E6 mRNA Research Articles

Clinical performance of multiplex high‐risk e6 mrna expression in comparison with hpv dna subtypes for the identification of women at risk of cervical cancer

We compared multiplex E6 messenger ribonucleic acid (mRNA) tests using real-time quantitative reverse transcriptase polymerase chain reactions (PCR) with human papillomavirus (HPV) DNA subtypes using a MY11/GP6+ PCR-based reverse-blot assay to identify cervical intraepithelial neoplasias of grade 2 or worse (CIN2+). In total, 684 women were studied, of whom 377 (55%) were diagnosed with CIN2+ histologically. The specificity of HPV mRNA to predict histological CIN2+ was higher than that of HPV DNA (81.3% vs. 44.2%). The odds ratios (ORs) to predict histological CIN2+ in women with positive for type 16, 18, 31, and 45 E6 mRNA or by HPV DNA detection were 7.1 (95% confidence interval [CI] 3.9-13.1) and 2.5 (95%CI 1.9-3.5), respectively, compared to those with negative for E6 mRNA or HPV DNA. The OR to predict histological CIN2+ in women with a cytological grade <CIN2+ and positive for type 16, 18, 31, and 45 E6 mRNA was 9.7 (95%CI 3.2-29.2), compared to those with a cytological grade <CIN2+, and negative for E6 mRNA (OR = 1), those with a cytological grade CIN2+, and negative for mRNA (OR = 6.9, 95%CI 4.4-10.8), and those with a cytological grade CIN2+ and positive for mRNA (OR = 28.0, 95%CI 9.8-79.6). As a HPV DNA positive triage, the OR to predict histological CIN2+ in women with a cytological grade <CIN2+ and positive for mRNA was higher than those with negative for mRNA (OR:12.8 [95%CI 3.6-5.4] vs. OR:1.6 [95%CI 0.9-2.9]). In conclusion, multiplex HPV E6 mRNA detection can be used as a triage for women with cytological grade <CIN2+.

Comparing triage algorithms using HPV DNA genotyping, HPV E7 mRNA detection and cytology in high-risk HPV DNA-positive women

BackgroundHigh-risk human papillomavirus (hrHPV) DNA positive women require triage testing to identify those with high-grade cervical intraepithelial neoplasia or cancer (≥CIN2). ObjectiveComparing three triage algorithms (1) E7 mRNA testing following HPV16/18/31/33/45/52/58 genotyping (E7 mRNA test), (2) HPV16/18 DNA genotyping and (3) cytology, for ≥CIN2 detection in hrHPV DNA-positive women. Study designhrHPV DNA-positive women aged 18–63 years visiting gynecology outpatient clinics were included in a prospective observational cohort study. From these women a cervical scrape and colposcopy-directed biopsies were obtained. Cervical scrapes were evaluated by cytology, HPV DNA genotyping by bead-based multiplex genotyping of GP5+6+-PCR-products, and presence of HPV16/18/31/33/45/52/58 E7 mRNA using nucleic acid sequence-based amplification (NASBA) in DNA positive women for respective HPV types. Sensitivities and specificities for ≥CIN2 were compared between E7 mRNA test and HPV16/18 DNA genotyping in the total group (n=348), and E7 mRNA test and cytology in a subgroup of women referred for non-cervix-related gynecological complaints (n=133). ResultsSensitivity for ≥CIN2 of the E7 mRNA test was slightly higher than that of HPV16/18 DNA genotyping (66.9% versus 60.9%; ratio 1.10, 95% CI: 1.0002–1.21), at similar specificity (54.8% versus 52.3%; ratio 1.05, 95% CI: 0.93–1.18). Neither sensitivity nor specificity of the E7 mRNA test differed significantly from that of cytology (sensitivity: 68.8% versus 75.0%; ratio 0.92, 95% CI: 0.72–1.17; specificity: 59.4% versus 65.3%; ratio 0.91, 95% CI: 0.75–1.10). ConclusionFor detection of ≥CIN2 in hrHPV DNA-positive women, an algorithm including E7 mRNA testing following HPV16/18/31/33/45/52/58 DNA genotyping performs similar to HPV16/18 DNA genotyping or cytology.

