• Asian Pacific Journal of Cancer Prevention
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• v.15 no.14
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• pp.5867-5872
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• 2014

Background: Cervical cancer is listed as one of high-incidence endemic diseases in Xinjiang. Our study aimed to evaluate the expression of TLR9 in uterine cervical tissues of Uyghur women and examine associations with clinicopathological variables. We further characterized the direct effects of TLR9 upon the selective silencing of human papillomavirus (HPV) E6 and E7 oncoprotein expression in HPV 16-positive human cervical carcinoma cells treated with siRNA in vitro. Materials and Methods: Immunohistochemistry was applied to evaluate TLR9 expression in 97 formalin-fixed paraffin-embedded cervical samples from Uyghur women; 32 diagnosed with cervical squamous cell carcinomas (CSCC), 14 with low-grade cervical intraepithelial neoplasias (CINI), 10 medium-grade (CINII), 24 high-grade (CINIII), and 17 chronic cervicitis. $BLOCK-iT^{TM}$ U6 RNAi Entry Vector $pENTR^{TM}$/U6-E6 and E7 was constructed and transfected the entry clone directly into the mammalian cell line 293FT. Then the HPV 16-positive SiHa human cervical carcinoma cell line was infected with RNAi recombinant lentivirus. RT-PCR and Western blotting were used to determine the expression of TLR9 in both SiHa and HPV 16 E6 and E7 silenced SiHa cells. Results: Immunohistochemical staining showed that TLR9 expression was undetectable (88.2%) or weak (11.8%) in chronic cervicitis tissues. However, variable staining was observed in the basal layer of all normal endocervical glands. TLR9 expression, which was mainly observed as cytoplasmic staining, gradually increased in accordance with the histopathological grade in the following order: chronic cervicitis (2/17, 11.8%)

• Korean Journal of Clinical Laboratory Science
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• v.49 no.4
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• pp.446-454
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• 2017

Human papillomaviruses (HPVs) are major causes of cervical cancer. Sixteen high risk HPVs, including HPV 16, HPV 18, HPV31, HPV 33, HPV 35, HPV 39, HPV 45, HPV 51, HPV 52, HPV 53, HPV 56, HPV 58, HPV 59, HPV 66, HPV 68, and HPV 69 are found in cervical cancer. HPVs 16 and 18 are mainly presented in 70% of cervical cancer. Therefore, identifying the presence of these high-risk HPVs is crucial. The objective of this study is to establish the HPV ViroCheck for detecting 16 HR-HPVs and genotypes of HPVs 16 and 18, as well as to analyze the analytical performance of HPV ViroCheck. We performed the analytical sensitivity of HPV E6 / E7 genes of 16 high risk HPVs to confirm the limit of detection. Then, a cross reactivity of HPV ViroCheck with microorganisms and viruses related to the cervix were analyzed for analytical specificity. Analytical sensitivity of high risk HPV genotypes ranged from 1 to 100 copies when using cloned DNAs. The limit of detection was 10 cells for both SiHa and HeLa cells. Cervical-related microorganisms and viruses did not show cross-reactivity to HPV DNA. Moreover, the intra- and inter-assay coefficient variations (CVs) were below 5%. In conclusion, HPV Virocheck will be useful for the detection of 16 HR HPVs, as well as HPV 16 and HPV 18 genotypes rapidly.

• YAKHAK HOEJI
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• v.46 no.6
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• pp.426-432
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• 2002

Cervical cancer is one of the leading causes of female death from cancer worldwide with about 500,000 deaths per year. A strong association between certain human papilloma viruses (HPV type 16 and 18) and cervical cancer has been well known. An extract of Saururus chinensis, named as PE-46, has been used to investigate whether this agent has the ability of inhibiting the oncogenes E6 and E7 of HPV type 16. PE-46 inhibited the proliferation of human cervical cancer cell lines in a dose response manner. PE-46 also inhibited the in vitro binding of E6 and E6AP which are essential for the binding and degradation of the tumor suppressor p53. In addition, PE-46 inhibited the in vitro binding of E7 and Rb which is essential tumor suppressor for the control of cell cycle. The levels of mRNA for E6 and E7 were also decreased by PE-46. SiHa cells treated with PE-46 induced G0/G1 arrest, resulting in inhibition of growth. Our study showed that the PE-46 can inhibit the cervical carcinomas via both inhibition of bindings between oncogenes and tumor suppressors, and inhibition of G1longrightarrowS transition. PE-46 inhibited the oncogenecity of E6 and E7 of HPV 16 type, thus could be used as a putative modulating agent for the treatment of cervical carcinomas caused by HPV.

