Dongrui Deng

RNA interference against HPV16 E7 oncogene leads to viral E6 and E7 suppression in cervical cancer cells and apoptosis via upregulation of Rb and p53

The simultaneous expression of human papillomavirus type 16 (HPV16) E6 and E7 oncogenes is pivotal for malignant transformation and maintenance of malignant phenotypes. Silencing these oncogenes is considered to be applicable in molecular therapies of human cervical cancer. However, it remains to be determined whether HPV16 E6 and E7 could be both silenced to obtain most efficient antitumor activity by using RNA interference (RNAi) technology. Herein, we designed a small interfering RNA (siRNA) targeting HPV16-E7 region to degrade either E6, or truncated E6 (E6*) and E7 mRNAs and to simultaneously knockdown both E6 and E7 expression. Firstly, the sequence targeting HPV16-E7 region was inserted into the shRNA packing vector pSIREN-DNR, yielding pSIREN-16E7 to stably express corresponding shRNA. HPV16-transformed SiHa and CaSki cells were used as a model system; RT-PCR, Western Blotting, MTT assay, TUNEL staining, Annexin V apoptosis assay and flow cytometry were applied to examine the effects of pSIREN-16E7. Our results indicated that HPV16-E7 specific shRNA (16E7-shRNA) induced selective degradation of E6 and E7 mRNAs and proteins. E6 silencing induced accumulation of cellular p53 and p21. In contrast, E7 silencing induced hypophosphorylation of retinoblastoma (Rb) protein. The loss of E6 and E7 reduced cell growth and ultimately resulted in massive apoptotic cell death selectively in HPV-positive cancer cells, compared with the HPV-negative ones. We demonstrated that 16E7-shRNA can induce simultaneous E6 and E7 suppression and lead to striking apoptosis in HPV16-related cancer cells by activating cellular p53, p21 and Rb. Therefore, RNAi using E7 shRNA may have the gene-specific therapy potential for HPV16-related cancers.

Changes in Prevalence and Clinical Characteristics of Cervical Cancer in the People's Republic of China: A Study of 10,012 Cases From a Nationwide Working Group

PURPOSE About one-third of the worlds total annual new cervical cancer cases are found in the Peoples Republic of China. We investigate the prevalence and clinical characteristics of cervical cancer cases in the Peoples Republic of China over the past decade. METHOD A total of 10,012 hospitalized patients with cervical cancer from regions nationwide were enrolled from 2000 to 2009. Demographic and clinical characteristics, therapeutic strategies, and outcomes were analyzed. RESULTS The mean age at diagnosis of all cervical cancer patients was 44.7 ± 9.5 years, which is 5-10 years younger than mean ages reported before 2000 in the Peoples Republic of China. The age distribution showed 16.0% of patients were ≤35 years old, 41.7% were 35-45 years old, and 41.7% were >45 years old. Early stage diagnoses were most prevalent: 57.3% were stage I, 33.9% were stage II, and 4.3% were stage III or IV. Most patients (83.9%) were treated with surgery, and only 9.5% had radiotherapy alone. Among 8,405 patients treated with surgery, 68.6% received adjuvant treatments, including chemotherapy (20.9%), radiotherapy (26.0%), and chemoradiotherapy (21.9%). Among stage IA patients, 16.0% were treated with corpus uteri preservation. The proportion of ovarian preservation was 42.0%. CONCLUSIONS Cervical cancer cases in the Peoples Republic of China show increasing prevalence in young patients and at early stages. In the past 10 years, surgery has become the dominant treatment and is increasingly combined with adjuvant chemotherapy for patients with stages I and II. Conservative surgical approaches are reasonable options for genital organ preservation in selected patients.

Matched-case comparison of neoadjuvant chemotherapy in patients with FIGO stage IB1-IIB cervical cancer to establish selection criteria.

