Normal Cervix Research Articles

Cytoprotective Activity of Neichitti (Vernonia cinerea) in Human Embryonic Kidney (HEK293) Normal Cells and Human Cervix Epitheloid Carcinoma (HeLa) Cells against Cisplatin Induced Toxicity: A Comparative Study

Introduction: Traditional Siddha Medicine literatures suggest to use the decoction of Vernonia cinerea (VC) to alleviate toxic effects caused by metals. Cisplatin, a metal used in cancer treatment is known to cause nephrotoxicity.Aim: To evaluate and compare protective activity of aqueous extract and fractions of VC in Human Embryonic Kidney (HEK293) cells and Human Cervix Epitheloid Carcinoma (HELA) cell lines against cisplatin induced cytotoxicity.Materials and Methods: The Crude Aqueous Extract (CAE) was obtained from whole plant of VC. CAE was fractioned using nonpolar to polar solvents, and Butanol Fraction (BF) as well as Aqueous Fractions (AF) were obtained. The cell lines were exposed with the IC50 dose of cisplatin, then treated with different doses of the extract or fractions. After 48 hours of incubation, viable cell growth was determined by MTT reduction assay in all the treatments by observing at absorbance of 540 nm. Dose response (% cell viability) curve was drawn and the values were statistically compared using One-Way ANOVA, followed by Dunnett’s (2-sided) post-hoc test.Results: Aqueous fraction showed 2.8 times higher cytoprotection (improvement in cell viability by 54%) than CAE against cisplatin induced toxicity in HEK293 cells. BF did not show protective activity in HEK293 cells. In HELA cells, AF showed 1.73 times higher cytoprotection than CAE and 1.55 times higher cytoprotection than BF against cisplatin, as evidenced by improvement in cell viability by 30%, 11% and 13% in AF, CAE and BF respectively. AF exhibited 80% higher cytoprotection against cisplatin cytotoxicity in normal HEK293 than cancer HELA cells. Whereas, CAE and BF showed lesser cytoprotective activity than AF.Conclusion: This study created scientific basis for using Vernonia cinerea against metal toxicities in Siddha system and suggests that the AF is suitable for further discovery of drug that selectively protect normal renal cell during cisplatin treatment in cancer patients.

Glycoprotein nonmetastatic melanoma protein B accelerates tumorigenesis of cervical cancer in vitro by regulating the Wnt/β-catenin pathway.

Cervical cancer is one of the most common cancers among women around the world. However, the underlying mechanism involved in cervical cancer progression is incompletely known. In the present study, we determined the role of glycoprotein nonmetastatic melanoma protein B (GPNMB) in tumorigenesis of cervical cancer. According to the GEO database, we found that GPNMB expression was significantly higher in cervical cancer than in normal cervix epithelium. A similar pattern was observed in GPNMB expression in cultured cervical cancer cells and normal cervical epithelial cells. Compared with the control, GPNMB knockdown significantly decreased the proliferation and migration capacity, but enhanced the apoptosis capacity of SiHa and HeLa cells. Additionally, the activity of MMP-2 and MMP-9 were aberrantly increased in SiHa and HeLa cells compared with normal cervical epithelial cells, whereas their activities were strongly inhibited by GPNMB siRNA. Furthermore, Wnt/β-catenin signaling was activated by GPNMB in SiHa and HeLa cells. Increased MMP-2/MMP-9 expression was suppressed by Dkk-1, inhibitor of Wnt/β-catenin signaling, while it was enhanced by stimulator BIO. The proliferation, migration, and apoptosis capacity of HeLa cells were found to be affected by Dkk-1 and BIO to different extents. In conclusion, we demonstrated that GPNMB contributed to the tumorigenesis of cervical cancer, at least in part, by regulating MMP-2/MMP-9 activity in tumor cells via activation of canonical Wnt/β-catenin signaling. This might be a potential therapeutic target for treating human cervical cancer.

Effect of hnRNP K and its interaction with HPV16 on cervical intraepithelial neoplasia

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.

Interaction between vaginal micro-environment alterations and HPV16 infection in cervical intraepithelial neoplasia

