| Objective"Periodontitis is a chronic infectious disease of periodontal tissue.It is one of the major diseases that affect human oral health.With up to 72cm2,the deep periodontal pockets in patients with severe periodontitis are reservoirs containing a large quantity of bacteria and inflammatory factors,causing near and distant organ diseases.A large number of studies have found that periodontitis is associated with a variety of systemic diseases,such as cardiovascular disease,diabetes,chronic obstructive pulmonary diseases,low birth weight infants,and so on.In the recent 20 years,some scholars have started to focus on researches on the relationship between periodontitis with its pathogens and malignant tumors.A number of studies have reported that there may be a correlation between periodontal diseases and malignant tumors,especially oral cancer.Virchow believed that cancer originated in the chronic inflammation sites which were rich in inflammatory cells,growth factors,active matrix,and promoters of DNA damage.In this environment,continuous cell proliferation might strengthen and improve the risk of cancer.As one of gram negative anaerobic bacteria,Porphyromonas gingivalis(P.gingivalis)is often detected in chronic periodontitis patients.It is one of the most virulent periodontal pathogens.P.gingivalis has many virulence factors that enable bacteria to adhere to the surface of the mouth and invade into the periodontal tissues of the host,and regulate the immune and inflammatory reaction of host cells.Studies have shown that intracellular P.gingivalis can influence certain cell cycle related molecules at different levels,such as cyclin D1,cyclinE,CDK4/6,and so on.It can shorten the cell cycle and promote the uncontrolled proliferation of cells.The most basic characteristic of malignant tumor is the uncontrolled proliferation of tumor cells.Oral cancer is one of the most common malignant tumors in head and neck,90%of which is oral squamous cell carcinoma(OSCC).P.gingivalis is mostly found in the oral periodontal tissues and can plant on the surface of the mouth.Some researchers found that P.gingivalis could promote the invasion and metastasis of OSCC cells and induce mesenchymal transformation of tumor cells,but the stimulation of P.gingivalis in Ca9-22 slightly inhibited cell proliferation.Others found that the combined action of P.gingivalisand Fusobacterium nucleatumon OSCC cells could promote proliferation of cells.In a cohort study of 1252 postmenopausal women,Mai found that there was no obvious correlation between periodontal pathogens in subgingival plaque and the incidence of oral cancerThe relation between P.gingivalis and the occurrence and development of OSCC remains controversial.In existing studies,only a few studies on large sample directly illustrated whether there was a correlation between P.gingivalis and OSCC.Therefore,this study detected P.gingivalis in squamous cell carcinoma first,observed the effect of P.gingivalis infection on the proliferation of OSCC cell line Tca8113,and researched the possible molecular mechanism involved.The objective of this study is to provide further evidence to make clear whether there is correlation between P.gingivalis and the occurrence and development of OSCC,and to provide a new direction for the prevention and treatment of OSCCMethods:This study concluded following three partsPart 1,Sixty-one cancer tissues and their adjacent paracancerous tissues as well as 30 normal oral tissues were obtained.Besides,participants in this experiment underwent a preoperative periodontal clinical examination.Then we analyzed the periodontal clinical indexes and tumor clinical pathological parameters of patients with squamous cell carcinoma.Fluorescence in situ hybridization was used to detect the positive rate of P.gingivalis in paraffin specimens and quantitative real-time PCR(qRT-PCR)were used to detect the content of P.gingivalis DNA in different tissues,and the clinical pathological parameters of squamous cell carcinoma were analyzed statistically.This stage was to make clear a certain correlation between periodontitis as well as the presence of P.gingivalis and oral squamous cell carcinomaPart 2,we infected oral squamous cell carcinoma cell line Tca8113 with P.gingivalis and observed the invasion of bacteria into cells by transmission electron microscope.CCK-8 method was used to detect cell proliferation and flow cytometry was applied for cell cycle.In addition,cyclin D1,the key factor in cell cycle,was detected by qRT-PCR and western blot for the mRNA and protein expression.Meanwhile,we studied the upstream transcription factor AP-1(c-Jun,c-Fos)and detected the expression of cyclin D1,c-Jun and c-Fos in tissue specimens using immunohistochemical methodPart 3,the mechanism of P.gingivalis infection in oral squamous cell carcinoma cell line Tca8113 was studied in this part.Above all,the microarray was applied for screening differentially expressed miRNA in oral squamous cell carcinoma Tca8113 cells after P.gingivalis infection.Further,the expression of miR-21was inhibited,and the expression of the target gene programmed cell death 4(PDCD4)was observed by qRT-PCR and Western blot;PDCD4 was over-expressed to observe its effect on the expression of transcription factor AP-1(c-Jun,c-Fos);c-Jun was silenced and qRT-PCR and Western blot were used to observe the effect on the expression of miR-21 and PDCD4.Then investigated the interaction mechanism,and observed the influence on the expression of mRNA and protein of cyclin D1.CCK-8 method was used to detect the influence of P.gingivalis infection on cell proliferation after miR-21,PDCD4 