Ning Ding;Wanwan Qi;Zihan Wu;Yaqin Zhang;Ruowei Xu;Qiannan Lin;Jin Zhu;Huilin Zhang
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.
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 types 16 and 18) and cervical cancer has been well known. An extract of natural products, named as Somatid, has been used to investigate whether this agent has the ability of inhibiting the oncogenes E6 and E7 of HPV type 16. This Somatid inhibited the proliferation of human cervical cancer cell lines (C-33A, SiHa, CaSki) and HaCaT keratinocytes in a dose response manner, In vitro binding assay and ELISA showed that Somatid inhibited the in vitro biding of E6 and E6AP which are essential for the binding and degradation of the tumor suppressor p53. In addition, Somatid 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 Somatid. Our data suggested that Somatid inhibited the oncogenecity of E6 and E7 of HPV 16 type, thus can be used as a putative anti-HPV agent for the treatment of cervical carcinomas caused by HPV.
Human papillomavirus (HPV) is the primary etiologic agent of cervical cancer. Consideration of safety and non human leukocyte antigen restriction, protein vaccine has become the most likely form of HPV therapeutic vaccine, although none have so far been reported as effective. Since tumor cells consistently express the two proteins E6 and E7, most therapeutic vaccines target one or both of them. In this study, we fabricated DC vaccines by transducing replication-defective recombinant adenoviruses expressing E6/E7 fusion gene of HPV-16, to investigate the lethal effects of specific cytotoxic T lymphocytes (CTL) against CaSki cells in vitro. Mouse immature dendritic cells (DC) were generated from bone marrow, and transfected with pAd-E6/E7 to prepare a DC vaccine and to induce specific CTL. The surface expression of CD40, CD68, MHC II and CD11c was assessed by flow cytometry (FCM), and the lethal effects of CTL against CaSki cells were determined by DAPI, FCM and CCK-8 methods. Immature mouse DC was successfully transfected by pAd-E6/E7 in vitro, and the transfecting efficiency was 40%-50%. A DC vaccine was successfully prepared and was used to induce specific CTL. Experimental results showed that the percentage of apoptosis and killing rate of CaSki cells were significantly increased by coculturing with the specific CTL (p <0.05). These results illustrated that a DC vaccine modified by HPV-16 E6/E7 gene can induce apoptosis of CaSki cells by inducing CTL, which may be used as a new strategy for biological treatment of cervical cancer.
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%)
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.
Kim, Joo-Ho;Cho, Il-Hoon;Seo, Sung-Min;Kim, Ji-Sook;Oh, Kyu-Ha;Kang, Heun-Soo;Kim, In-Gyu;Paek, Se-Hwan
Bulletin of the Korean Chemical Society
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v.30
no.12
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pp.2999-3005
/
2009
Among the more than 120 different types of human papillomavirus (HPV), types 16 and 18 have been known to be high risk agents that cause cervical cancer. We examined, in an immuno-chromatographic analysis, the potential of using the early gene product, E7 protein, as a diagnostic marker of cervical cancer caused by HPV. We developed monoclonal antibodies specific to HPV-16 and 18 E7 proteins that were produced from bacterial cells using gene recombinant technology. For each E7 protein, the optimal antibody pair was selected using the immuno-chromatographic sandwichtype binding system based on the lateral flow through membrane pores. Under these conditions, this rapid testing assay had a detection capability as low as 2 ng/mL of E7 protein. Furthermore, since viral analysis required the host cell to be lysed using chemicals such as detergents, it was possible that the E7 protein was structurally damaged during this process, which would result in a decrease in detection sensitivity. Therefore, we examined the detrimental effects caused by different detergents on the E7 protein using HeLa cells as the host. In these experiments, we found that the damage caused by the detergent, nonylphenylpolyethylene glycol (NP-40), was minimal relative to Triton X-100 commonly used for the cell lysis. Temperature also affected the stability of the E7 protein, and we found that the E7 protein was stabilized at 4$^{\circ}C$ for about 2 h, which was 4 times longer than at room temperature. Finally, a HPV-infected cervical cancer cell line, which was used as a real sample model, was treated using the optimized conditions and the presence of E7 proteins were analyzed by immuno-chromatography. The results of this experiment demonstrated that this rapid test could specifically detect HPV-infected samples.
Primary human dermal papilla cells (HDPCs) are often preferred in studies on hair growth and regeneration. However, primary HDPCs are limited by their reduced proliferative capacity, decreased hair induction potential, and extended doubling times at higher passages. To overcome these limitations, pTARGET vectors containing human papillomavirus16 (HPV16) E6/E7 oncogenes were transfected into HDPCs and selected using G-148 to generate immortalized cells here. HPV16 E6/E7 oncogenes were efficiently transfected into primary HDPCs. Immortalized HDPC showed higher proliferative activity than primary HDPC, confirming an increased proliferation rate. Expression of p53 and pRb proteins was downregulated by E6 and E7, respectively. E6/E7 expressing HDPC cells revealed that cyclin-dependent kinase (CDK) inhibitor p21 expression was decreased, while cell cycle-related genes and proteins (CDK2 and cyclin E) and E2F family genes were upregulated. Immortalized HDPCs maintained their responsiveness to Wnt/β-catenin pathway and hair follicle formation capability, as indicated by their aggregative properties and stemness. E6/E7 immortalized HDPCs may facilitate in vitro hair growth and regeneration studies.
Background: The study was aimed to evaluate the prevalence and genotype distribution of HPV infection in vulvar squamous cell carcinoma (SCC) in northern Thailand and the clinicopathological difference with regard to HPV infection status. Materials and Methods: Formalin-fixed paraffin-embedded tissue samples of vulvar SCC diagnosed between January 2006 and December 2012 were collected. HPV infection was detected by nested polymerase chain reaction (PCR) with primers MY09/11 and GP5+/6+. HPV genotyping was performed using the Linear Array Genotyping Test, followed by type-specific PCR targeting the E6/E7 region of HPV16/18/52 if the Linear Array test was negative. The histologic slides of vulvar lesions and the medical records were reviewed. Results: There were 47 cases of vulvar SCC included in the study (mean patient age $57.9{\pm}13.2$ years). HPV infection was detected in 29 cases (62%), all of which had single HPV infections. HPV16 accounted for 23 (49%). The patients with HPV-positive SCC had a significantly younger mean age than those with HPV-negative tumors (52.7 years vs 66.2 years, p<0.001). There was no significant difference in tumor stage distribution with regard to the status of HPV infection. The presence of vulvar intraepithelial neoplasia (VIN) of usual type (basaloid or warty) was significantly more frequent in HPV-positive cases compared with HPV-negative cases (62% vs 6%, p<0.001), whereas differentiated-type VIN was more common in HPV-negative cases (24% vs 0%, p=0.019). Conclusions: HPV infection was detected in 62% of vulvar SCC in northern Thailand. HPV16 was the predominant genotype similar to the data reported from other regions. HPV-positive SCC occurred in younger patients compared with HPV-negative SCC, and was associated with usual-type VIN. Vaccination against HPV16/18 may potentially prevent almost one half of vulvar SCC in northern Thailand.
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.
Proceedings of the Korean Society of Developmental Biology Conference
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2003.10a
/
pp.106-106
/
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.
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