Association of MicroRNA-10b Expression and P16 with Pathological Features in Various Stages of Cervical Precancerous Lesions.

Cervical cancer is the fourth most prevalent cancer for females with 14,100 new cases each year globally. Efficient screening and intervention at the precancerous stage is the key point to the prevention and treatment of cervical cancer. However, no widely recognized biomarkers have been discovered yet. We investigated the expression of miR-10b in cervical cells and its correlation with clinicopathological features in different pathological grades of cervical precancerous lesions. The expression of miR-10b in cervical cytology samples from 20 cases of LSIL, 22 cases of HSIL, 18 cases of early-stage cervical cancer, and 20 cases of cervicitis controls were assessed using qPCR. From the same cervical cytology samples, the human papillomavirus (HPV) load was assessed using semi-PCR and the lesion size, and gland involvement levels from the same subjects were assessed during the cervical examination. The correlation between miR-10b expression and different pathological grades of cervical lesions was analyzed. We also calculated the correlation between HPV load, lesion size, gland involvement, P16 expression, and different pathological grades. The expression of miR-10b exhibited a step-decreasing manner from cervicitis control (4.23(4.00,4.71)) to LSIL (2.67(2.52,2.90)), HSIL (1.49(1.30,1.80)) and cervical cancer group (0.65(0.55,0.80)). There is a significant difference (P<0.001) between cervicitis and HSIL, cervicitis and cervical cancer, ISIL and HSIL, as well as ISIL and cervical cancer but not between the cervicitis group and the LSIL group. In addition, more severe pathological grades were correlated with a bigger rate of gland involvement (P＜0.001). We also found that different pathological grades were correlated with the intensity of P16 expression (P=0.001), and the intensity of P16 expression is positively correlated with different pathological grades (P<0.05). Repressed expression of miR-10b is related to the progression of cervical precancerous lesions. Increased gland involvement rate and increased intensity of P16 expression are risk factors for developing cervical cancers. Our result showed that miR-10b may be a potential biomarker for the screening and ranking of cervical precancerous lesions.

Analysis of eEF1A2 gene expression and copy number in cervical carcinoma.

OBJECTIVE: To explore and analyze the expression of eukaryotic translation elongation factor 1 alpha 2 (eEF1A2) gene in cervical cancer tissues, its relationship with patient survival, gene mutations, and changes in copy number in cervical cancer and chronic cervicitis tissues. METHODS: The expression of the eEF1A2 gene in cervical cancer and its relationship with patient survival were analyzed using gene expression profile interactive analysis. Changes in eEF1A2 expression in cervical cancer tissues were analyzed using cBioPortal, a portal for cancer genomics analysis. The eEF1A2 copy number in cervical cancer tissues and chronic cervicitis tissues was determined by real-time fluorescence quantitative polymerase chain reaction. The relationship between the expression of eEF1A2 protein and the clinical stage, pathological grade, and patient survival of cervical cancer was analyzed by the database: The Human Protein Atlas, an integrated repository portal for tumor-immune system interactions. RESULTS: Gene expression profile interactive analysis database analysis showed no significant differences in the expression of eEF1A2 between cervical cancer and normal cervical tissues (P > .05). The eEF1A2 gene expression level was not correlated with the survival of cervical cancer patients (P > .05). Analysis of the cBioPortal database showed that 18 of 297 cervical cancer patients had eEF1A2 gene changes, including missense mutation, splice mutation, amplification, and messenger RNA increase. There was no significant difference in eEF1A2 gene copy number between cervical cancer and chronic cervicitis (P > .05). The Human Protein Atlas and an integrated repository portal for tumor-immune system interactions database analysis of immunohistochemical data showed that eEF1A2 protein expression was no significant difference in clinical stage, pathological grade and patient survival of cervical cancer (P > .05). CONCLUSION: The eEF1A2 gene was mutated in cervical cancer tissues. The eEF1A2 gene copy number was not associated with changes in the expression of the eEF1A2 gene in cervical cancer tissues.

C/EBPß expression decreases in cervical cancer and leads to tumorigenesis.