and c-Jun interferenceStatistical analysis:statistical software SPSS17.0 was used for statistical analysis.The mean of measurement data was expressed by mean ± SD.Independent samples t test was used for comparisonin twogroups,and variance analysis was used in multiple groups;Categorical data were compared using X2 test.Spearman and pearson correlation analysis was used for correlation analysis.P<0.05 was considered statistically significant.Results:Concludedfollowing three partsPart 1,the periodontal clinical indicators PD,AL and the number of remaining teeth of OSCC patients were associated with lymph node metastasis.In other words,the more severe the periodontitis was,the worse the degree of tissue differentiation was and the higher the incidence of lymph node metastasis would be.The result of fluorescence in situ hybridization showed that in oral squamous cell carcinoma,adjacent tissues and normal tissues,the positive rates of P.gingivalis were 60.7%,32.8%and 13.3%,respectively.Differences between groups were statistically significant(P<0.05).It was clear that the rate in oral squamous cell carcinoma was much higher than that in adjacent tissues and normal tissues and the rate in paracancerous tissues was higher than that in normal tissues.In the tissue sections,bacteria mainly invaded into the epithelial layer,and a few of them invaded into the deep muscular layer.At the same time,we extracted bacterial genomic DNA from tissues,detected the copy number of P.gingivalis DNA in various tissues using absolute quantitative Realtime-PCR method.What’s more,we found that the content of P.gingivalis DNA in squamous cell carcinoma was much higher than that in adjacent tissues and normal tissues,and the content in adjacent tissues was higher than that in normal tissues.The differences were statistically significant(P<0.05)The positive rate of P.gingivalisin patients with poor tissue differentiation and lymph node metastasis was highPart 2,Tca8113 cells were observed by scanning electron microscopy after P.gingivalis infection.The results showed that bacteria could adhere to and invade into oral squamous cell carcinoma cells.The cell proliferation was detected by CCK-8 assay.The cell proliferation was associated with multiplicity of infection(MOI)of P.gingivalis and the infection time.When MOI was 50:1,the cell proliferation was the most obvious and sustained the longest time.It was chosen as the MOI value for the follow-up study.At the same time,the changes of cell cycle could be seen,including the increasing percentage of cells in S phase,the decreasing proportion of cells in G1 phase,and the ability to go through G1 phase rapidly.The expression of cyclin D1,c-Jun and c-Fos mRNA and protein,which were associated with cell cycle and tumor proliferation,increased.The increasing expression of cyclin D1,c-Jun and c-Fos protein detected in oral squamous cell carcinoma tissues was positively correlated with P.gingivalis infectionPart 3,in this part,we applied microarray for screening differentially expressed microRNA in oral squamous cell carcinoma Tca8113 cells after P.gingivalis infection,involving several pathways associated with the occurrence and the development of tumors,including Ras signaling pathway,pathways in cancer,proteoglycans in cancer,MAPK signaling pathway and PI3K-Akt signaling pathway and so on.The target genes involved were mainly associated with transcription regulation,epidermal growth factor signaling pathway,cell adhesion and so on.These signaling pathways played an important role in the occurrence and development of tumors.Furthermore,we investigated differentially expressed miR-21.The expression of miR-21 in oral squamous cell carcinoma was significantly higher than that in adjacent tissues and normal tissues(P<0.05).We down-regulated the expression of miR-21 and found that it could reversely regulate the programmed cell death factor PDCD4 and inhibit cell proliferation induced by P.gingivalis infection.Overexpression of PDCD4 could make activation of c-Jun and c-Fos disappear,and inhibit the proliferation of Tca8113 cells and the expression of cyclin cyclin D1 induced by P.gingivalis infection.Silence of c-Jun by siRNA could significantly reduce the expression of miR-21,and inhibit cell proliferation as well as cyclin D1 expression.Therefore,the cell proliferation of Tca8113 and the increasing expression of cyclin D1 induced by P.gingivalis infection might be the regulatory role of negative feedback loops of miR-21,PDCD4 and AP-1Conclusion:this study discovered that the periodontal clinical indicators PD,AL and the number of remaining teeth of oral squamous cell carcinoma patients were associated with lymph node metastasis.The positive rate and the content of P.gingivalis in oral squamous cell carcinoma were significantly higher than that in normal tissues,and were associated with tissue differentiation and lymph node metastasis.The expression of miR-21,c-Jun,c-Fos and cyclin D1 was positively correlated with the positive expression of P.gingivalis in oral squamous cell carcinoma.The expression of miR-21 in oral squamous cell carcinoma was higher than in adjacent tissues and normal tissues Meanwhile,it was revealed that P.gingivalis infection could regulate the proliferation of oral squamous cell carcinoma Tca8113 cells through miR-21/PDCD4/AP-1 negative feedback loop.These results suggested that P.gingivalis infection was closely related to the occurrence and development of oral squamous cell carcinoma,which provided a new perspective for the prevention and treatment of oral squamous cell carcinoma. |
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