MicroRNA-16 modulates HuR regulation of cyclin E1 in breast cancer cells.

RNA binding protein (RBPs) and microRNAs (miRNAs or miRs) are post-transcriptional regulators of gene expression that are implicated in development of cancers. Although their individual roles have been studied, the crosstalk between RBPs and miRNAs is under intense investigation. Here, we show that in breast cancer cells, cyclin E1 upregulation by the RBP HuR is through specific binding to regions in the cyclin E1 mRNA 3' untranslated region (3'UTR) containing U-rich elements. Similarly, miR-16 represses cyclin E1, dependent on its cognate binding sites in the cyclin E1 3'UTR. Evidence in the literature indicates that HuR can regulate miRNA expression and recruit or dissociate RNA-induced silencing complexes (RISC). Despite this, miR-16 and HuR do not affect the other’s expression level or binding to the cyclin E1 3'UTR. While HuR overexpression partially blocks miR-16 repression of a reporter mRNA containing the cyclin E1 3'UTR, it does not block miR-16 repression of endogenous cyclin E1 mRNA. In contrast, miR-16 blocks HuR-mediated upregulation of cyclin E1. Overall our results suggest that miR-16 can override HuR upregulation of cyclin E1 without affecting HuR expression or association with the cyclin E1 mRNA.

A novel dithiocarbamate derivative induces cell apoptosis through p53-dependent intrinsic pathway and suppresses the expression of the E6 oncogene of human papillomavirus 18 in HeLa cells.

Dithiocarbamates (DTCs) exhibit a broad spectrum of antitumor activities, however, their molecular mechanisms of antitumor have not yet been elucidated. Previously, we have synthesized a series of novel dithiocarbamate derivatives. These DTCs were examined for cytotoxic activities against five human cancer cell lines. In this study, one of dithiocarbamate (DTC1) with higher potential for HeLa cells was chosen to investigate molecular mechanisms for its anti-tumor activities. DTC1 could inhibit proliferation, and highly induce apoptosis in HeLa cells by activating caspase-3, -6 and -9; moreover, activities of caspase-3, -6 and -9 were inhibited by pan-caspase inhibitor, Z-VAD-FMK. Furthermore, DTC1 decreased the levels of Bcl-2 and Bcl-xL, and increased expression of cytosol cytochrome c, Bak, Bax and p53 in a time-dependent manner but had no effect on the level of Rb. It was shown that DTC1 induced HeLa cells apoptosis through a p53-dependent pathway as tested by the wild type p53 inhibitor, pifithrin-α. Additionally, the relative expression of E6 and E7 were evaluated in HPV18-positive (HeLa cells) by real-time PCR and western blotting. The results firstly demonstrated that DTC1 suppressed both expression of E6 mRNA and E6 oncoprotein, but had no effect on the expression of E7 mRNA and protein in HPV18. Our results suggested that DTC1 may serve as novel chemotherapeutic agents in the treatment of cervical cancer and potential anti-HPV virus candidates that merit further studies.

Development of anti-E6 pegylated lipoplexes for mucosal application in the context of cervical preneoplastic lesions

Cervical cancer induced by human papillomavirus (HPV) is the fourth highest mortality causing cancer in women despite the use of prophylactic vaccines. E6 targeting represents an attractive strategy to treat this cancer. Indeed, oncoprotein E6 is produced by keratinocytes infected by HPV and is partially responsible for carcinogenesis. E6 interferes with the apoptosis process in stressed cells by degradation of p53 tumor suppressor gene. Our strategy consists in using E6 siRNA complexed with pegylated lipoplexes. The addition of hydrophilic polymer around the nanoparticles is crucial to use them by vaginal application on account of cervicovaginal mucus. Physicochemical characteristics were evaluated and in vitro assays were performed to evaluate transfection potential, E6 mRNA extinction and p53 re-expression. Cationic liposomes DOTAP/Cholesterol/DOPE 1/0.75/0.5 (N/P 2.5) with or without 50% DSPE-PEG2000 and associated with siE6 have demonstrated good physicochemical characteristics in terms of complexation, size, surface charge and stability. Both lipoplexes have been tested on CaSki cell line (HPV 16+) with 50nM and 100nM of siE6. Lipoplexes formulations induce 30–40% of E6 mRNA extinction and induce the re-expression of p53. In conclusion, pegylated anti-E6 lipoplexes have demonstrated their efficiency to cross the cellular membrane and to release siRNA into the cytoplasm confirmed by final p53 protein production.