• Journal of Microbiology and Biotechnology
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• v.33 no.8
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• pp.1091-1100
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• 2023

Human papillomavirus (HPV) types 16 and 18 are the major causes of cervical lesions and are associated with 71% of cervical cancer cases globally. However, public health infrastructures to support cervical cancer screening may be unavailable to women in low-resource areas. Therefore, sensitive, convenient, and cost-efficient diagnostic methods are required for the detection of HPV16/18. Here, we designed two novel methods, real-time ERA and ERA-LFD, based on enzymatic recombinase amplification (ERA) for quick point-of-care identification of the HPV E6/E7 genes. The entire detection process could be completed within 25 min at a constant low temperature (35-43℃), and the results of the combined methods could be present as the amplification curves or the bands presented on dipsticks and directly interpreted with the naked eye. The ERA assays evaluated using standard plasmids carrying the E6/E7 genes and clinical samples exhibited excellent specificity, as no cross-reaction with other common HPV types was observed. The detection limits of our ERA assays were 10⁰ and 10¹ copies/µl for HPV16 and 18 respectively, which were comparable to those of the real-time PCR assay. Assessment of the clinical performance of the ERA assays using 114 cervical tissue samples demonstrated that they are highly consistent with real-time PCR, the gold standard for HPV detection. This study demonstrated that ERA-based assays possess excellent sensitivity, specificity, and repeatability for HPV16 and HPV18 detection with great potential to become robust diagnostic tools in local hospitals and field studies.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.106-106
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• 2003

Human papillomavirus type 16(HPV16) has been known to the major factor for the development of uterine cervical carcinomas. We have extended these studies to investigate the in vivo activities of HPV-16 E6/E7 when expressed in squamous epithelia of transgenic mice. Grossly, hK14HPV16E6/E7 transgenic mice had multiple phenotypes, including wrinkled skin that was apparent prior to the appearance of hair on neonates, thickened ears, and loss of hair in adults. In the transgenic mice, the wrinkled skin phenotype on the body and legs died at the age of 3-4 weeks. Histological analysis of demonstrated that E6/E7 causes epidermal hyperplasia in multiple transgenic lineages with high penetrance. This epithelial hyperplasia was characterized by an expansion of the proliferating compartment and an expansion of the keratinocyte and was associated with hyperkeratosis. These transgenic mice expressed E6/E7 transgene mainly in skin, heart, pancreas and kidney. Hyperplasia was found at the skin. The enzyme activities of GR, GPx and CuZnSOD were measured from the transgene cause keratinocyte at the skin. The specific enzyme activities were significantly higher in transgenic mice skin compared to the normal mice skin. Thus these transgenic mice may be useful for the develpment of antioxidant enzymes or other therapies for HPV-associated hyperkeratosis.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.3
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• pp.1151-1157
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• 2015

Human papillomavirus (HPV) is responsible for one of the most frequent sexually transmitted infections. The first phylogenetic analysis was based on a LCR region fragment. Nowadays, 4 variants are known: African (Af-1, Af-2), Asian-American (AA) and European (E). However the existence of sub-lineages of the European variant havs been proposed, specific mutations in the E6 and LCR sequences being possibly related to persistent viral infections. The aim of this study was a phylogenetic study of HPV16 sequences of endocervical samples from C${\acute{o}}$rdoba, in order to detect the circulating lineages and analyze the presence of mutations that could be correlated with malignant disease. The phylogenetic analysis determined that 86% of the samples belonged to the E variant, 7% to AF-1 and the remaining 7% to AF-2. The most frequent mutation in LCR sequences was G7521A, in 80% of the analyzed samples; it affects the binding site of a transcription factor that could contribute to carcinogenesis. In the E6 sequences, the most common mutation was T350G (L83V), detected in 67% of the samples, associated with increased risk of persistent infection. The high detection rate of the European lineage correlated with patterns of human migration. This study emphasizes the importance of recognizing circulating lineages, as well as the detection of mutations associated with high-grade neoplastic lesions that could be correlated to the development of carcinogenic lesions.

• YAKHAK HOEJI
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• v.48 no.6
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• pp.328-334
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• 2004

Cervical cancer is one of the leading causes of female death. Viral oncoproteins E6 and E7 are selectively retained and expressed in carcinoma cells infected with HPV (Human pa pilloma virus) type 16 and cooperated in immotalization and transformation of primary keratinocyte. E6 and E7 oncoproteins interfere the functions of tumor suppressor proteins p53 and retinoblasoma protein (pRb), respectively. Among a lots of natural products, Mentha arvensis Linne var.piperascens have inhibitory effects on bindings between E6 oncoprotein and tumor suppressor p53, E3 ubiqutin- protein ligase (E6AP). HPV oncoprotein inhibitors from Mentha piperita L. were isolated by solvent partition and column chromatography (Silica gel, RP-18) and inhibitory compounds were finally purified by HPLC using an ELISA screening system based on binding between E6 and E6AP. The aim of this study is to identify the structure of inhibitory compounds and to investigate whether these compounds have inhibitory effects on functions of E6 oncoprotein. We investigated whether caffeic acid methyl ester (CAM) extracted from Mentha piperita L. could inhibit the function of E6 oncoprotein. CAM inhibited the in vitro binding of E6 and E6AP which are essential for the binding and degradation of the tumor suppressor p53 and also inhibited the proliferation of human cervical cancer cell lines (SiHa and CaSKi) in a dose response manner. These results suggest that CAM inhibited the function of E6 oncoprotein, suggesting that it can be used as a potential drug for the treatment of cervical cancers infected with HPV.