BACKGROUND: Cervical cancer is currently estimated to be the fourth most common cancer among women worldwide and the leading cause of cancer-related deaths in some of the world's poorest countries. C/EBPß has tumor suppressor effects because it is necessary for oncogene-induced senescence. However, C/EBPß also has an oncogenic role. The specific role of C/EBPß in cervical cancer as a tumor suppressor or oncoprotein is unclear. OBJECTIVE: To explore the role of the C/EBPß protein in cervical tumorigenesis and progression. METHODS: Quantitative RT-PCR was used to analyze C/EBPß (15 cervical cancer tissue samples and 15 corresponding normal cervical tissue samples), miR-661, and MTA1 mRNA expression in clinical samples (10 cervical cancer tissue samples and 10 corresponding normal cervical tissue samples). Immunohistochemistry was used to analyze C/EBPß (381 clinical samples), Ki67 (80 clinical samples) and PCNA ( 60 clinical samples) protein expression. MALDI-TOF MassARRAY was used to analyze C/EBPß gene methylation (13 cervical cancer tissues and 13 corresponding normal cervical tissues). Cell proliferation was analyzed by CCK-8 in cervical cancer cell lines. Western blotting and immunohistochemistry were performed to detect C/EBPß protein expression levels, and mRNA expression was analyzed by quantitative RT-PCR analysis. Flow cytometry was performed to measure cell cycle distribution and cell apoptosis. Colony formation, Transwell, cell invasion, and wound healing assays were performed to detect cell migration and invasion. RESULTS: C/EBPß protein expression was significantly reduced in cervical cancer tissues compared with cervicitis tissues (P < 0.01). Ki67 protein and PCNA protein expression levels were significantly higher in cervical cancer tissues compared with cervicitis tissues. The rate of C/EBPß gene promoter methylation of CpG12, 13, 14 and CpG19 in cervical cancer tissues was significantly increased compared with normal cervical tissue (P < 0.05). In addition, C/EBPß was overexpressed in cervical cancer cells and this overexpression inhibited cell proliferation, migration, invasion, arrested cells in S phase, and promoted apoptosis. CONCLUSIONS: We have demonstrated that C/EBPß decreased in cervical cancer tissues and overexpression of the C/EBPß gene in cervical cancer cells could inhibit proliferation, invasion and migration.

Detection of DKK-1 gene methylation in exfoliated cells of cervical squamous cell carcinoma and its relationship with high risk HPV infection.

PURPOSE: To detect the methylation of Dickkopf-associated protein 1 (DKK-1) gene promoter in cervical exfoliated cells and to study its clinical significance in cervical squamous cell carcinoma (CSCC) and its relationship with high-risk HPV infection. METHODS: Methylation-specific PCR (MSP) was utilized to detect the methylation of DKK-1 gene promoter in cervical exfoliated cells from 40 patients with CSCC and 40 patients with chronic cervicitis in the Affiliated Hospital of Inner Mongolia Medical University. The methylation rate of DKK-1 gene promoter in different clinicopathological factors and its relationship with high-risk HPV infection was compared, and different detection methods were compared. RESULT: The degree of methylation of DKK-1 gene promoter in CSCC group was significantly higher than that in cervicitis group (P < 0.05). In CSCC group, the degree of methylation was significantly different in high-risk HPV infection, histological differentiation, tumor size, lymph node metastasis and the International Federation of Gynecology and Obstetrics (FIGO) staging (all P < 0.05). The degree of methylation is not related to the type of high-risk HPV infection (P > 0.05). The one-year survival rate of CSCC patients with high-risk HPV positive and DKK-1 gene promoter methylation is relatively low, only 74.1%. The sensitivity, specificity and accuracy of DKK-1 gene methylation combined with high-risk HPV detection were 96.7%, 78.0% and 85.0%, respectively. CONCLUSION: Methylation of DKK-1 gene promoter in cervical exfoliated cells of patients with CSCC is related to high-risk HPV infection and different clinicopathological factors, but the degree of methylation of DKK-1 gene is not related to the type of high-risk HPV infection. It may become an indicator different from HPV typing detection, which may play a shunt role in suggesting whether further invasive cervical examination is needed and reduce cervical invasive examination and overtreatment. It may be related to the survival rate of patients, which can be used to estimate the development and prognosis of CSCC and may play a good role in early warning in follow-up monitoring of CSCC after treatment. DKK1 gene methylation combined with HPV detection can improve the sensitivity, specificity and accuracy of diagnosis, which may improve the detection rate of early CSCC and make up for the deficiency of HPV and TCT detection. That may become a non-invasive screening method for CSCC.