Frequency and prognostic significance of p16(INK4A) protein overexpression and transcriptionally active human papillomavirus infection in laryngeal squamous cell carcinoma.

Background:Human papillomavirus (HPV) infection is a powerful prognostic biomarker in a subset of head and neck squamous cell carcinomas, specifically oropharyngeal cancers. However, the role of HPV in non-oropharyngeal sites, such as the larynx, remains unconfirmed.Methods:We evaluated a cohort of 324 laryngeal squamous cell carcinoma (LSCC) patients for the expression of p16INK4A (p16) protein by immunohistochemistry (IHC) and for high-risk HPV E6 and E7 mRNA transcripts by RNA in situ hybridisation (ISH). p16 expression and HPV status were correlated with clinicopathological features and outcomes.Results:Of 307 patients assessable for p16 IHC, 20 (6.5%) were p16 positive. Females and node-positive patients were more likely to be p16 positive (P<0.05). There were no other significant clinical or demographic differences between p16-positive and -negative cases. There was no difference in overall survival (OS) between p16-positive and -negative patients with 2-year survival of 79% in each group (HR=0.83, 95% CI 0.36–1.89, P=0.65). There was no statistically significant difference in failure-free survival (FFS) with 2-year FFS of 79% and 66% for p16-positive and -negative patients, respectively (HR=0.60, 95% CI 0.26–1.36, P=0.22). Only seven cases were found to be HPV RNA ISH positive, all of which were p16 IHC positive. There was no statistically significant difference in OS between patients with HPV RNA ISH-positive tumours compared with -negative tumours with 2-year survival of 86% and 71%, respectively (HR=0.76, 95% CI 0.23–2.5, P=0.65). The 2-year FFS was 86% and 59%, respectively (HR=0.62, 95% CI 0.19–2.03, P=0.43).Conclusions:p16 overexpression is infrequent in LSCC and the proportion of cases with high-risk HPV transcripts is even lower. There are no statistically significant correlations between p16 IHC or HPV RNA ISH status and OS or disease outcomes.

Redox-regulated suppression of splenic T-lymphocyte activation in a model of sympathoexcitation.

Sympathoexcitation, increased circulating norepinephrine, and elevated levels of reactive oxygen species are driving forces underlying numerous cardiovascular diseases, including hypertension. However, the effects of elevated norepinephrine and subsequent reactive oxygen species production in splenic T-lymphocytes during hypertension are not currently understood. We hypothesized that increased systemic levels of norepinephrine inhibits the activation of splenic T-lymphocytes via redox signaling. To address this hypothesis, we examined the status of T-lymphocyte activation in spleens of a mouse model of sympathoexcitation-driven hypertension (ie, norepinephrine infusion). Splenic T-lymphocytes from norepinephrine-infused mice demonstrated decreased proliferation accompanied by a reduction in interferon gamma and tumor necrosis factor-α production as compared with T-lymphocytes from saline-infused mice. Additionally, norepinephrine directly inhibited splenic T-lymphocyte proliferation and cytokine production ex vivo in a dose-dependent manner. Furthermore, norepinephrine caused an increase in G1 arrest in norepinephrine-treated T-lymphocytes, and this was accompanied by a decrease in pro-growth cyclin D3, E1, and E2 mRNA expression. Interestingly, norepinephrine caused an increase in cellular superoxide, which was shown to be partially causal to the inhibitory effects of norepinephrine, as antioxidant supplementation (ie, Tempol) to norepinephrine-infused mice moderately restored T-lymphocyte growth and proinflammatory cytokine production. Our findings indicate that suppression of splenic T-lymphocyte activation occurs in a norepinephrine-driven model of hypertension due to, at least in part, an increase in superoxide. We speculate that further understanding of how norepinephrine mediates its inhibitory effects on splenic T-lymphocytes may elucidate novel pathways for therapeutic mimicry to suppress T-lymphocyte-mediated inflammation in an array of diseases.

