ABSTRACT
Objective To explore the role of toll-like receptor (TLR)/nitric oxide (NO) pathway in cervical tumor with high risk human papillomavirus (hrHPV) infection.Methods (1)Study was based on 36 women with nonmalignantcervical tissue as control group and 36 women with squamous cell cervical cancer (SCC),all with hrHPV infection which were assessed by using 14 types hrHPV E6/E7 mRNA real-time PCR kit.The amount of NO was detected by Griess reaction,the expression of inducible nitric oxide synthase (iNOS) was detected by immunohistochemistry (IHC).The mRNA expression of TLR3,TLR4,TLR7,TLR8,TLR9,nuclear factor-κB (NF-κB) p65 and iNOS in control and SCC epithelium which was captured by laser capture microdissection (LCM) were determined.(2)The expressions of TLR4 in CaSki,HeLa and C33a were detected by cell immunofluorescence method.The mRNA and protein expression of TLR/NO pathway transduction molecules including TLR4,NF-κBp65 and iNOS in CaSki,HeLa and C33a cell lines were detected by real-time PCR and western blot.Results (1)The level of NO was much higher in SCC group than that in control group [(42.92±0.36) μmol/L vs (15.49±0.24) μmol/L;P＜0.05].iNOS was detected in 75％ (27 cases) of patients with squamous cervical carcinoma,while only 6％ (2 cases) of normal controls were confirmed with positve result (P＜0.05).TLR/NO pathway maybe activated in SCC,for the mRNA levels of TLR3,TLR4,TLR7,TLR8,NF-κBp65 and iNOS increased significantly when compared to control group (all P＜0.05),and the greatest change in the expression level of TLR in SCC was spotted on TLR4(7.41±0.39 vs 1.86±0.21).(2)The results of immunofluorescence showed that TLR4 was located at plasma membrance of hrHPV positive HeLa and CaSki cells,while the integral optical density of TLR4 in HeLa cells (3 599±427) or CaSki cells (2 080±456) were higher than that in C33a cells (730±96;P＜0.05).The mRNA and protein level of TLR4,NF-κBp65 and iNOS in HeLa and CaSki cells were higher than those of C33a cells (P＜0.05).Conclusion TLR4/NO pathway is highly expressed in cervical cancer with hrHPV infection,while the pathway may be involved in cervical tumorigenesis with hrHPV infection.