BACKGROUND: Cervical squamous cell carcinoma (CESC), the most common subtype of cervical cancer, is primarily caused by the high-risk human papillomavirus (HPV) infection and genetic susceptibility. Single-cell RNA sequencing (scRNA-seq) has been widely used in CESC research to uncover the diversity of cell types and states within tumor tissues, enabling a detailed study of the tumor microenvironment (TME). This technology allows precise mapping of HPV infection in cervical tissues, providing valuable insights into the initiation and progression of HPV-mediated malignant transformation. METHODS: We performed the scRNA-seq to characterize gene expression in tumor tissues and paired adjacent para-cancerous tissues from four patients with early-stage CESC using the 10× Genomics platform. The HPV infection and its subtypes were identified using the scRNA data and viral sequence mapping, and trajectory analyses were performed using HPV+ or HPV- cells. Interactions between different types of keratinized cells and their interactions with other cell types were identified, and pathways and specificity markers were screened for proliferating keratinized cells. The Cancer Genome Atlas (TCGA) dataset was used to verify the prognostic correlation between tumor-specific PI3+S100A7+ keratinocyte infiltration and CESC, and the localization relationship between PI3+S100A7+ keratinocytes and macrophages was verified by immunofluorescence staining. RESULTS: Various types of keratinocytes and fibroblasts were the two cell types with the most significant differences in percentage between the tumor tissue samples and paired adjacent non-cancerous tissue samples in the early stages of CESC. We found that PI3+S100A7+ keratinocytes were associated with early HPV-positive CESC, and PI3+S100A7+ keratinocytes were more abundant in tumors than in adjacent normal tissues in the TCGA-CESC dataset. Analysis of clinical information revealed that the infiltration of PI3+S100A7+ keratinocytes was notably higher in tumors with poor prognosis than in those with good prognosis. Additionally, multiplex immunofluorescence analysis showed a specific increase in PI3+S100A7+ expression within tumor tissues, with PI3+S100A7+ keratinocytes and CD163+ macrophages being spatially very close to each other. In the analysis of cell-cell interactions, macrophages exhibited strong crosstalk with PI3+S100A7+ proliferating keratinocytes in HPV-positive CESC tumors, mediated by tumor necrosis factor (TNF), CCL2, CXCL8, and IL10, highlighting the dynamic and tumor-specific enhancement of macrophage-keratinocyte interactions, which are associated with poor prognosis and immune modulation. Using CIBERSORTx, we discovered that patients with high infiltration of both PI3+S100A7+ proliferating keratinocytes and macrophages had the shortest overall survival. In the analysis of cell-cell interactions, PI3+S100A7+ proliferating keratinocytes and macrophages were found to be involved in highly active pathways that promote differentiation and structure formation, including cytokine receptor interactions, the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway, and TNF signaling pathway regulation. Further subtyping of fibroblast populations identified four subtypes. The C1 group, characterized by its predominance in tumor tissues, is a subtype enriched with cancer-associated fibroblasts (CAFs), whereas the C3 group is primarily enriched in adjacent non-cancerous tissues and consists of undifferentiated cells. Moreover, the distinct molecular and cellular differences between HPV16- and HPV66-associated tumors were demonstrated, emphasizing the unique tumor-promoting mechanisms and microenvironmental influences driven by each HPV subtype. CONCLUSIONS We discovered a heterogeneous population of keratinocytes between tumor and adjacent non-cancerous tissues caused by HPV infection and identified macrophages and specific CAFs that play a crucial role during the early stage in promoting the inflammatory response and remodeling the cancer-promoting TME. Our findings provide new insights into the transcriptional landscape of early-stage CESC to understand the mechanism of HPV-mediated malignant transformation in cervical cancer.
Humans ; Female ; Papillomavirus Infections/genetics* ; Uterine Cervical Neoplasms/genetics* ; Carcinoma, Squamous Cell/pathology* ; Keratinocytes/metabolism* ; Single-Cell Analysis/methods* ; Tumor Microenvironment/genetics*