Honeybee venom possesses anticancer and antiviral effects by differential inhibition of HPV E6 and E7 expression on cervical cancer cell line

Bee venom (BV) therapy is a type of alternative medical treatment used to treat various diseases in oriental medicine. The mechanisms underlying the effects of BV remain poorly understood. In the present study, we evaluated the antiviral effect of BV on cervical carcinoma cell lines (CaSki, HeLa, C33A and TC-1). BV treatments resulted in a more significant suppression of cell growth in HPV16-infected cells (CaSki) and a lesser suppression in HPV18-infected cells (HeLa). However, less suppression was observed in HPV-negative C33A cells. In 10µg/ml BV-treated CaSki cells, the mRNA expression and protein levels of HPV16 E6 and E7 were significantly decreased by BV, while HPV18 E6 and E7 mRNA expression levels were not significantly altered by 10µg/ml BV-treated HeLa cells. The antitumor effects of BV were in accordance with invitro data, in restricting tumor growth invivo and were much more effective on the suppression of tumor growth. Furthermore, the mRNA and protein expression levels of HPV16 E6 and E7 were decreased by BV in TC-1 tumors. These findings demonstrated the antiviral effects of BV in HPV-infected cervical cancer cells and the anticancer effects of BV in HPV16 E6/E7-expressed TC-1 tumors. Collectively, BV plays a differential role in suppressing HPV16-infected cells (CaSki cells) and HPV18-infected cells (HeLa cells) by the downregulation of E6/E7 protein of HPV16/18.

Polymorphisms and functional analysis of the intact human papillomavirus16 e2 gene.

High risk human papillomavirus (HR-HPV) E2 proteins play roles in transcriptional regulation and are commonly functionally disrupted when the HPV genome integrates into host chromosomes. Some 15-40% of cancer cases, however, contain an intact E2 gene or episomal HPV. In these cases, polymorphism of the E2 gene might be involved. This study aimed to determine polymorphisms of the E2 gene in episomal HPV16 detected in high grade squamous intraepithelial lesions and squamous cell carcinomas and altered functions compared to the E2 prototype. The E2 gene was amplified and sequenced. Two expression vectors containing E2 gene polymorphisms were constructed and transfected in SiHa and C33A cells, then E6 gene as well as Il- 10 and TNF-α expression was determined by quantitative RT-PCR. Expression vectors and reporter vectors containing the HPV16 long control region (LCR) were co-transfected and transcriptional activity was determined. The results showed that a total of 32 nucleotides and 23 amino acids were changed in all 20 cases of study, found in the transactivation (TA) domain, hinge (H) region and DNA binding (DB) domain with 14, 5 and 13 nucleotide positions. They mostly caused amino acid change. The expressing vectors containing different E2 gene polymorphisms showed E6 mRNA suppression, TNF-α mRNA suppression and IL-10 induction but no statistically significant differences when compared to the E2 prototype. Moreover, promoter activity in HPV16 LCR was not affected by E2 protein with different gene polymorphisms, in contrast to nucleotide variations in LCR that showed an effect on transcription activity. These results demonstrated that E2 gene polymorphisms of episomal HPV16 did not affect transcriptional regulation and suggested that nucleotide variation as well as epigenetic modification of the LCR might play a role in inducing malignant transformation of cells containing episomal HPV16.