OBJECTIVE Neoadjuvant chemotherapy (NACT) for cervical cancer still remains controversial. NACT was evaluated to establish selection criteria. METHODS A matched-case comparison was designed for the NACT group (n=707) and primary surgery treatment (PST; n=707) group to investigate short-term responses and high/intermediate risk factors (HRFs/IRFs). The 5-year disease-free survival (DFS) and overall survival (OS) rates were stratified by NACT response, HRFs/IRFs, International Federation of Gynecology and Obstetrics (FIGO) stage and tumour size, respectively. RESULTS The clinical and pathological response rates were 79.3% and 14.9% in the NACT group. In comparison to the PST group, IRFs but not HRFs were significantly decreased (P<0.05), and the 5-year DFS rate was significantly improved in the NACT group (88.4% versus 83.1%, P=0.021). Moreover, the 5-year DFS and OS rates were favourably increased in the clinical responders in comparison to the PST group and the clinical non-responders (P<0.05). Compared to those of clinical non-responders, the 5-year DFS and OS rates of clinical responders, with or without HRFs, were also significantly increased (P<0.01). In stage IB2, the 5-year DFS and OS rates were significantly increased, whereas operation duration declined in the NACT group (P<0.05). For patients with stage IB tumours of 2-5 cm, the 5-year DFS and OS rates of clinical responders were significantly improved (P<0.05). CONCLUSIONS NACT is a suitable option for patients with cervical cancer, especially for NACT responders and patients with stage IB, which provides a new concept of fertility preservation for young patients.

Triggering of death receptor apoptotic signaling by human papillomavirus 16 E2 protein in cervical cancer cell lines is mediated by interaction with c-FLIP

Human papillomavirus (HPV) E2 gene disruption is one of the key features of HPV-induced cervical malignant transformation. Though it is thought to prevent progression of carcinogenesis, the pro-apoptotic function of E2 protein remains poorly understood. This study shows that expression of HPV16 E2 induces apoptosis both in HPV-positive and -negative cervical cancer cell lines and leads to hyperactivation of caspase-8 and caspase-3. Activation of these signaling factors is responsible for the observed sensitivity to apoptosis upon treatment with anti-Fas antibody or TNF-α. In addition, immunoprecipitation experiments clearly show an interaction between HPV16 E2 and c-FLIP, a key regulator of apoptotic cell death mediated by death receptor signaling. Moreover, c-FLIP and a caspase-8 inhibitor protect cells from HPV16 E2-mediated apoptosis. Overexpression of c-FLIP rescues cervical cancer cells from apoptosis induced by HPV16 E2 protein expression. The data suggest that HPV16 E2 abrogates the apoptosis-inhibitory function of c-FLIP and renders the cell hypersensitive to the Fas/FasL apoptotic signal even below threshold concentration. This suggests a novel mechanism for deregulation of cervical epithelial cell growth upon HPV-induced transformation, which is of great significance in developing therapeutic strategies for intervention of cervical carcinogenesis.