Association of TLR4 and TLR9 gene polymorphisms and haplotypes with cervicitis susceptibility.

BACKGROUND: Cervicitis is one of the major health problems amongst women caused by infection of various pathogens including Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV) as well as human papillomavirus (HPV), and persistent cervical inflammation is one of the etiologic agents of cervical cancer. Toll-like receptors (TLRs) play an important role in the recognition and subsequent elimination of these pathogens. Variations in the Toll-like receptor genes influence susceptibility to pathogens as well as disease progression independently. METHODS: Ten single nucleotide polymorphisms, five each of TLR4 and TLR9 genes were analyzed among 130 cervicitis patients and 150 controls either using polymerase chain reaction-restriction fragment length polymorphism or allele specific-PCR. RESULTS: T. vaginalis infection was found at the highest frequency (30.7%) as compared to C. trachomatis (1.5%), N. gonorrhoeae (2.3%) and HPV (4.6%) infections in cervicitis patients. TLR4 rs11536889 CC (age-adjusted OR, 2.469 [95% CI, 1.499 to 4.065]; p < 0.001) and TLR9 rs187084 TC (age-adjusted OR, 2.165 [95% CI, 1.267-3.699]; p = 0.005) genotypes showed the higher distribution in cervicitis patients compared to controls. In addition, TLR4 rs11536889 C allele was shown to increase the risk of cervicitis (age-adjusted OR, 1.632 [95% CI, 1.132 to 2.352]; p = 0.009) compared to controls. The TLR4 haplotype GCA (OR, 0.6 [95% CI, 0.38-0.95]; p = 0.0272) and TLR9 haplotype GTA (OR, 1.99 [95% CI, 1.14-3.48]; p = 0.014) were found to be associated with decreased and increased risk of cervicitis respectively. CONCLUSIONS: TLR4 and TLR9 polymorphisms, as well as haplotypes were shown to modulate the cervicitis risk.

Expression of AMHR2 and C-KIT in cervical lesions in Uyghur Women of Xinjiang, China.

BACKGROUND: Cervical cancer is one of the most common malignant tumors in women. Anti-Müllerian hormone receptor 2 (AMHR2) and C-Kit were two members of protein kinase which were reported increased in some cancers like ovarian carcinoma and breast cancer. The present study aimed to assess the expression of AMHR2 and c-Kit in cervical cancer of different differentiated degrees as well as in cervicitis sections. METHODS: All the lesions were collected randomly during clinical observations in hospitals located in Xinjiang, China. Polymerase chain reaction (PCR) and immunohistochemical staining were used to detect AMHR2 and c-Kit expression in cervical samples from women who had been infected with human papilloma virus (HPV)16. The expression rate was compared between cervical cancer of well, moderately and poorly differentiated and cervicitis. RESULTS: The average age of the patients was 45 years; ranged from 23 to 80. For AMHR2, all 17 cervicitis samples ranged from (++) to (++++), while cervical cancer showed 11 (+), 9 (++), 15 (+++),9 (++++), and 8 (-), which showed AMHR2 expression was lessen with the poorer of differentiation degree of cervical cancer (P < .05). For c-Kit, 18 cervicitis samples mainly expressed as (-) with none showed (+++) or (++++), while cervical cancer samples showed 7 (-), 6 (+), 1 (++), 2 (+++), and 8 (++++), which indicated c-Kit's expression increased with the reduction of cervical cancer's differentiation degree (P < .05). CONCLUSION: AMHR2 might have some correlation with self defense of our body, while c-Kit might link with the potential invasive capacity of cervical cancer.

MMP-9/RECK Imbalance: A Mechanism Associated with High-Grade Cervical Lesions and Genital Infection by Human Papillomavirus and Chlamydia trachomatis.