BACKGROUND: Head and neck cancers (HNCs) are a heterogeneous group of tumors that progress owing to varied enviromental and genetic risk factors. Viral infections are threatening and adept at altering the expression of cellular transcription factors such as nuclear factor kappa B (NF-κB) and deregulation of other cellular proteins like NF kappa B inhibitor alpha (IκBα). The present study was conducted to detect high-risk genotypes of human papillomavirus (HPV) and protein expression of NF-κB signaling pathway in HNC patients with HPV infection. METHODS: For HPV detection, genomic DNA from 152 HNC tumors was extracted formalin-fixed paraffin-embedded tissue DNA kit. For genotyping, polymerase chain reaction (PCR) using a general primer, HPV type-specific primers and agarose gel electrophoresis were performed. Immunohistochemistry (IHC) was also performed on 4-μm thick tissue sections using HPV E6 monoclonal antibody. Protein expression analysis of NF-κB signaling pathway including p50, p65, and IκBα was performed using IHC. RESULTS: PCR analysis showed that 24.3% (37/152) of HNC cases were HPV positive. Among HPV positive, 86.5% (32/37) were tobacco users, while among HPV negative, 66.9% (77/115) were tobacco users. A significant association of HPV positivity and tobacco user was observed by univariate analysis [ P   <  0.01; odds ratio (OR): 0.310, 95% confidence interval (CI): 0.110 to 0.870]. More HPV positive patients were with poor oral hygiene (78.3%) when compared with patients with good oral hygiene (21.6%) [ P  < 0.03, OR: 2.440, 95% CI: 1.650 to 3.600]. The results of the logistic regression analysis showed that age, tobacco use and oral hygiene are significant predictors ( P  < 0.02). PCR and IHC staining results confirmed that HPV16 was predominant among HNC cases (64.8%) when compared with HPV18 (35.2%). Expression of NF-κB proteins (p50, p65, and IκBα inhibitor) were also observed in HPV and non-HPV infected HNC tissues. IHC expression of p50, and p65 showed nuclear staining, while IκBα inhibitor showed cytoplasmic staining. Protein expression in HPV cases was higher as compared to HPV naive cases ( P  < 0.05). CONCLUSIONS From the study, it can be established that the use of tobacco, oral hygiene, and HPV infection may be synergistically involved in modulating the expression of NF-κB signaling pathway for the development and progression of HNC in the Pakistani population.
Alphapapillomavirus ; Antibodies, Monoclonal ; DNA ; DNA, Viral/genetics* ; Formaldehyde ; Head and Neck Neoplasms ; Humans ; NF-KappaB Inhibitor alpha/genetics* ; NF-kappa B/metabolism* ; Oral Hygiene ; Pakistan ; Papillomaviridae/metabolism* ; Papillomavirus Infections/metabolism* ; Signal Transduction ; Tobacco ; Tobacco Use ; Transcription Factors/metabolism*

OBJECTIVE: "Multi-targeting" drugs can prove fruitful to combat drug-resistance of multifactorial disease-cervical cancer. This study envisioned to reveal if Thuja homeopathic mother tincture (MT) and its bioactive component could combat human papillomavirus (HPV)-16-infected SiHa cervical cancer cells since it is globally acclaimed for HPV-mediated warts. METHODS: Thuja MT was studied for its antiproliferative and antimigratory properties in SiHa cells followed by microscopic determination of reactive oxygen species (ROS) generation by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining and loss in mitochondrial membrane potential (MtMP) by rhodamine 123 (Rh123) staining. Apoptosis and autophagy inductions were studied by acridine orange/ethidium bromide (AO/EB) staining and immunoblot analyses of marker proteins. The bioactive component of Thuja MT detected by gas chromatography-mass spectrometry was studied for antiproliferative and antimigratory properties along with in silico prediction of its cellular targets by molecular docking and oral drug forming competency. RESULTS: Thuja MT showed significant antiproliferative and antimigratory potential in SiHa cells at a 50% inhibitory concentration (IC₅₀) of 17.3 µL/mL. An increase in DCFDA fluorescence and loss in Rh123 fluorescence prove that Thuja MT acted through the burst of ROS and loss in MtMP respectively. AO/EB-stained cells under the microscope and immunoblot analyses supported Thuja-induced cellular demise via dual pathways-apoptosis and autophagy. Immunoblots showed cleavage of caspase-3 and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) along with upregulation of Beclin-1, microtubule-associated protein 1 light chain 3B (LC3B)-II, and p62 proteins. Hence, the apoptotic cascade followed a caspase-3-dependent pathway supported by PARP-1 cleavage, while autophagic death was Beclin-1-dependent and mediated by accumulation of LC3BII and p62 proteins. Thujone, detected as the bioactive principle of Thuja MT, showed greater anti-proliferative and anti-migratory potential at an IC₅₀ of 77 µg/mL, along with excellent oral drug competency with the ability for gastrointestinal absorption and blood-brain-barrier permeation with nil toxicity. Molecular docking depicted thujone with the strongest affinity for mammalian target of rapamycin, phosphoinositide 3-kinase, and protein kinase B followed by B-cell lymphoma 2, murine double minute 2 and adenosine monophosphate-activated protein kinase, which might act as upstream triggers of apoptotic-autophagic crosstalk. CONCLUSION Robust "multi-targeting" anticancer potential of Thuja drug and thujone for HPV-infected cervical cancer ascertained its therapeutic efficacy for HPV infections.
Animals ; Apoptosis ; Autophagy ; Beclin-1/pharmacology* ; Bicyclic Monoterpenes ; Caspase 3 ; Cell Line, Tumor ; Female ; Humans ; Mammals/metabolism* ; Mice ; Molecular Docking Simulation ; Papillomavirus Infections/drug therapy* ; Phosphatidylinositol 3-Kinases ; Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use* ; Reactive Oxygen Species/metabolism* ; Thuja/metabolism* ; Uterine Cervical Neoplasms/pathology*