Surface modification of tri-calcium phosphate nanoparticles by DOPE and/or anti-E6 antibody to enhance uptake of antisense of E6 mRNA

The main aim of this study was to evaluate the uptake of E6 mRNA antisense into cervical cancer cells, induced by human papilloma virus (HPV). In this study, the carrier of the antisense was tri-calcium phosphate nanoparticles (TCP NPs) conjugated with dioleoyl phosphatidyl ethanolamine (DOPE) and/or anti-E6 antibody. At first, TCP NPs were synthesized, coated with carboxy-polyethylene glycol, and then conjugated with anti-E6 antibody and/or DOPE by carbodiimide cross-linker. Then, a single stranded DNA, which was complementary (antisense) of E6 mRNA, was attached to each one. Finally, the uptake of conjugated and unconjugated TCP NPs into HelaS3 cells was separately evaluated by Fourier transform infrared spectroscopy, optical microscopy, and fluorescent microscopy. Also, the cytotoxicity of these carriers was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Overall, 4 types of TCP NPs were used in this study, including 1) TCP NPs conjugated with DOPE (TCP NPs/DOPE), 2) TCP NPs conjugated with DOPE and antibody (TCP NPs/DOPE/Anti-E6 Ab), 3) TCP NPs conjugated with antibody (TCP NPs/Anti-E6 Ab), and 4) TCP NPs which not conjugated with DOPE and antibody (unconjugated TCP NPs). Uptake tests showed that although all types of TCP NPs could transfer antisense of E6 mRNA into HelaS3 cells, TCP NPs/DOPE and TCP NPs/DOPE/Anti-E6 Ab had more uptake than TCP NPs/Anti-E6 Ab and unconjugated TCP NPs. Moreover, MTT assay showed that TCP NPs/DOPE was more toxic than TCP NPs/DOPE/Anti-E6 Ab, TCP NPs/Anti-E6 Ab, and unconjugated TCP NPs. It can be concluded that TCP NPs/DOPE/Anti-E6 Ab is a good choice for oligonucleotide delivery, because of higher uptake and less toxicity, compared with other formulations.

Human papillomavirus does not have a causal role in colorectal carcinogenesis.

To investigate the presence of human papillomavirus (HPV) DNA along with the integration, the quantification and the expression of the HPV16 in colorectal cancers. A prospective series of colorectal tumors were genotyped for HPV DNA. The clinical and pathological variables of the HPV-positive tumors were compared to those of HPV-negative samples. The integration status of HPV16 was evaluated by calculating E2/E6 ng ratios. HPV16-positive tumors were also evaluated for (1) E2, E4, E5, E6 and E7 viral gene ng quantification; (2) relative quantification compared to W12 cells; and (3) viral E2, E4, E5, E6 and E7 mRNA transcripts by real-time polymerase chain reaction. HPV infection was detected in 16.9% of all tumors examined, and HPV16 was the most frequent type detected (63.6% of positive tissues). Notably, the clinical and pathological features of HPV-positive colorectal cancers were not significantly different than those of HPV-negative cancers (χ (2) and t-test for all clinical and pathological features of HPV-positive vs HPV-negative colorectal cancers: p ns). HPV16 DNA was present exclusively in episomal form, and the HPV16 E2, E4, E5, E6 and E7 genes were detected in trace nanogram quantities. Furthermore, the HPV16 genes ranged from 10(-3) to 10(-9) compared to W12 cells at an episomal stage. Although the extractions were validated by housekeeping gene expression, all the HPV16 positive tissues were transcriptionally inactive for the E2, E4, E5, E6 and E7 mRNAs. Based on our results, HPV is unlikely involved in colorectal carcinogenesis.

The E3 ubiquitin ligase seven in absentia homolog 1 may be a potential new therapeutic target for Parkinson's disease.