HPV16 E5 peptide vaccine in treatment of cervical cancer in vitro and in vivo

SummaryHuman papillomavirus (HPV)-induced cervical cancer is the second most common cancer among women worldwide. Despite the encouraging development of the preventive vaccine for HPV, a vaccine for both prevention and therapy or pre-cancerous lesions remains in high priority. Thus far, most of the HPV therapeutic vaccines are focused on HPV E6 and E7 oncogene. However these vaccines could not completely eradicate the lesions. Recently, HPV E5, which is considered as an oncogene, is getting more and more attention. In this study, we predicted the epitopes of HPV16 E5 by bioinformatics as candidate peptide, then, evaluated the efficacy and chose an effective one to do the further test. To evaluate the effect of vaccine, rTC-1 (TC-1 cells infected by rAAV-HPV16E5) served as cell tumor model and rTC-1 loading mice as an ectopic tumor model. We prepared vaccine by muscle injection. The vaccine effects were determined by evaluating the function of tumor-specific T cells by cell proliferation assay and ELISPOT, calculating the tumor volume in mice and estimating the survival time of mice. Our in vitro and in vivo studies revealed that injection of E5 peptide+CpG resulted in strong cell-mediated immunity (CMI) and protected mice from tumor growth, meanwhile, prolonged the survival time after tumor cell loading. This study provides new insights into HPV16 E5 as a possible target on the therapeutic strategies about cervical cancer.Human papillomavirus (HPV)-induced cervical cancer is the second most common cancer among women worldwide. Despite the encouraging development of the preventive vaccine for HPV, a vaccine for both prevention and therapy or pre-cancerous lesions remains in high priority. Thus far, most of the HPV therapeutic vaccines are focused on HPV E6 and E7 oncogene. However these vaccines could not completely eradicate the lesions. Recently, HPV E5, which is considered as an oncogene, is getting more and more attention. In this study, we predicted the epitopes of HPV16 E5 by bioinformatics as candidate peptide, then, evaluated the efficacy and chose an effective one to do the further test. To evaluate the effect of vaccine, rTC-1 (TC-1 cells infected by rAAV-HPV16E5) served as cell tumor model and rTC-1 loading mice as an ectopic tumor model. We prepared vaccine by muscle injection. The vaccine effects were determined by evaluating the function of tumor-specific T cells by cell proliferation assay and ELISPOT, calculating the tumor volume in mice and estimating the survival time of mice. Our in vitro and in vivo studies revealed that injection of E5 peptide+CpG resulted in strong cell-mediated immunity (CMI) and protected mice from tumor growth, meanwhile, prolonged the survival time after tumor cell loading. This study provides new insights into HPV16 E5 as a possible target on the therapeutic strategies about cervical cancer.

Correlation between physical status of human papilloma virus and cervical carcinogenesis

The prevalence of human papilloma virus (HPV)-16 in patients with cervical cancer, the physical status of HPV-16 in patients with cervical lesions, and the role of HPV-16 integration in cervical carcinogenesis were investigated. HPV genotyping was performed by using PCR approach with the primer GP5+/GP6+ and type-specific primer on biopsy specimens taken operatively from 198 women. Multiple PCR was done to detect physical status of HPV-16 in a series of cervical liquid-based cytology samples and biopsy specimens obtained from different cervical lesions with HPV-16 infection, including 112 specimens with cervical cancer, 151 specimens with CIN I, 246 specimens with CIN and 120 specimens with CINIII. The results showed that there were 112 cervical cancer samples (56.57% of total cervical cancer patients) with HPV-16 infection. The frequency of HPV-16 pure integration was 65.18% (73/112), 56.57% (47/120), 23.58% (58/246) and 7.95% (12/151) in cervical cancer, CINIII, CINII and CINI patients respectively. In situ hybridization was performed on some paraffin-embedded sections of CINII, CINIII and cervical cancer to verify the physical status of HPV-16 infection. Significant difference was observed between cervical cancer and CIN I, CINII, CINIII in the frequency of HPV-16 integration (P<0.01). It is suggested that HPV-16 is the most prevalent type and is associated with cervical cancer. In the case of HPV-16 infection there are close associations between the severity of cervical lesions and the frequency of HPV-16 integration. The application of testing HPV genotyping and physical status based on detection of HC-II HPV DNA would be in favor of predicting the prognosis of cervical precancerosis and enhancing the screening accuracy of cervical cancer.SummaryThe prevalence of human papilloma virus (HPV)-16 in patients with cervical cancer, the physical status of HPV-16 in patients with cervical lesions, and the role of HPV-16 integration in cervical carcinogenesis were investigated. HPV genotyping was performed by using PCR approach with the primer GP5+/GP6+ and type-specific primer on biopsy specimens taken operatively from 198 women. Multiple PCR was done to detect physical status of HPV-16 in a series of cervical liquid-based cytology samples and biopsy specimens obtained from different cervical lesions with HPV-16 infection, including 112 specimens with cervical cancer, 151 specimens with CIN I, 246 specimens with CIN and 120 specimens with CINIII. The results showed that there were 112 cervical cancer samples (56.57% of total cervical cancer patients) with HPV-16 infection. The frequency of HPV-16 pure integration was 65.18% (73/112), 56.57% (47/120), 23.58% (58/246) and 7.95% (12/151) in cervical cancer, CINIII, CINII and CINI patients respectively. In situ hybridization was performed on some paraffin-embedded sections of CINII, CINIII and cervical cancer to verify the physical status of HPV-16 infection. Significant difference was observed between cervical cancer and CIN I, CINII, CINIII in the frequency of HPV-16 integration (P<0.01). It is suggested that HPV-16 is the most prevalent type and is associated with cervical cancer. In the case of HPV-16 infection there are close associations between the severity of cervical lesions and the frequency of HPV-16 integration. The application of testing HPV genotyping and physical status based on detection of HC-II HPV DNA would be in favor of predicting the prognosis of cervical precancerosis and enhancing the screening accuracy of cervical cancer.