Objective: To investigate the effect of heterogeneous nuclear ribonucleoprotein K (hnRNP K) and its interaction with human papillomavirus 16 (HPV16) on cervical intraepithelial neoplasia (CIN). Methods: The participants included 67 women with normal cervix (NC), 69 women with CINⅠ and 68 women with CINⅡ/Ⅲ in a community cohort of pathologically diagnosed women established in Jiexiu of Shanxi province, from June 2014 to June 2015. A structured questionnaire was used to collect the demographic data of the subjects and the related factors of cervical lesions. Cervical exfoliated cells and cervical tissues from biopsy or surgery were selected. The infection status of HPV16 was detected by flow-through hybridization. The protein expression levels of hnRNP K were evaluated by Western blot. SPSS 23.0 software was used to collate and analyze the data. To study the differences in demographic characteristics, related factors, hnRNP K protein and HPV16 infection among NC, CINⅠand CINⅡ/Ⅲgroups, χ(2) test, trend χ(2) test, and Kruskal-Wallis H test were conducted. Multiple comparisons of hnRNP K protein in three groups were completed by using the Bonferroni method. The OR and its 95%CI of hnRNP K, HPV16 and CIN were calculated by using the unconditional logistic regression models. Two-way interactions between hnRNP K protein and HPV16 infection on CIN were analyzed by using additive model and related indicators. Results: HPV16 infection rates were 10.4% in women with normal cervix, 14.5% in women with CINⅠ and 41.2% in women with CINⅡ/Ⅲ, respectively. The differences among three groups were significant (P<0.001). Moreover, the infection rates of HPV16 gradually increased with the increasing severity of CIN (trend χ(2)=18.512, P<0.001). The differences in protein expression of hnRNP K among three groups were significant (H=48.138, P<0.001) and the expressionincreased with the development of cervical lesionss (trend χ(2)=21.765, P<0.001). Results from the interaction analysis indicated that there were additive effects between high expression of hnRNP K protein and HPV16 in CINⅡ/Ⅲ group compared with normal group (API=0.639, 95%CI: 0.083-1.196). In contrast, no such additive effect was found in CINⅠ group. Conclusions: HPV16 infection and over-expression of hnRNP K protein were associated with the increased risk of cervical intraepithelial neoplasia. There might be interaction between hnRNP K protein overexpression and HPV16 infection existed on the progress of CINⅡ/Ⅲ.
Case-Control Studies ; Disease Progression ; Female ; Gene Expression Regulation, Neoplastic ; Heterogeneous-Nuclear Ribonucleoprotein K/metabolism* ; Human papillomavirus 16 ; Humans ; Papillomavirus Infections ; Uterine Cervical Neoplasms/virology* ; Uterine Cervical Dysplasia/virology*

OBJECTIVETo explore the viral etiology of human breast cancer to determine whether there are novel molecular targets for gene therapy of breast cancer and provide evidence for the research of gene therapy and vaccine development for breast cancer.

METHODSPCR was used to screen HPV16 and HPV18 oncogenes E6 and E7 in the SKBR3 cell line and in 76 paraffin embedded breast cancer tissue samples. RNA interference was used to knock down the expression of HPV18 E6 and E7 in SKBR3 cells, then the changes in the expression of cell-cycle related proteins, cell viability, colony formation, metastasis, and cell cycle progression were determined.

RESULTSHPV18 oncogenes E6 and E7 were amplified and sequenced from the SKBR3 cells. Of the patient samples, 6.58% and 23.68% were tested to be positive for HPV18 E6 and HPV18 E7. In the cell culture models, the knockdown of HPV18 E6 and E7 inhibited the proliferation, metastasis, and cell cycle progression of SKBR3 cell. The knockdown also clearly affected the expression levels of cell cycle related proteins.

CONCLUSIONHPV was a contributor to virus caused human breast cancer, suggesting that the oncogenes in HPV were potential targets for gene therapy of breast cancer.