In this study, we investigated the effect of an antibody against E3 ubiquitin ligase seven in absentia homolog 1 (SIAH-1) in PC12 cells. 1-Methyl-4-phenylpyridinium (MPP+) treatment increased α-synuclein, E1 and SIAH-1 protein levels in PC12 cells, and it reduced cell viability; however, there was no significant change in light chain 3 expression. Treatment with an SIAH-1 antibody decreased mRNA expression levels of α-synuclein, light chain 3 and SIAH-1, but increased E1 mRNA expression. It also increased cell viability. Combined treatment with MPP+ and rapamycin reduced SIAH-1 and α-synuclein levels. Treatment with SIAH-1 antibody alone diminished α-synuclein immunoreactivity in PC12 cells, and reduced the colocalization of α-synuclein and light chain 3. These findings suggest that the SIAH-1 antibody reduces the monoubiquitination and aggregation of α-synuclein, promoting its degradation by the ubiquitin-proteasome pathway. Consequently, SIAH-1 may be a potential new therapeutic target for Parkinson's disease.

Prevalence of human papillomavirus in laryngeal and hypopharyngeal squamous cell carcinomas in northern Spain

BackgroundRecent studies support a role for human papillomavirus (HPV) in oropharyngeal squamous cell carcinomas (SCCs); however, the significance of HPV in non-oropharyngeal head and neck cancers is uncertain. The aim of this study was to determine the prevalence of HPV in a large cohort of laryngeal and hypopharyngeal SCCs in northern Spain. Materials and methodsClinical records and paraffin-embedded tumor specimens of 124 consecutive patients surgically treated for laryngeal (62 cases) and hypopharyngeal (62 cases) SCCs between 2002 and 2007 were retrieved. All cases were histologically evaluated, and presence of HPV was assessed by p16-immunohistochemistry followed by GP5+/6+-PCR-based DNA detection. Samples positive in both assays were subjected to HPV genotyping and HPV E6 transcript analysis. ResultsSeventeen cases (14%) were positive for p16 immunostaining, of which 2 (1 larynx, 1 hypopharynx, 1.6% of total series) were found positive for HPV DNA by subsequent GP5+6+-PCR. Both SCCs contained HPV type 16 and showed HPV16 E6 mRNA expression. ConclusionsHPV is only occasionally involved in laryngeal and hypopharyngeal SCC patients in northern Spain.

Persistence of Human Papillomavirus, Overexpression of p53, and Outcomes of Patients After Endoscopic Ablation of Barrett's Esophagus

We investigated the role of high-risk human papillomavirus (hr-HPV) in patients with Barrett's dysplasia and adenocarcinoma (EAC). Clearance vs persistence of HPV (DNA, E6 or E7 mRNA, and p16INK4A protein) and overexpression or mutation of p53 were determined for 40 patients who underwent endotherapy for Barrett's dysplasia or EAC. After ablation, dysplasia or neoplasia was eradicated in 34 subjects (24 squamous, 10 intestinal metaplasia). Six patients had detectable lesions after treatment; 2 were positive for transcriptionally active hr-HPV, and 4 had overexpression of p53. Before endotherapy, 15 patients had biologically active hr-HPV, 13 cleared the infection with treatment, and dysplasia or EAC was eliminated from 12 patients. One patient who cleared HPV after ablation acquired a p53 mutation, and their cancer progressed. Of 13 patients with overexpression of p53 before treatment, 10 cleared the p53 abnormality after ablation with eradication of dysplasia or neoplasia, whereas 3 of 13 had persistent p53 mutation-associated dysplasia after endotherapy (P = .004). Immunohistochemical and sequence analyses of p53 produced concordant results for 36 of 40 samples (90%). Detection of dysplasia or neoplasia after treatment was associated with HPV persistence or continued p53 overexpression.

Regulation of cell cycle of hepatocellular carcinoma by NF90 through modulation of cyclin E1 mRNA stability.