BACKGROUND: Matrix metalloproteinases (MMP) are important enzymes in the tumor microenvironment associated with progression of cervical intraepithelial neoplasia (CIN) toward squamous cell carcinoma (SCC) of the cervix. However, the role of MMPs in the inflammatory process associated with Chlamydia trachomatis infection concomitant with the carcinogenic process driven by HPV has not yet been addressed. In the present study, we analyzed the state of the MMP-9-RECK axis in cervical carcinogenesis. METHODS: The levels of MMP-9 and RECK expression were analyzed by immunocytochemistry in liquid-based cytology samples from 136 women with high-grade cervical lesions (CIN2/CIN3) and cervical SCC diagnosed by LLETZ, and in 196 women without cervical neoplasia or CIN1. Real-time qPCR was performed to analyze expression of MMP-9 and RECK in 15 cervical samples. The presence of HPV-DNA and other genital pathogens was evaluated by PCR. RESULTS: We found a higher expression of MMP-9 [OR, 4.2; 95% confidence interval (CI), 2.2-7.8] and lower expression of RECK (OR, 0.4; 95% CI, 0.2-0.7) in women with CIN2/CIN3/SCC when compared with women from the control group (no neoplasia/CIN1). A statistically significant association was also found between MMP-9/RECK imbalance and infection by alpha-9 HPV and C. trachomatis. The prevalence of C. trachomatis infection was significantly higher in women with high-grade cervical disease (OR, 3.7; 95% CI, 1.3-11.3). CONCLUSIONS: MMP-9/RECK imbalance in cervical smears is significantly associated with high-grade cervical diseases and infection by alpha-9 HPV and C. trachomatis. IMPACT: MMP-9/RECK imbalance during cervical inflammation induced by C. trachomatis might play a role in HPV-mediated cervical carcinogenesis.

Up-Regulation of miR-21 Is Associated with Cervicitis and Human Papillomavirus Infection in Cervical Tissues.

MicroRNA-21 (miR-21) is recognized as an oncomir and shows up-regulation in many types of human malignancy. The aim of this study was to investigate the association of miR-21 expression associated with HPV infection in normal and abnormal cervical tissues. Cervical tissue samples with different cytological or histopathological grades were investigated for HPV by PCR and for miR-21 and programmed cell death, protein 4 (PDCD4) expression using quantitative real-time PCR (qRT-PCR). Laser capture microdissection (LCM) of stromal and epithelial tissues and in situ hybridization (ISH) using locked nucleic acid (LNA) probes were performed on a subset of fixed specimens. Cell line experiments were conducted on fibroblasts stimulated in culture media from HeLa cells, which were then assessed for miR-21, PDCD4, IL-6 and α-SMA expression by qRT-PCR. Twenty normal cervical cell, 12 cervicitis, 14 cervical intraepithelial neoplastic I (CIN I), 22 CIN II-III and 43 cervical squamous cell carcinoma (SCC) specimens were investigated. miR-21 levels were significantly lower in normal than in abnormal tissues. The expression of miR-21 in HPV negative normal cytology was significantly lower than in HPV positive samples in abnormal tissue and SCC. The miR-21 expression was significantly higher in HPV negative cervicitis than HPV negative normal cells. LCM and ISH data showed that miR-21 is primarily expressed in the tumor-associated stromal cell microenvironment. Fibroblasts treated with HeLa cell culture media showed up-regulated expression of miR-21, which correlated with increased expression of α-SMA and IL-6 and with down-regulation of PDCD4. These results demonstrate that miR-21 is associated with HPV infection and involved in cervical lesions as well as cervicitis and its up-regulation in tumor-stroma might be involved in the inflammation process and cervical cancer progression.

MEKK3 and survivin expression in cervical cancer: association with clinicopathological factors and prognosis.

Mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 3 (MEKK3) is an important protein kinase and a member of the MAPK family, which regulates cellular responses to environmental stress and serves as key integration points along the signal transduction cascade that not only link diverse extracellular stimuli to subsequent signaling molecules but also amplify the initiating signals to ultimately activate effector molecules and induce cell proliferation, differentiation and survival. To explore the relationship between MEKK3 and cell apoptosis, clinicopathology and prognosis, we characterize the expression of MEKK3 and survivin in cervical cancer. MEKK3 and survivin expression was measured by RT-PCR and Western blotting of fresh surgical resections from 30 cases of cervical cancer and 25 cases of chronic cervicitis. Protein expression was detected by tissue microarray and immunochemistry (En Vision) in 107 cases of cervical cancer, 86 cases of cervical intraepithelial neoplasia (CIN), and 35 cases of chronic cervicitis. Expression patterns were analyzed for their association with clinicopathological factors and prognosis in cervical cancer. Expression of MEKK3 and survivin mRNA was significantly higher in cervical cancer than in the controls (p<0.05). MEKK3 and survivin expression differed significantly between cervical carcinoma, CIN, and cervicitis (p<0.05) and correlated with clinical stage, infiltration depth, and lymph node metastasis (p<0.05). MEKK3 expression was positively correlated with survivin (p<0.05). Kaplan-Meier survival analysis showed that MEKK3 and survivin expression, lymph node metastasis, depth of invasion, and FIGO stage reduce cumulative survival. Cox multivariate regression analysis showed that MEKK3, survivin, and clinical staging are independent prognostic factors in cervical cancer (p<0.05). Expression of MEKK3 and survivin are significantly increased in cervical cancer, their overexpression participating in the occurrence and development of cervical cancer, with protein expression and clinical staging acting as independent prognostic factors for patients with cervical cancer.

Expression and prognostic value of aquaporin 1, 3 in cervical carcinoma in women of Uygur ethnicity from Xinjiang, China.

BACKGROUND: Overexpression of several aquaporins has been reported in different types of human cancer but the role of aquaporins in carcinogenesis has not yet been clearly defined. There is few report concerning role of aquaporins in human cervical carcinogenesis so far. Here, we determined the expression and prognostic value of aquaporin 1, 3 in cervical carcinoma in Chinese women of Uygur ethnicity. METHODS AND RESULTS: Real-time PCR analyses demonstrated aquaporin 1, 3 mRNA were differentially expressed in cervical carcinoma, CIN 2-3 and mild cervicitis. Immunofluorescent and immunohistochemical analyses demonstrated aquaporin 1 was predominantly localized to stromal endothelial cells in cervical lesions. Aquaporin 3 was localized to the membrane of normal squamous epithelium, CIN and carcinoma cells. Aquaporin 1 and 3 were upregulated in cervical cancer compared to mild cervicitis and CIN2-3 (P<0.05); Tumor expression of aquaporin 1, 3 significantly increased in advanced stage disease, and patients with deeper tumor infiltration, lymph node metastases or larger tumor volume (P<0.05). Multivariate analysis demonstrated that aquaporin 1, 3 were not independent prognostic factors in cervical carcinoma. CONCLUSION: Aquaporins may participate in the initiation and progression of cervical carcinoma by promoting tumor growth, invasion or lymph node metastasis. Further study is required to determine whether aquaporins have potential as prognostic factors in cervical cancer.

Significance and expression of aquaporin 1, 3, 8 in cervical carcinoma in Xinjiang Uygur women of China.

Overexpression of several aquaporins (AQPs) has been reported in different types of human cancer but their role in carcinogenesis, for example in the cervix, have yet to be clearly defined. In this study, expression of AQPs in cervical carcinomawas investigated by real-time PCR, immunofluorescent and immunohistochemical assays and evaluated for correlations with clinicopathologic variables. AQP1, 3, 8 exhibited differential expression in cervical carcinoma, corresponding CIN and mild cervicitis. AQP1 was predominantly localized in the microvascular endothelial cell in the stroma of mild cervicitis, CIN and cervical carcinoma. AQP3 and AQP8 were localized in the membrane of normal squamous epithelium and carcinoma cells, local signals being more common than diffuse staining. AQP1 and AQP3 expression was remarkably stronger in cervical cancer than in mild cervicitis and CIN2-3 (P<0.05). AQP8 expression was highest in CIN2-3 (91.7%), but levels in cervical carcinoma were also higher than in mild cervicitis. AQP1, AQP3, AQP8 expression significantly increased in advanced stage, deeper infiltration, metastatic lymph nodes and larger tumor volume (P<0.05). Our findings showed that AQPs might play important roles in cervical carcinogenesis and tumour progression in Uygur women.

[Relationship between TAP gene promoter methylation and cervical lesions with HPV infection in Uyghur women].