Adult ; Aged ; Aged, 80 and over ; Base Sequence ; Breast Neoplasms ; genetics ; therapy ; Female ; Genetic Therapy ; methods ; Humans ; Middle Aged ; Oncogene Proteins, Viral ; genetics ; metabolism ; Papillomaviridae ; physiology ; Papillomavirus Infections ; genetics ; therapy ; Sequence Alignment

We wished to screen the cell line that stably expresses the HPV18E5 protein, and to ascertain the influence of HPV18E5 protein on cell proliferation and the cell cycle. The HPV18E5 gene was amplified by the polymerase chain reaction. Then, the His-tag pSecTag-HPV18E5 eukaryotic expression vector was constructed by digestion ligation and connection. The recombinant plasmid was transfected into Balb/c3T3 cells with lipofectamine, and positive cell lines were screened by a culture medium containing bleomycin. HPV18E5 expression in cells was confirmed by western blotting and immuno-enzymatic methods. The influence of HPV18E5 on cell proliferation and the cell cycle were detected by Cell Counting Kit-8 and flow cytometry, respectively. The pSecTag-HPV18E5 eukaryotic expression vector was constructed. After 21-day selection in a culture medium containing 400 μg/mL bleomycin, stably expressing HPV18E5 protein cells were harvested. Compared with control groups, cell proliferation in HPV18E5 stably expressed cells was obviously increased, as was the S phase in the cell cycle. Our results suggested that HPV18E5 influences cell proliferation and the cell cycle. Our study has laid the foundation of the biologic properties of HPV18E5 protein, which will aid further studies on the mechanism of action of carcinogenesis.
Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Human papillomavirus 18 ; genetics ; metabolism ; Humans ; Oncogene Proteins, Viral ; genetics ; metabolism ; Papillomavirus Infections ; physiopathology ; virology ; Transfection

Prevention of infection by the human papillomavirus (HPV) has become a hot research topic since the relationship between the HPV and cervical cancer was confirmed. Persistent infection with HPV and early expression of proteins has an important role in the pathogenesis of cervical cancer. Vaccines that protect against four high-risk types of HPV (-6, -11, -16, -18) have been used worldwide. A bivalent vaccine (HPV-16 and -18) developed by Walvax is in clinical trials. This study reviews progress in ascertainment of the structure and function of the HPV genome, the molecular mechanism of carcinogenesis, and vaccines based on virus-like particles.
Animals ; Carcinogenesis ; Female ; Humans ; Papillomaviridae ; genetics ; immunology ; metabolism ; Papillomavirus Infections ; pathology ; prevention & control ; virology ; Papillomavirus Vaccines ; genetics ; immunology ; Uterine Cervical Neoplasms ; pathology ; prevention & control ; virology ; Viral Proteins ; genetics ; immunology ; metabolism

OBJECTIVE: To explore the differential expression of microRNA (miRNA) in HPV16-positive squamous carcinoma of the cervix in the Uygur of southern Xinjiang and to predict the target genes of the miRNAs.
 METHODS: Samples of HPV16-positive squamous carcinoma of the cervix from 5 Uygurs were collected for miRNA microarray assay. The differentially expressed miRNAs were selected for further verification by real-time quantitative RT-PCR. The software, including targetscan, miRwalk, miRanda and pictar, were used to predict the target genes of the verified miRNAs.
 RESULTS: Eighteen differentially expressed miRNAs were identified by miRNA microarray assay. The significantly differentially expressed miRNA-138 and miRNA-720 were verified by real-time quantitative RT-PCR. According to the prediction, the target genes for miRNA-138 were EZH2, LYPLA1, ARHGEF3, CLNS1A, EIF4EBP1, GNAI2, LIMK1, RHOC, ROCK2, SLC20A1, TERT, and H2AFX, while for miRNA-720 were EZH2, AGAP2, SPOCK2, FGF14, HNRNPA2B1, QKI, FOXG1, ACVR1B, DNMT3A, EPHB2, LATS2, KRAS, CCND2, NBN, ENAM, AMELX, PRNP, and CALB1.
 CONCLUSION miR-138 and miR-720 are the down-regulated target miRNAs in HPV16-positive squamous carcinoma of the cervix in the Uygur of southern Xinjiang. The common target gene for miR-138 and miR-720 is EZH2, which might be related to cervical squamous carcinoma invasion and metastasis.
Carcinoma, Squamous Cell ; metabolism ; virology ; China ; Down-Regulation ; Female ; Human papillomavirus 16 ; isolation & purification ; Humans ; MicroRNAs ; metabolism ; Papillomavirus Infections ; metabolism ; Real-Time Polymerase Chain Reaction ; Uterine Cervical Neoplasms ; metabolism ; virology

OBJECTIVEInvestigating the distribution of anti-hepatitis E virus (HEV-IgG), anti-human papillomavirus (HPV L1-IgG) and risk factors among female residents in Xinmi County, to explore the influencing factors of HPV vaccine study using HEV vaccinated population as a control.