Activation of cyclin E1, a key regulator of the G1/S cell-cycle transition, has been implicated in many cancers including hepatocellular carcinoma (HCC). Although much is known about the regulation of cyclin E1 expression and stability, its post-transcriptional regulation mechanism remains incompletely understood. Here, we report that nuclear factor 90 (NF90), a double-stranded RNA (dsRNA) binding protein, regulates cyclin E1 in HCC. We demonstrate that NF90 is upregulated in HCC specimens and that suppression of NF90 decreases HCC cell growth and delays G1/S transition. We identified cyclin E1 as a new target of NF90 and found a significant correlation between NF90 and cyclin E1 expression in HCC. The mRNA and protein levels of cyclin E1 were downregulated upon NF90 knockdown. Suppression of NF90 caused a decrease in the half-life of cyclin E1 mRNA, which was rescued by ectopic expression of NF90. Furthermore, NF90 bound to the 3' untranslated regions (3'UTRs) of cyclin E1 mRNA in vitro and in vivo. Knockdown of NF90 also inhibited tumor growth of HCC cell lines in mouse xenograft model. Moreover, we showed that inhibition of NF90 sensitized HCC cells to the cyclin-dependent kinase 2 (CDK2) inhibitor, roscovitine. Taken together, downregulation of NF90 in HCC cell lines can delay cell-cycle progression, inhibit cell proliferation, and reduce tumorigenic capacity in vivo. These results suggest that NF90 has an important role in HCC pathogenesis and that it can serve as a novel therapeutic target for HCC.

SAHA inhibits the transcription initiation of HPV18 E6/E7 genes in HeLa cervical cancer cells

High risk human papillomavirus (HPV) is a well recognized causative agent of cervical cancer. Suberoylanilide hydroxamic acid (SAHA) is a potential anti-cervical cancer drug; however, its effect on the expression of HPV E6 and E7 genes remains unclear. Here, we show that, in SAHA treated HeLa cells, HPV18 E6 and E7 mRNA and protein levels were reduced, HPV18 promoter activity was decreased, and the association of RNP II with HPV18 promoter was diminished, suggesting that SAHA inhibited the transcription initiation of HPV18 E6 and E7 genes. In SAHA-treated HeLa, although the level of lysine 9-acetylated histone H3 in the whole cell extracts increased obviously, its enrichment on HPV18 promoter was significantly reduced which is correlated with the down-regulation of HPV E6 and E7.

A systems biology analysis of the changes in gene expression via silencing of HPV-18 E1 expression in HeLa cells.

Previous studies have reported the detection of a truncated E1 mRNA generated from HPV-18 in HeLa cells. Although it is unclear whether a truncated E1 protein could function as a replicative helicase for viral replication, it would still retain binding sites for potential interactions with different host cell proteins. Furthermore, in this study, we found evidence in support of expression of full-length HPV-18 E1 mRNA in HeLa cells. To determine whether interactions between E1 and cellular proteins play an important role in cellular processes other than viral replication, genome-wide expression profiles of HPV-18 positive HeLa cells were compared before and after the siRNA knockdown of E1 expression. Differential expression and gene set enrichment analysis uncovered four functionally related sets of genes implicated in host defence mechanisms against viral infection. These included the toll-like receptor, interferon and apoptosis pathways, along with the antiviral interferon-stimulated gene set. In addition, we found that the transcriptional coactivator E1A-binding protein p300 (EP300) was downregulated, which is interesting given that EP300 is thought to be required for the transcription of HPV-18 genes in HeLa cells. The observed changes in gene expression produced via the silencing of HPV-18 E1 expression in HeLa cells indicate that in addition to its well-known role in viral replication, the E1 protein may also play an important role in mitigating the host's ability to defend against viral infection.

The cellular uptake of antisense oligonucleotid of E6 mRNA into cervical cancer cells by DOPE-modified hydroxyapatite nanoparticles