A novel “priming-boosting” strategy for immune interventions in cervical cancer

Despite the encouraging development of a preventive vaccine for human papillomavirus (HPV), it cannot improve ongoing infections. Therefore, a new vaccine is urgently needed that can prevent and treat cervical cancer, and cure pre-cancerous lesions. In this study, we constructed two peptide-based vaccines. The first was a short-term, long-peptide (ST-LP) vaccine that simultaneously targeted three key carcinogenic epitopes (E5-E6-E7) on HPV16. We tested this vaccine in murine TC-1 cells infected with a recombinant adeno-associated virus (rAAV) fused with HPV16E5 DNA (rTC-1 cells), which served as a cell model; we also tested it in immune-competent mice loaded with rTC-1 cells, which served as an ectopic tumor model. The ST-LP injections resulted in strong, cell-mediated immunity, capable of attacking and eliminating abnormal antigen-bearing cells. Furthermore, to prolong immunogenic capability, we designed a unique rAAV that encoded the three predicted epitopes for a second, long-term, long-peptide (LT-LP) vaccine. Moreover, we used a new immune strategy of continuous re-injections, where three ST-LP injections were performed at one-week intervals (days 0, 7, 14), then one LT-LP injection was performed on day 120. Our in vitro and in vivo studies revealed that this strategy could boost the immune response to produce longer and stronger protection against target cells, and mice were thoroughly protected from tumor growth. Our results showed that priming the immune system with the ST-LP vaccine, followed by boosting the immune system with the LT-LP vaccine could generate a rapid, robust, durable cytotoxic T-lymphocyte response to HPV16-positive tumors.

Association of 42 SNPs with genetic risk for cervical cancer: an extensive meta-analysis

BackgroundA large number of single nucleotide polymorphisms (SNPs) associated with cervical cancer have been identified through candidate gene association studies and genome-wide association studies (GWAs). However, some studies have yielded different results for the same SNP. To obtain a more comprehensive understanding, we performed a meta-analysis on previously published case–control studies involving the SNPs associated with cervical cancer.MethodsElectronic searches of PubMed and Embase were conducted for all publications about the association between gene polymorphisms and cervical cancer. One-hundred and sixty-seven association studies were included in our research. For each SNP, three models (the allele, dominant and recessive effect models) were adopted in the meta-analysis. For each model, the effect summary odds ratio (OR) and 95% CI were calculated. Heterogeneity between studies was evaluated by Cochran’s Q test. If the p value of Q test was less than 0.01, a random effect model was used; otherwise, a fixed effect model was used.ResultsThe results of our meta-analysis showed that: (1) There were 8, 2 and 8 SNPs that were significantly associated with cervical cancer (P < 0.01) in the allele, dominant and recessive effect models, respectively. (2) rs1048943 (CYP1A1 A4889G) showed the strongest association with cervical cancer in the allele effect model (1.83[1.57, 2.13]); in addition, rs1048943 (CYP1A1 A4889G) had a very strong association in the dominant and recessive effect model. (3) 15, 11 and 10 SNPs had high heterogeneity (P < 0.01) in the three models, respectively. (4) There was no published bias for most of the SNPs according to Egger’s test (P < 0.01) and Funnel plot analysis. For some SNPs, their association with cervical cancer was only tested in a few studies and, therefore, might have been subjected to published bias. More studies on these loci are required.ConclusionOur meta-analysis provides a comprehensive evaluation of cervical cancer association studies.