METHODSA screening study of cervical cancer in Xinmi County, Henan Province, was performed. The information of demographic characteristics and risk factors was collected using standard questionnaire. Nine ml blood was drawn from each woman for enzyme-linked immunosorbent assay to detect HEV-IgG and HPV L1-IgG antibody. Percentile, histogram and binary logistic regression model were used to describe the distribution of risk factors and their correlation to HPV and HEV infection.

RESULTSThe average age of the Xinmi female residents was 47.2 years, their positive rate of HPV L1 antibody was 26.8%, and that of HEV-IgG antibody was 31.0%, both of which were raised with age (P < 0.001). Single factor analysis showed that non-education, low-income and growing age were associated with HEV-IgG antibody positivity, and non-education, lowering ages of first sexual life and growing age were associated with HPV L1-IgG antibody positivity. Multivariable analysis showed that growing age, low-income and work as peasantry were independent risk factors for HEV-IgG antibody positivity, and lowering ages of first sexual life, non-education and growing age were independent risk factors for HPV L1-IgG antibody positivity.

CONCLUSIONSBoth the HEV-IgG and HPV L1-IgG antibodies positive rates increase with age. Age is the common risk factor of HEV-IgG and HPV L1-IgG antibodies in female residents in Xinmi County. The risk factors of HEV-IgG and HPV L1-IgG antibodies have no statistical association, neither cross reaction was found in the HEV-IgG and HPV L1-IgG detection.

Antibodies ; Antibodies, Viral ; China ; Enzyme-Linked Immunosorbent Assay ; Female ; Hepatitis E ; blood ; epidemiology ; Hepatitis E virus ; Humans ; Immunoglobulin G ; metabolism ; Middle Aged ; Papillomaviridae ; Papillomavirus Infections ; blood ; epidemiology ; Papillomavirus Vaccines ; Risk Factors ; Uterine Cervical Neoplasms ; diagnosis

OBJECTIVEThe aim of this study was to investigate the characteristics of p16 and PR immunoreactivity and HPV infection in endocervical adenocarcinoma.

METHODSParaffin blocks of 62 patients with endocervical adnocarcinoma treated in the Cancer Institute and Hospital, Chinese Academy of Medical Sciences from year 2005 to year 2010 were collected. p16 and PR immunostaining and HPV detecting by SPF-10 PCR were conducted on all cases.

RESULTSHPV infection rate of the 62 endocervical adnocarcinoma cases was 74.2% with four cases combined with CIN3. Among the 46 HPV-positive cases, there were 22 cases of HPV18 infection (47.8%), 14 cases of HPV16 infection (30.4%), one case of HPV59 infection (2.2%). and nine multiple HPV infection cases (19.6%). The mean age of the 16 HPV-negative cases was (49.6 ± 10.5)year, while the mean age of the 46 HPV-positive cases was (42.8 ± 9.7)year, showing a significant difference between the two subgroups (P = 0.022). The positive rate of p16 infection was 80.6%. Association analysis showed that the results of p16 and HPV test were independent to each other (P = 0.077). The positive rate of PR was 3.2%. Among the 62 cases, there were 24 cases containing normal cervical glands, with 19 cases PR-positive in the normal cervical glands and the positive rate was 79.2%. The difference of PR positivity between neoplastic glands and normal glands was statistically significant by Chi-square test (P < 0.01) .

CONCLUSIONSThe HPV infection rate of endocervical adnocarcinoma is 74.2%, and the major subtypes were HPV16 and HPV18 infection. p16 immunoreactivity in endocervical adenocarcinoma maybe not the proof of high-risk HPV-related neoplasm. PR staining can be used as a reference designator to differentiate between neoplastic and normal cervical glands.

Adenocarcinoma ; metabolism ; pathology ; virology ; Adult ; Cervical Intraepithelial Neoplasia ; metabolism ; pathology ; virology ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Female ; Human papillomavirus 16 ; isolation & purification ; Human papillomavirus 18 ; isolation & purification ; Humans ; Immunohistochemistry ; Middle Aged ; Papillomaviridae ; isolation & purification ; Papillomavirus Infections ; virology ; Receptors, Progesterone ; metabolism ; Retrospective Studies ; Uterine Cervical Neoplasms ; metabolism ; pathology ; virology