• The Journal of Korean Society of Virology
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• v.28 no.1
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• pp.53-62
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• 1998

Human papillomavirus (HPV) 16, E7 proteins derived from the prototype (Bac73) and natural variant (Bac101) E7 open reading frame were produced in Sf9 insect cells. The variant E7 gene occurred naturally by substitution mutation at the position of 88 nucleotide, resulting serine instead of asparagine. Using E7 specific monoclonal antibody (VD6), both E7 proteins were identified in recombinant baculovirus infected SF9 cells. Radiolabelling and immunoprecipitation analysis revealed that both E7 proteins were phosphoproteins. Immunostaining result showed that E7 proteins were mainly localized in the cytoplasm. Nuclear form of E7 proteins was also detected after a sequential fractionation procedure for removing chromatin structure. Considering that the VD6 recognition site in E7 protein is located within 10 amino acid at the N-terminus, this region appears to be blocked by the nuclear component. Western blot analysis revealed that nuclear form was more abundant than cytoplasmic E7 proteins. Time course immunostaining showed that the primary location of E7 protein was the nucleus and exported to the cytoplasm as proteins were accumulated. These events occurred similarly in both Bac73 and Bac101 infected Sf9 cells, suggesting that these two proteins may have similar biological functions.

• Korean Journal of Bronchoesophagology
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• v.4 no.2
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• pp.197-204
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• 1998

Human papillomavirus(HPV) is epitheliotrophic virus invading the anogenital tract and the upper aerodigestive tract HRV produces a diversity of benign and maljgnant tumors. In this study, the author determined the frequency of association of human papillomavirus(HPV) and laryngeal carcinomas and investigated the significance of HRV infection of different subtypes in the tumorigenesis of laryngeal carcinoma. Laryngeal squamous cell cancinomas from 34 patients who did not have preexisting papillomas by clinical history were retrieved from formalin-fixed, paraffin-embedded blocks and analyzed for HPV. Nineteen cases were tumors of the true vocal folds, 11 were supraglottic and 4 were transglottic. HPV detection was dane using polymerase chain reaction amplification with HPV L$_1$consensus primer. HPV type was determined by the same method using HPV-6, 11 and 16,-18 type-specific E6 primers. The results were as follows : 1) HPV DNA was detected in 7 cases among the 34 patients(20.6%). According to the type of HPV DNA HPV-11 was detected in 3 cases, HPV-16 was detected in 2 cases and HPV-6 and HPV-18 were detected in 1 case, respectively. 2) These 7 HPV-positive patients were advanced cancinoma cases. From these results, we concluded that HPV was thought to be the etiological factor of laryngeal squamous cell carcinomas.

• Journal of Periodontal and Implant Science
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• v.32 no.3
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• pp.603-614
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• 2002

Human gingival fibroblasts have proven to useful as a species specific cell culture system in various system on periodontal disease and regeneration. However, their use is limited, since they are hard to obtain and lifespan is short due to replicative senescence. To overcome these disadvantages, we transfected primary human gingival fibroblasts by the E6 and E7 genes of the Human papilloma virus(HPV) 16. The full length of HPV 16 E6 and E7 was cloned from the pBR322 into BamHl and Sal I of a pBabe vector including hygromycin B resistance. Before pBabeE6/E7 plasmid transfection, peak 8 GFP including G418 resistance was transfected into primary GF to check the transfection efficency. PBabe E6/E7 plasmid was transfected using Lipofectamine plus following manufacter's instruction into primary normal human gingival fibroblasts in 60mm dishes with FBS free DMEM. After 2 days of transfection, the cells were treated with hygromycin for 2 weeks until the transfected control cells died. The resulting hygromycin resistant colonies were pooled, and clonned, and sucessful　transfection was established for immortalized gingival fibroblast cell lines. Immoralized GF cells showed stellate shape, that is similar to that of orange grains, and more rapid growth and higher proliferation than that of primary gingival fibroblasts. This cell lines overcame crisis and could be cultured over 30 subcultured, could be use for three dimentional culture, epithelial-mesenchymal interaction study.