OBJECTIVE: To study the relationship between TAP (transporter associated with antigen processing) gene promoter regional methylation level and cervical lesions with HPV infection in Uyghur women. METHODS: A specialized software was used to design specific primers of CpG island fragments of TAP1 and TAP2 gene promoter for PCR amplification, bisulfitemodified SiHa cancer cell DNA for PCR amplification, cloning and sequencing analysis to obtain the relevant information on the gene base sequence methylation of CpG sites. Seventy-eight fresh cervical tissue samples from Uyghur women with cervicitis (number = 15), cervical intraepithelial neoplasia (CIN, number = 30) and cervical squamous cell carcinoma (number = 33) were collected. The methylation level of TAP1 and TAP2 gene promoter regions was detected using MassArray DNA technology. HPV infection status was determined by HPV gene chips. The relationship between CpG-island methylation of gene promoter regions and HPV infection was then analyzed. RESULTS: Each TAP1 and TAP2 gene corresponding target fragment contained 23 and 8 CpG sites. There were 5 and 8 CpG sites methylation occurred in SiHa cervical cancer cells genomic DNA respectively. The TAP1 methylation level increased steadily with the severity of cervical lesions. The methylation levels in cervical squamous cell carcinoma and CIN (0.048 ± 0.039 and 0.037 ± 0.026, respectively) were higher than that of normal cervical tissue (0.035 ± 0.029, P < 0.05). Although TAP2 gene methylation level also demonstrated similar changes, the difference however was not statistically significant (P > 0.05). HPV gene chip detected 13 HPV genotypes, with HPV16 infection rate being 66.7% (52/78). The methylated proportion of TAP1 positively correlated with HPV16 infection (χ(2) = 6.08, P = 0.039). CONCLUSION: TAP1 methylation is a remarkable phenomenon occurring in a range of cervical lesions and significantly associated with cervical HPV infection.

[Death-associated protein kinase promoter (DAPK) hypermethylation in uterine cervical cancer and intraepithelial neoplasia in Uyghur nationality women].

OBJECTIVE: To investigate the methylation levels of death-associated protein kinase (DAPK) in Uyghur female patients with different cervical lesions in Xinjiang, and to discuss the relationship of the expression and significance of DAPK in normal cervix, chronic cervicitis, cervical intraepithelial neoplasia (CINI, CIN II/III) and invasive cervical squamous cell carcinoma. METHODS: 30 cases of normal cervix and chronic cervicitis, 30 cases of CINI, 30 cases of CINII/III and 30 cases of cervical squamous cell carcinoma were tested by methylation specific PCR (MSP). Expressions of DAPK in 30 cases of normal cervix and chronic cervicitis, 30 cases of CINI, 30 cases of CINII/III and 30 cases of cervical squamous cell carcinoma were assayed using immunohistochemical SP staining. RESULTS: The methylation rate of DAPK gene in normal cervix and chronic cervicitis was 3.33%, 10% in cervical intraepithelial neoplasia CINI, 36.7% in CINII/III, and 63.3% in invasive cervical squamous cell carcinoma. The methylation rate of DAPK in the SCC group was significantly higher than that in the other groups (P < 0.05). Aberrant promoter methylation of the DAPK gene was positively correlated with the degree of cervical lesions. The positive rate of DAPK protein in normal cervix and chronic cervicitis was 93.3%, 83.3% in cervical intraepithelial neoplasia CINI, 60.0% in CINII/III, and 33.3% in invasive cervical squamous cell carcinoma. The expression of DAPK in the SCC group was significantly lower than that in the other groups (P < 0.05). The positive rate of DAPK protein was negatively correlated with the degree of cervical lesions (r(s) = -0.603, P < 0.001). CONCLUSIONS: Methylation of DAPK is involved in the cervical carcinogenesis and DAPK gene promoter methylation occurs in the early development of cervical cancer in Uyghur women in Xinjiang. Detection of DAPK gene methylation may provide a basis for use in early detection of cervical cancer. DAPK protein expression is decreasing even disappears along with the progression of cervical lesions.

In captive rhesus macaques, cervicovaginal inflammation is common but not associated with the stable polymicrobial microbiome.