Objective(s): Although several chemical and physical methods for gene delivery have been introduced, their cytotoxicity, non-specific immune responses and the lack of biodegradability remain the main issues. In this study, hydroxyapatite nanoparticles (NPs) and 1,2-dioleoyl-sn-glycero-3-phosphoethanol​amine (DOPE)-modified hydroxyapatite NPs was coated with antisense oligonucleotide of E6 mRNA, and their uptakes into the cervical cancer cell line were evaluated. Materials and Methods: Calcium nitrate and diammonium phosphate were used for the synthesis of the hydroxyapatite nanoparticle. Thus, they were coated with polyethylene glycol (PEG), DOPE and antisense oligonucleotide of E6 mRNA using a cross-linker. Then, hydroxyapatite NPs and DOPE-modified hydroxyapatite NPs were incubated 48 hours with cervical cancer cells and their uptakes were evaluated by fluorescent microscopy. Results: The hydroxyapatite NPs had different shapes and some agglomeration with average size of 100 nm. The results showed DOPE-modified hydroxyapatite NPs had higher uptake than hydroxyapatite NPs (P<0.05). Conclusions: Hydroxyapatite NPs conjugated with DOPE are a good choice for gene delivery and silencing of viral genes in cervical cancer cells, but their efficacy should be addressed more in future studies.

Abstract 1378: The histone methyltransferase adaptor WDR5 is a novel cofactor in neuroblastoma

Abstract Introduction: Neuroblastoma is the most common extracranial solid malignancy in early childhood. WDR5 is an essential component of the MLL and SET1/COMPASS histone H3 lysine 4 (H3K4) methyltransferase complex and plays a key role in the trimethylation of H3K4. However, there is no previous study establishing the role of WDR5 in neuroblastoma. Results: Affymetrix gene array studies showed that knocking-down WDR5 expression with siRNAs reduced the expression of the cell cycle genes cyclin E1. Consistently, RT-PCR and immunoblot confirmed that WDR5 siRNAs reduced cyclin E1 mRNA and protein expression, ChIP assays showed that WDR5 siRNAs reduced histone H3K4 tri-methylation at the cyclin E1 gene promoter, and Alamar blue assays demonstrated that WDR5 induced neuroblastoma cell proliferation. Additionally, high levels of WDR5 gene expression in human neuroblastoma tissues predict poor patient survival independent of patient age and disease stage. Conclusions: Our data demonstrate WDR5 as a novel co-factor in neuroblastoma oncogenesis, and provide critical evidence to support the application of WDR5 inhibitors for the therapy of neuroblastoma patients. Citation Format: Yuting Sun, Pei Yan Liu, Daniel Carter, Nicolas Sokolowski, Karen L. MacKenzie, Glenn M. Marshall, Tao Liu. The histone methyltransferase adaptor WDR5 is a novel cofactor in neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1378. doi:10.1158/1538-7445.AM2014-1378

MiR-122 directly inhibits human papillomavirus E6 gene and enhances interferon signaling through blocking suppressor of cytokine signaling 1 in SiHa cells.

Human Papillomavirus (HPV) 16 infection is considered as one of the significant causes of human cervical cancer. The expression of the viral oncogenes like E6 and E7 play an important role in the development of the cancer. MiR-122 has been reported to exhibit a strong relationship with hepatitis viruses and take part in several tumor development, while the effects of miR-122 on HPV infection and the HPV viral oncogenes expression still remain unexplored. In this study, using RNAhybrid software, the potential binding sites between miR-122 and HPV16 E6 and E7 mRNAs were identified. Over and loss of miR-122 function showed that miR-122 could directly bind with HPV16 E6 mRNA and significantly inhibit its expression in SiHa cells, which was further confirmed by constructing the miR-122-E6-mu to eliminate the miR-122 binding effects with E6. The increase of the expression of type I interferon (IFN) and its classical effective molecules and the phosphorylation of signal transducers and activators of transcription (STAT1) protein indicated that miR-122 might enhance type I interferon in cervical carcinoma cells, which explained the significant reduction of HPV16 E7 and E6*I mRNA expression. This might be due to the binding between miR-122 and suppressor of cytokine signaling 1 (SOCS1) mRNA, which is the suppressor of interferon signaling pathway. Moreover, it was identified that the miR-122 binding position was nt359-nt375 in SOCS1 mRNA. Taken together, this study indicated that HPV16 could be effectively inhibited by miR-122 through both direct binding with E6 mRNA and promoting SOCS1-dependent IFN signaling pathway. Thus, miR-122 may serve as a new therapeutic option for inhibiting HPV infection.