Study On The Correlation Between TLR9-MyD88 Signal Pathway And Condyloma Acuminatum

Objective: In this study, we will observe the expression of TLR9 and some important signal transduction molecules such as My D88, IRF-7,which are in My D88-dependent pathway, in different prognostic condyloma acuminatum lesions. To explore the role of TLR9-My D88 signaling pathway in the pathogenesis of condyloma acuminatum as well as the relationship between them and the occurrence and development of condyloma acuminatum. We expect that the results of the study can provide a new strategy for the prevention and treatment of condyloma acuminatum.Methods: According to the inclusion and exclusion criteria, 15 cases of primary and 15 cases with recurrent condyloma acuminatum were selected as the experimental group, and 12 healthy people were served as the control group. Cut out the lesions of experimental group after routine disinfection and2% lidocaine local anesthesia. In the control group, the foreskin was obtained by circumcision. After 10% neutral formalin fixation, dehydration, clearing,paraffin, embedding and other steps,all tissue samples were made into paraffin block. At the protein levels,the expression of TLR9, My D88 and IRF-7 were detected by immunohistochemical method in primary, recurrent condyloma acuminatum lesions and normal control group. At the gene level, RNA was extracted by FFPE sample RNA Isolation Kit, and then the expression ofTLR9,My D88,IRF-7 in different prognostic CA lesions and normal control group was tested by real-time fluorescent quantitative PCR method respectively. All of the experimental data were evaluated by SPSS20.0software package for statistical description and analysis.Result:1.The results of immunohistochemistry: TLR9, My D88 and IRF-7 respectively had different levels of expression in primary, recurrent genital warts and normal control group. In the control group, TLR9 was weakly positively expressed in the basal layer; in primary group, TLR9 was mainly concentrated in the epidermal basal layer, spinous layer and granular layer, with positive or strongly positive expression; however, in recurrence group, TLR9 showed positive or strongly positive expression in each layer of epidermal. In the control group, My D88 was weakly positive and was mainly concentrated in the basal layer; My D88 was strongly expressed in the layers above the spine and around vacuole cells in primary group, moderately in the basal layer;however, in the recurrence group, My D88 was strongly expressed in the basal layer and spinous layer, moderately in granular layer. IRF-7 was mainly expressed in the cytoplasm of the basal layer in normal foreskin tissues, which was weakly positive; the spinous layer and stratum corneum were showed strongly positive in the primary group, moderately in the basal layer and granular layer; while IRF-7 was strongly expressed in the spinous layer,granular layer and stratum corneum, moderately in basal layer in therecurrence group. Statistical analysis showed that the expression of TLR9,My D88 and IRF-7 in the three groups were significantly different(P<0.001) at the protein level.The expression of TLR9, My D88, IRF-7 in the primary and recurrent group was significantly higher than that in the control group(P<0.05), but there was no significant difference between the primary and recurrent group(P>0.05). Spearman correlation analysis showed that TLR9 and My D88, TLR9 and IRF-7, My D88 and IRF-7 were positively correlated,and the correlation degree is high(rs = 0.907, 0.916, 0.876, respectively).2.The results of real-time fluorescence quantitative PCR: The concentration of the RNA extracted by Amoy Dx RNA kit was between 58ug/ml-118ug/ml,OD260/OD230 ratio and OD260/OD280 ratio were both between 1.8~2.0, and1% agarose gel electrophoresis analysis showed that the extracted RNA had good integrity. The real-time PCR amplification curve of gene β-actin, TLR9,My D88 and IRF-7 was S-shaped and the baseline was smooth. The melting curve showed a single and sharp peak. Statistical analysis showed: the expression of β-actin in each group was relatively constant, and there was no significant difference among the three groups(P>0.05).At the gene level, the expression of TLR9, My D88 and IRF-7 in the control group was significantly lower than that of the primary group and relapse group, the difference was statistically significant(P < 0.05), but there was no statistical difference in the primary group and recurrence group(P > 0.05). Pearson correlation analysis showed that TLR9 and My D88, TLR9 and IRF-7, My D88 and IRF-7 had apositive correlation with a high correlation(r= 0.868, 0.872, 0.781,respectively).Conclusion:1.The concentration and purity of the RNA, which was extracted by Amoy Dx RNA kit, were both good, the integrity as well as the quality was good.2.The expression of β-actin was relatively constant in each group so thatβ-actin can be used as reference gene for detection the expression of TLR9,My D88 and IRF-7 among the three groups.3.Whether at the protein level or at the genetic level, TLR9, My D88 and IRF-7in the CA lesions were abnormally expressed, and the levels of expression were apparently higher than the normal control group. This indicates that the high expression of TLR9 may serve as HPV recognition receptors, TLR9 may identify the Cp G-DNA sequence of HPV virus and then activate the My D88 dependent pathway, trigger signal transduction, activate IRF-7 and other transcription factors, regulate the expression of interferon, and participate in the aspect of the local immune response against HPV. But the expression in the primary and recurrent groups was not statistically different, which suggested that TLR9, My D88, IRF-7 may have no significant relationship with the course and prognosis of Condyloma acuminatum, it may also be related to the immune escape and the persistent infection of HPV, which could not further enhance the activation of the signal transduction pathway. In addition, the result may also be related to the less amounts of specimens.4.Abnormal expression of TLR9, My D88 and IRF-7 in the CA lesions, may be closely related to the body’s immune response against HPV, My D88 dependent signaling pathway may play an important role in the pathogenesis of CA, but specific causal relationship and the exact immunological mechanisms are not yet entirely clear, which needs further study.