Vaginal inoculation of rhesus macaques (RM) with simian immunodeficiency virus (SIV) has been used to study the biology of HIV transmission. Although the results of vaginal SIV transmission experiments could be affected by vaginal inflammation, studies to date have been conducted without regard to levels of pre-existing genital inflammation present in RM. We collected cevicovaginal secretions (CVS) from 33-36 RM during the mid menstrual cycle (day 10-20) at 2 time points approximately 8 months apart and characterized the mRNA and protein levels of inflammatory cytokines, chemokines and interferon-stimulated genes. There was extreme variability in the levels of inflammatory mediators (IFN-α, IFN-γ, IL-6, TNF, IL-1b, IP-10, MIG, IL-12 and IL-17). In most animals, the mRNA levels of the inflammatory mediators were similar in the 2 CVS samples collected 8 months apart, suggesting that genital inflammation is stable in a subset of captive female RM. At both time points the cervicovaginal microbiota had low levels of Lactobacillus and was relatively diverse with an average of 13 genera in the samples from the first time point (median 13, range 7-21) and an average of 11.5 genera in the samples from the second time point (median 11, range 5-20). Many of the macaques had similar microbiota in the samples collected 8 months apart. However, we found no correlation between specific bacterial genera and the mRNA or protein levels of the inflammatory mediators in the genital tract of RM in this study. It seems likely that results of published vaginal SIV transmission experiments in RM have been influenced by pre-existing inflammation in the animals used for the experiments.

CCAAT/enhancer-binding protein delta mediates tumor necrosis factor alpha-induced Aurora kinase C transcription and promotes genomic instability.

Epidemiologic and clinical research indicates that chronic inflammation increases the risk of certain cancers, possibly through chromosomal instability. However, the mechanism of inflammation-dependent chromosomal instability associated with tumorigenesis is not well characterized. The transcription factor CCAAT/enhancer-binding protein δ (C/EBPδ, CEBPD) is induced by tumor necrosis factor α (TNFα) and expressed in chronically inflamed tissue. In this study, we show that TNFα promotes aneuploidy. Loss of CEBPD attenuated TNFα-induced aneuploidy, and CEBPD caused centromere abnormality. Additionally, TNFα-induced CEBPD expression augmented anchorage-independent growth. We found that TNFα induced expression of aurora kinase C (AURKC) through CEBPD, and that AURKC also causes aneuploidy. Furthermore, high CEBPD expression correlated with AURKC expression in inflamed cervical tissue specimens. These data provide insight into a novel function for CEBPD in inducing genomic instability through the activation of AURKC expression in response to inflammatory signals.

[Detection of human telomerase RNA component gene by fluorescent in situ hybridization for screening of cervical lesions].

OBJECTIVE: To investigate the value of fluorescence in situ hybridization (FISH) detection of human telomerase RNA component (hTERC) gene amplification in screening of cervical lesions. METHODS: A total of 146 post-thinPrep cytology test (TCT) samples were analyzed using FISH by two-color interphase probe targeting hTERC gene at chromosome 3q26 and the data were compared with the cytological and histological results. RESULTS: FISH analysis was successful in 120 cases (20 cases of normal and 100 abnormal cases by TCT). Gene amplification of hTERC by FISH had a positive correlation with the cytological (r = 0.465, P < 0.01) and histological grade results (r = 0.610, P < 0.01). Extra copies of hTERC were seen in 28.6% (6/21) of CINI, 61.1% (11/18) of CINII, 75.0% (18/24) of CINIII and 91.7%(22/24) of squamous cell carcinoma, respectively. None (0/13) of the inflammation cases showed hTERC amplification. The sensitivity and specificity for detecting high grade lesions by FISH were 77.3% (51/66) and 82.4% (28/34); and the positive and negative predictive values were 89.5% and 65.1%, respectively. The rate of hTERC gene gain in high grade lesions was significantly higher than that in the low grade lesions (χ(2) = 32.550, P < 0.01). Combined with the high copy numbers, the sensitivity for detecting high grade lesions was increased to 81.2%. CONCLUSIONS: Detection of hTERC gene amplification by FISH improves the screening efficiency of high-risk cervical epithelial lesions. The presence of high copy numbers of hTERC correlates with the presence of high grade cervical dysplasia.

[Correlation between human papillomavirus type 16 infection and human leukocyte antigen class I expression in cervical cancers of Uighur women].