Objective: To explore the effect of vaginal micro-environment alterations and HPV16 infection and their interaction in the progression of cervical intraepithelial neoplasia. Methods: The participants of this study came from the cervical lesions study cohort in Shanxi province, including 623 women with normal cervical (NC), 303 patients with pathogenically diagnosed low-grade cervical intraepithelial neoplasia (CINⅠ) and 93 patients with pathogenically diagnosed high-grade cervical intraepithelial neoplasia (CINⅡ/Ⅲ). The data of the demographic characteristics of the study subjects and factors related to cervical intraepithelial neoplasia were collected, and HPV16 infection were detected by using flow-through hybridization technology and H(2)O(2), β-glucuronidase, clotting enzyme, neuraminidase and leucocyte esterase in vaginal secretions were detected by using the combined detection kit of aerobic vaginitis and bacterial vaginosis. pH value and vaginal cleanliness were also detected at the same time. The database was established and analyzed by SPSS statistical software (version 22.0). Results: The HPV16 infection rate (trend χ(2)=55.45, P<0.001) and the abnormal rates of H(2)O(2) (trend χ(2)=26.19, P<0.001), pH (trend χ(2)=5.06, P=0.024), vaginal cleanliness (trend χ(2)=19.55, P<0.001), β-glucuronidase (trend χ(2)=17.52, P<0.001) and neuraminidase (trend χ(2)=14.90, P<0.001) increased gradually along with the severity of cervical intraepithelial neoplasia, but the abnormal rates of clotting enzyme and leucocyte esterase showed no same trend. The results of GMDR model analysis showed that there was interaction between HPV16 infection and abnormalities of H(2)O(2), β-glucuronidase, clotting enzyme and neuraminidase in CINⅠ group, and the interaction between HPV16 infection and the abnormalities of vaginal cleanliness, H(2)O(2), β-glucuronidase and neuraminidase in CIN Ⅱ/Ⅲ group. Conclusion: Our findings indicated that the vaginal micro-environment alterations and HPV16 infection could increase the risk of cervical intraepithelial neoplasia, and they might have an important synergistic effect in the progression of cervical intraepithelial neoplasia.

Elevated CTHRC1 expression is an indicator for poor prognosis and lymph node metastasis in cervical squamous cell carcinoma

Collagen triple helix repeat containing 1 (CTHRC1) is overexpressed in different kinds of cancer tissues and may thus promote tumor formation. Thus, we aimed to explore whether CTHRC1 could be a predictor of clinical significance including survival in cervical squamous cell carcinoma (CSCC). Western blot analysis was conducted in 20 frozen tissue specimens of CSCC and 10 frozen tissue sections of normal cervix. Immunohistochemistry was performed in 130 tissues with CSCC. The relationships of CTHRC1 expression with clinicopathological variables, and prognosis of CSCCs were explored. Statistical analyses were carried out using χ2 test, multivariate Cox proportional-hazard model, Kaplan-Meier method, and univariate and multivariate logistic regression. The expression of the CTHRC1 protein was significantly higher in tumors than in normal tissues (P < .001). CTHRC1 overexpression was strongly associated with the International Federation of Gynecology and Obstetrics stage (P = .038), histologic grade (P < .001), stromal infiltration depth (P < .001), lymphovascular space invasion (P = .023), lymph node metastasis (P = .001), and recurrence (P = .021). The multivariate proportional-hazard model revealed that CTHRC1 overexpression was an independent predictor of overall survival and disease-free survival (P = .034 and P = .025, respectively). Multivariate logistic regression analysis revealed that high CTHRC1 expression was positively correlated with lymph node metastasis (odds ratio: 2.658; 95% confidence interval: 1.120-6.305; P = .020). Therefore, CTHRC1 may be a valuable biomarker for predicting the prognosis and metastasis of CSCC and a potential therapeutic target for treatment of the disease.

Maternal embryonic leucine zipper kinase: A novel biomarker and a potential therapeutic target of cervical cancer.

Maternal embryo leucine zipper kinase (MELK) is highly expressed in a variety of malignant tumors and involved in cell cycle regulation, cell proliferation, apoptosis, tumor formation etc However, the biological effects of MELK in cervical cancer are still uninvestigated. This study aimed to explore the expression of MELK in cervical cancer, as well as its effects on the proliferation, apoptosis, DNA damage repair on cervical cancer cell line in vitro and to provide novel ideas for further improving the clinical efficacy of cervical cancer. Immunohistochemistry, Western blot, RT‐qPCR, CCK8, and immunofluorescence techniques were used to detect the expression of MELK in cervical cancer tissues, paracancerous tissues, and cervical cancer cell lines. Several cervical cancer cell lines were treated with MELK knockdown by siRNA and MELK selective inhibitor OTSSP167. The effects on proliferation, apoptosis, and colony formation capacity, and tumor cell DNA damage repair‐related factor were detected in cell lines. Our data showed that the high expression rate of MELK in cervical cancer patients was 56.92%. MELK expression in cervical cancer samples was significantly higher than that in paraneoplastic tissues. Highly expressed MELK correlated with the cervical histopathological grading and greatly increased with the cervical histopathological grading, from normal cervix and cervical intraepithelial neoplasia to cervical cancer. Moreover, the abnormal expression of MELK was related to cervical cancer metastasis at early stage. The knockdown of MELK with siRNA and OTSSP167 had strong inhibition effects on the proliferation, apoptosis, and colony formation of cervical cancer cells. MELK knockdown could also aggravate the DNA damage of cervical cancer cells possibly by homologous recombination repair pathway. Therefore, MELK may be a predicting marker of poor prognosis of cervical cancer and may also be a new therapeutic target for cervical cancer, providing ideas for improving the therapeutic effect of cervical cancer.

