TLR9 Expression in Uterine Cervical Lesions of Uyghur Women Correlate with Cervical Cancer Progression and Selective Silencing of Human Papillomavirus 16 E6 and E7 Oncoproteins in Vitro

Background: Cervical cancer is listed as one of high-incidence endemic diseases in Xinjiang. Our study aimedto evaluate the expression of TLR9 in uterine cervical tissues of Uyghur women and examine associations withclinicopathological variables. We further characterized the direct effects of TLR9 upon the selective silencing ofhuman papillomavirus (HPV) E6 and E7 oncoprotein expression in HPV 16-positive human cervical carcinomacells treated with siRNA in vitro. Materials and
Methods: Immunohistochemistry was applied to evaluate TLR9expression in 97 formalin-fixed paraffin-embedded cervical samples from Uyghur women; 32 diagnosed withcervical squamous cell carcinomas (CSCC) , 14 with low-grade cervical intraepithelial neoplasias (CINI), 10medium-grade (CINII), 24 high-grade (CINIII), and 17 chronic cervicitis. BLOCK-iT™ U6 RNAi Entry VectorpENTR™/U6-E6 and E7 was constructed and transfected the entry clone directly into the mammalian cell line293FT. Then the HPV 16-positive SiHa human cervical carcinoma cell line was infected with RNAi recombinantlentivirus. RT-PCR and Western blotting were used to determine the expression of TLR9 in both SiHa and HPV16 E6 and E7 silenced SiHa cells.
Results: Immunohistochemical staining showed that TLR9 expression wasundetectable (88.2%) or weak (11.8%) in chronic cervicitis tissues. However, variable staining was observed inthe basal layer of all normal endocervical glands. TLR9 expression, which was mainly observed as cytoplasmicstaining, gradually increased in accordance with the histopathological grade in the following order: chroniccervicitis (2/17, 11.8%) <CINI (4/19, 28.6%) <CINII (3/10, 30.0%) <CINIII (12/24, 50.0%) <CSCC (17/32, 53.1%)(p<0.05), but not with tumor differentiation. RT-PCR and Western blotting showed that TLR9 expression wasup-regulated in HPV16 E6 and E7 silenced SiHa cells at both mRNA and protein levels.
Conclusions: TLR9expression increases according to the histopathological grade of cervical pathological process. HPV E6 and E7oncoprotein have negative effects on the expression and function of TLR9.