OBJECTIVE: To explore the relationship between human papillomavirus(HPV) infection and expression of human leukocyte antigen class I (HLA-I) family genes (HLA-A, B and C) in cervical cancers of Uighur women, and to investigate their effect on cervical cancer progression. METHODS: Fresh tissue samples of 78 Uighur women with cervical squamous carcinoma, cervical intraepithelial neoplasia (CIN) or benign cervicitis were selected. HLA-A, B and C expression and HPV infection were analyzed using RT-PCR and HPV gene chips, respectively. RESULTS: There was a tendency of increasing the total loss of HLA-A, B and C mRNA as the cervical lesions became more aggressive. Loss of HLA-I mRNA in CIN (I, II and III) and cervical squamous carcinoma was 70.0% (14/20) and 84.8% (39/46) respectively. Poorly differentiated cervical carcinomas had the highest HLA-I expression loss (90.6%). In contrast, HLA-I mRNA loss was seen in only 8% of cases of cervicitis. Moreover, it was found that high risk HPV 16 infection was strongly correlated with the loss HLA-I mRNA expression (r = 0.803, P < 0.01). CONCLUSIONS: The loss of HLA-I gene expression is strongly correlated with HPV-16 infection, and may serve as a biomarker of cervical cancer progression in Uighur women.

The association of p16(INK4A) and fragile histidine triad gene expression and cervical lesions.

OBJECTIVE: This cross-sectional study was intended to assess the association between immunohistochemical analysis of p16 and fragile histidine triad (FHIT) and the presence of precancerous cervical lesions. MATERIALS AND METHODS: Women seen at Pérola Byington Hospital, São Paulo, Brazil, with histologically confirmed cervicitis (n = 31), cervical intraepithelial neoplasia (CIN) 1 (n = 30), CIN 2,3 (n = 30), and cervical cancer (n = 7) had also cervical material collected for liquid-based cytology, human papillomavirus Hybrid Capture 2 (HC2) test, and p16 and FHIT immunohistochemical reactions. RESULTS: p16 and FHIT reactions were scored as the following: <1%, 1% to 5%, >5% to 25%, and >25%. Receiver operating curve analysis was used to select p16 and FHIT score cutoffs for further categorical analyses. All but one of the 37 CIN 2,3/cancer cases had a p16 score of greater than 1% to 5%. Among the 61 cervicitis/CIN 1 cases, 46 (75%) had a p16 score lower than 1% to 5%. In contrast, no association of FHIT expression and severity of cervical lesions could be demonstrated in this data set. Receiver operating curve analyses suggested the score of 1% to 5% for p16 as the cutoff that best discriminates CIN 2,3/cancer from cervicitis/CIN 1. No cutoff for FHIT scores could be suggested with data set. CONCLUSIONS: p16, but not FHIT expression, has the potential to be used as complementary diagnostic tool to investigate human papillomavirus-induced cervical lesions, if these results are confirmed in larger studies.

[Study of p16(INK4A) expression and DNA ploidy in HPV-negative cervical cancers and precursors].

OBJECTIVE: To investigate the clinicopathological significance of p16(INK4A) expression and DNA ploidy status in HPV-negative uterine cervical cancers and their precursors. METHODS: HPV-negative cervical lesions, including 20 cases of cervicitis, 20 cases of cervical intraepithelial neoplasm (CIN), 3 cases of cervical glandular intraepithelial neoplasm (CGIN), 38 cases of invasive squamous cell carcinoma (SCCs) and 15 cases of invasive adenocarcinoma were selected and subject to screening for HPV infection by PCR method. The p16(INK4A) protein expression and DNA ploidy status were studied by immunohistochemistry and flow cytometry respectively. RESULTS: Specific expression of p16(INK4A) was seen in both the nucleus and cytoplasm of the dysplastic and malignant cells of CIN, CGIN, cervical SCC and adenocarcinoma. In contrast, no expression was present in normal and inflammatory squamous or glandular epithelium. DNA aneuploidy was significantly more frequent in invasive SCCs and adenocarcinomas than in CIN (P < 0.01). Aneuploid was also more frequent in the lymph node positive group than lymph node negative group, although no statistic significance was found. Among the 8 cases of p16(INK4A) negative SCCs, two showed DNA aneuploidy. CONCLUSIONS: Immunohistochemical detection for p16(INK4A) can be an early diagnostic marker for HPV-negative cervical SCC and adenocarcinoma. DNA ploidy analysis may further assist the diagnosis of cervical malignancies.