Upregulation of the Tim-3/Gal-9 pathway and correlation with the development of preeclampsia

OBJECTIVE It is well documented that an imbalance in immune tolerance at the maternal-fetal interface is likely to play an essential role in the etiology of preeclampsia. However, the mechanisms underlying immune tolerance during preeclampsia are still poorly understood. Tim-3, a Th1-specific cell surface molecule, is a relatively newly described molecule with important immunological functions. It can regulate Th1 responses with its ligand galectin-9 (Gal-9), and has become an attractive candidate for exploring the pathogenesis of preeclampsia. STUDY DESIGN Twenty normal pregnancies and 20 preeclamptic pregnancies were enrolled in the present study. We examined the expression and function of Tim-3/Gal-9 in decidual tissue at the RNA and protein levels. In order to analyze their correlation with the development of preeclampsia, we measured the expression of Tim-3 on peripheral blood leukocytes using flow cytometry. IFN-γ, IL-10, and IL-17 in the peripheral blood plasma were measured by ELISA. RESULTS Tim-3/Gal-9 was upregulated in decidual tissue of preeclamptic vs. normotensive pregnancies. There was a significantly increased Th1 and Th17 response in PE as demonstrated by the upregulated levels of IFN-γ/IL-17, whereas IL-10 secreted by Th2 cells was sharply reduced. CONCLUSIONS The present study showed that an abnormal Tim-3/Gal-9 pathway was able to facilitate the development of preeclampsia. Our data uncovered a novel mechanism by which the Tim-3/Gal-9 pathway regulates immune responses, and we now identify this pathway as a potential therapeutic target in preeclampsia.

Up-regulated expression of Tim-3/Gal-9 at maternal-fetal interface in pregnant woman with recurrent spontaneous abortion

SummaryThe relationship between T cell immunoglobulin domain and mucin domain protein 3 (Tim-3)/Galectin (Gal)-9 pathway and recurrent spontaneous abortion (RSA) was studied. Thirty-one pregnant women with RSA and 27 normal early gravidas were investigated to detect the levels of Tim-3 and Gal-9 in villi and deciduas by Western blotting. Meanwhile, the concentration of interleukin (IL)-4 and IL-12 in peripheral blood plasma was determined by ELISA in 25 healthy fertile non-pregnant controls, the normal early gravidas and pregnant women with RSA mentioned above, respectively. It was found that the relative expression levels of Tim-3 and Gal-9 in villi and deciduas were significantly increased in pregnant women with RSA as compared with those in the normal early gravidas. The concentration of IL-4 in peripheral blood plasma of pregnant women with RSA was lower than that of the normal early gravidas (P<0.05) and healthy fertile non-pregnant controls (P<0.05), but that of IL-2 in pregnant women with RSA was significantly higher than that of the normal early gravidas (P<0.05) and healthy fertile non-pregnant controls (P<0.05). It was suggested that the overexpression of Tim-3/Gal-9 pathway may be related to the pathogenesis of RSA.The relationship between T cell immunoglobulin domain and mucin domain protein 3 (Tim-3)/Galectin (Gal)-9 pathway and recurrent spontaneous abortion (RSA) was studied. Thirty-one pregnant women with RSA and 27 normal early gravidas were investigated to detect the levels of Tim-3 and Gal-9 in villi and deciduas by Western blotting. Meanwhile, the concentration of interleukin (IL)-4 and IL-12 in peripheral blood plasma was determined by ELISA in 25 healthy fertile non-pregnant controls, the normal early gravidas and pregnant women with RSA mentioned above, respectively. It was found that the relative expression levels of Tim-3 and Gal-9 in villi and deciduas were significantly increased in pregnant women with RSA as compared with those in the normal early gravidas. The concentration of IL-4 in peripheral blood plasma of pregnant women with RSA was lower than that of the normal early gravidas (P<0.05) and healthy fertile non-pregnant controls (P<0.05), but that of IL-2 in pregnant women with RSA was significantly higher than that of the normal early gravidas (P<0.05) and healthy fertile non-pregnant controls (P<0.05). It was suggested that the overexpression of Tim-3/Gal-9 pathway may be related to the pathogenesis of RSA.