Fluorescence in situ hybridization(FISH)was applied to verify the different abundance of F.nucleatum in the two group tissue species.2.HIOECs and SCC-9 cells were used to establish the in vitro infection model by F.nucleatum ATCC25586 and S.gordonii Challis CH1 alone.CCK-8 method was used to detect the cell proliferation.Cell cycle distribution and cell apoptosis were analyzed by flow cytometry.Scratch assay was applied to investigate cell migration ability,and MMP-2 and MMP-9 expression in the cell supernatant were determined by gelatin zymography assay.3.HIOECs and SCC-9 cells were infected with F.nucleatum,and the epithelialmesenchymal transition markers E-cadherin,N-cadherin,Vimentin and SNAI1 were visualized by immunofluorescence staining assay.Additionally,the localization of Ecadherin at 2h to 12 h after F.nucleatum infection was observed.Changes in m RNA and protein levels of E-cadherin,N-cadherin,Vimentin and SNAI1 were detected by q RT-PCR and Western blot,respectively.Heat-inactivated F.nucleatum and purified Fad A protein at a concentration of 10 μg/m L were used to treat HIOECs and SCC-9 cells,respectively,and the m RNA expressions of the four EMT markers mentioned above were determined by q RT-PCR.4.High throughput sequencing was performed to identify the aberrantly expressed genes including lnc RNAs and m RNAs expressed by F.nucleatum-infected HIOECs.Cluster analysis and KEGG signal pathway analysis of some genes related to epithelial mesenchymal transition were performed,and several selected differentially expressed genes were validated by q RT-PCR.5.SiRNA interference technology was used to inhibit the expression of MIR4435-2HG,and the m RNA and protein expressions of SNAI1 were evaluated after MIR4435-2HG knockdown with or without subsequent F.nucleatum infection by q RT-PCR and Western blot assays.Based on the online databases and bioinformatics analyses,the mi RNAs that could bind to MIR4435-2HG and SNAI1 were predicted and the corresponding binding sites were obtained.Then the expressions of the mi RNAs were verified by q RT-PCR and mi R-296-5p was selected as the candidate gene.q RT-PCR was used to detect mi R-296-5p expression after MIR4435-2HG knockdown or in cells co-transfected with MIR4435-2HG si RNAs and mi R-296-5p inhibitor.And the m RNA and protein expression of SNAI1 in cells co-transfected with MIR4435-2HG and mi R-296-5p inhibitor were detected by q RTPCR and western blot,respectively.Dual luciferase reporter gene assays were performed to verify the binding between MIR4435-2HG and mi R-296-5p or mi R-296-5p and SNAI1.6.The m RNA expression of Akt2 was detected in HIOECs and SCC-9 cells infected with F.nucleatum and also in cells co-transfected with MIR4435-2HG si RNA and mi R-296-5p inhibitor by q RT-PCR.The protein expression of Akt2 in HIOECs and SCC-9 cells transfected with MIR4435-2HG si RNA or mi R-296-5p inhibitor was determined by Western blot assay.Further,SNAI1 expression at both m RNA and protein levels were detected after Akt2 knockdown by si RNA interference.Results: 1.F.nucleatum was highly abundant in OSCC species,while fewer F.nucleatum was observed in the normal tissues,suggesting a correlation between F.nucleatum infection and OSCC.2.F.nucleatum infection had no significant effect on cell proliferation,while S.gordonii infection can inhibit cell proliferation slightly.The flow cytometry assays showed that F.nucleatum infection did not accelerate cell cycle progression,but increased the cell apoptosis compared with uninfected control cells.The scratch assay showed that F.nucleatum but not S.gordonii infection enhanced the cell migration ability.The activity of MMP-9 of HIOECs and SCC-9 cells infected with F.nucleatum were increased detected by gelatin zymography assay.3.The mRNA and protein expressions of N-cadherin,Vimentin and SNAI1 were increased,while the expression of E-cadherin was decreased in HIOECs and SCC-9 cells infected with F.nucleatum.Immunofluorescence staining revealed that E-cadherin was mainly distributed on the cell membrane,and the cells remain closely adjacent in the non-infected HIOECs and SCC-9 cells,while it appeared disorganized and punctiform in the cytoplasm after F.nucleatum infection.These results indicated that F.nucleatum can initiate or promote epithelial-mesenchymal transition in non-cancerous and cancerous oral epithelial cells.In addition,q RT-PCR results showed that heat-inactivated F.nucleatum and Fad A protein could induce abnormal expression of the four epithelial-mesenchymal transiton markers mentioned above.4.A total of 164 lncRNAs were aberrantly expressed according to the high throughput sequencing for non-infected HIOECs and F.nucleatum infected HIOECs,of which 74 lnc RNAs were upregulated and 90 lnc RNAs were downregulated.Moreover,887 m RNAs were upregulated and 1866 m RNAs were downregulated(fold change≧4,P value < 0.05,FDR<0.05).A cluster of differentially expressed genes were involved in EMT,and KEGG analysis showed that these genes are mainly enriched in the adherens junction signaling pathway.5.F.nucleatum infection upregulated the expression of lnc RNA MIR4435-2HG.And the m RNA and protein expressions of SNAI1 were decreased when inhibiting the expression of MIR4435-2HG by si RNA transfection,while F.nucleatum infection could attenuate the inhibitory effect of MIR4435-2HG knockdown on SNAI1 and rescue the expression of SNAI1.According to the results of online prediction and q RT-PCR validation,mi R-296-5p was found to contain binding sites with MIR4435-2HG and SNAI1,and the expression of mi R-296-5p was significantly decreased in HIOECs and SCC-9 cells infected with F.nucleatum.Further,MIR4435-2HG knockdown with si RNA transfection could upregulate the expression of mi R-296-5p,and transfecting mi R-296-5p inhibitor could increase the expression of SNAI1.The dual luciferase reporter gene assay verified that MIR4435-2HG could bind to mi R-296-5p,while mi R-296-5p could not bind with SNAI1.6.F.nucleatum infection upregulated the m RNA expression of Akt2.MIR4435-2HG knockdown suppressed the expression of Akt2 at both m RNA and protein levels,and the expression of Akt2 was upregulated when transfected with mi R-296-5p inhibitor.Akt2 knockdown with si RNA could reduce the m RNA and protein expressions of SNAI1.Conclusion: 1.F.nucleatum was enriched in OSCC tissues,suggesting the correlation between F.nucleatum infection and OSCC.2.F.nucleatum infection had no significant effect on the proliferation and cell cycle progression of HIOECs and SCC-9 cells,but promoted cell apoptosis and increased the cell migration ability significantly.3.F.nucleatum infection could increase the expressions of N-cadherin,Vimentin and SNAI1,and cause a reduced expression and functional loss of E-cadherin,indicating that F.nucleatum could initiate or promote epithelial-mesenchymal transition changes in nontumorous and tumorous oral epithelial cells.Moreover,heat-inactivated F.nucleatum and Fad A protein exhibited a similar effect on promoting EMT as live F.nucleatum.4.F.nucleatum infection could trigger or promote epithelial-mesenchymal transition in both non-cancerous and cancerous oral epithelial cells through regulation of lnc RNA MIR4435-2HG/mi R-296-5p/Akt2/SNAI1 signaling pathway.