Xiao Jingjing, Guo Qisang, Wang Xianzheng, Xie Feng, Zhang Hongwei, Sui Long
Medical Center for Diagnostics and Treatment of Cervical Diseases, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, China.
Email:
Zhonghua Fu Chan Ke Za Zhi. 2015 Jan;50(1):41-7.
To explore the role of toll-like receptor (TLR)/ nitric oxide (NO) pathway in cervical tumor with high risk human papillomavirus (hrHPV) infection.
(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.
(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).
TLR4/NO pathway is highly expressed in cervical cancer with hrHPV infection, while the pathway may be involved in cervical tumorigenesis with hrHPV infection.
探讨Toll样受体（TLR）/一氧化氮（NO）通路在高危型人乳头瘤病毒（hrHPV）感染的宫颈肿瘤中的作用。
（1）研究以36例非恶性宫颈组织女性为对照组，36例宫颈鳞状细胞癌（SCC）女性为研究组，所有患者均感染hrHPV，采用14种hrHPV E6/E7 mRNA实时荧光定量PCR试剂盒进行评估。采用Griess反应检测NO含量，免疫组织化学法（IHC）检测诱导型一氧化氮合酶（iNOS）的表达。通过激光捕获显微切割（LCM）获取对照组和SCC上皮组织，检测TLR3、TLR4、TLR7、TLR8、TLR9、核因子κB（NF-κB）p65和iNOS的mRNA表达。（2）采用细胞免疫荧光法检测CaSki、HeLa和C33a细胞中TLR4的表达。采用实时荧光定量PCR和蛋白质印迹法检测CaSki、HeLa和C33a细胞系中TLR/NO通路转导分子TLR4、NF-κB p65和iNOS的mRNA和蛋白表达。
（1）SCC组NO水平显著高于对照组[（42.92±0.36）μmol/L比（15.49±0.24）μmol/L；P<0.05]。75%（27例）宫颈鳞状细胞癌患者检测到iNOS，而正常对照组仅6%（2例）呈阳性结果（P<0.05）。SCC中TLR/NO通路可能被激活，与对照组相比，SCC中TLR3、TLR4、TLR7、TLR8、NF-κB p65和iNOS的mRNA水平显著升高（均P<0.05），SCC中TLR表达水平变化最大的是TLR4（7.41±0.39比1.86±0.2）。（2）免疫荧光结果显示，TLR4位于hrHPV阳性的HeLa和CaSki细胞的细胞膜上，HeLa细胞（3 599±427）或CaSki细胞（2 080±456）中TLR4的积分光密度高于C33a细胞（）730±96；P<0.05）。HeLa和CaSki细胞中TLR4、NF-κB p65和iNOS的mRNA和蛋白水平高于C33a细胞（P<0.05）。
TLR4/NO通路在hrHPV感染的宫颈癌中高表达，该通路可能参与hrHPV感染的宫颈肿瘤发生。