Visual inspection of cervix with acetic acid as a feasible screening test for cervical neoplasia among women attending at OPD in Rajshahi Medical College Hospital

Background: Visual inspection of cervix after application of 3-5% acetic acid (VIA) is a potential alternative to Pap smear cytology for screening of cervical cancer in resource poor settings.Objectives: This study was to evaluate the performance of visual inspection based screening approach in the detection of precancerous and early cancerous lesions of the cervix.Materials &amp; Methods: VIA was carried out in 540 eligible women attending Gynae OPD. Detection of well-defined, opaque, acetowhite lesion close to squamocolumnar junction or in transitional zone of the cervix constituted positive VIA. All screened women evaluated by colposcopy and biopsy were taken from colposcopically suspected areas. The final diagnosis was based on histology.Results: Out of 540 patients, 328 were VIA negative and 212 were VIA positive. Colposcopy showed normal results in 340 cases, low grade CIN in 138 cases, high grade CIN in 44 cases and cancer in 18 cases. There were biopsy proven chronic cervicitis and metaplastic changes in 423 cases, CIN I in 66 cases, CIN II in 25 cases, CIN III / carcinoma-in-situ in 5 cases. The sensitivity of VIA was 74.36%, specificity 70.45%, positive predictive value 41.04%, &amp; negative predictive value 90.85%.Conclusion: VIA can differentiate a normal cervix from a precancerous cervix with reasonable accuracy. As it is low cost and simple method, it can be set in any hospital or any health care centre of rural or urban areas of poor resource settings.KYAMC Journal Vol. 9, No.-2, July 2018, Page 56-60

MR targetedimaging for the expression of tenascin-C in cervicalcancer.

To detect cervical cancer and compare tumor invasiveness using a molecular targeted probe. Tenascin-C expression was evaluated in 15 specimens. Five of them were cervical cancer with node metastasis (group A), five were cervical cancer without node metastasis (group B), and another five were normal cervix tissues (group C). Superparamagnetic iron oxide (SPIO) nanoparticles and tenascin-C antibody were conjugated as an MR probe. After the fresh tissues incubated with the probe for 24 h, MR was performed to analysis the tissue signal changes. Cervical cancer tissues with node metastasis showed highest tenascin-C expression, while normal cervix showed little expression. For the non-metastatic cervical cancer patients, tenascin-C showed moderate expression. Tenascin-C was found diffusely in the stromal surrounding malignant tumor cells. After MR, the image signal changes (contrast-to-noise ratio) kept consistent with tenascin-C expression and showed statistical difference between the three groups (A: 3.87 ± 1.45 vs B: 2.33 ± 1.04 vs C: 0.66 ± 0.31; p = 0.002). Tenascin-C expression can help to detect cervical cancer. MRI with SPIO-antitenascin-C may be used to evaluate the preoperative cervical cancer patients with node metastasis. Tenascin-C expression can help to detect cervical cancer and compare cancer invasiveness. Protein expression difference can be captured and compared on MR with SPIO-antitenascin-C.

SOX17 restrains proliferation and tumor formation by down-regulating activity of the Wnt/\u03b2-catenin signaling pathway via trans-suppressing \u03b2-catenin in cervical cancer

The SRY-box containing gene 17 (SOX17) is considered as a regulator in stemness maintenance and a suppressor in some malignant tumors. However, the biological function and molecular mechanism of SOX17 in the process of initiation and progression of cervical cancer remain obscure. In this study, immunohistochemistry showed that the expression of SOX17 was high in the normal cervix, moderate in the high-grade squamous intraepithelial lesion, and low in the cervical cancer. SOX17 inhibited the proliferation and viability of cervical cancer cells in vitro as well as tumor formation in vivo. Additionally, SOX17 induced the cell cycle arrest at the transition from the G0/G1 phase to the S phase. The TOP/ FOP-Flash reporter assay and Western blotting showed SOX17 inhibited the activity of the Wnt/β-catenin signaling pathway in cervical cancer. Further, firefly luciferase reporter assay and quantitative chromatin immunoprecipitation (qChIP) assays confirmed that SOX17 trans-suppressed the expression of β-catenin by directly binding to the specific region of the β-catenin promoter. Together, our data demonstrated that SOX17 restrained the proliferation and tumor formation by down-regulating the activity of the Wnt/β-catenin signaling pathway via trans-suppression of β-catenin in cervical cancer.

Association between high risk human papillomavirus DNA load and cervical lesions in different infection status

Objective: To explore the relationship between high risk HPV (HR-HPV) DNA load and cervical lesions in HR-HPV single/ multiple infections. Methods: Two thousand six hundred and forty-six women from Shanxi, Henan and Xinjiang were recruited into a cervical cancer screening program. Cervical exfoliated cell specimens collected from all of the participants were detected by hybrid capture Ⅱ (HC2), cytological diagnosis was performed according to the Bethesda System, and pathological diagnosis was interpreted using cervical intraepithelial neoplasia (CIN) terminology.Totally 571 cervical specimens were selected and retested to ascertain the HPV types and single/ multiple infections by liner array, a PCR-based method. Semi-quantitative result of HR-HPV DNA load (pg/ml) was estimated by HR HC2.According to the taxonomy of "International Human Papillomavirus Reference Center" , 13 HR-HPVs, including HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68, which could be detected by HR HC2 were divided into 4 subgroups. Results: The positive rate of HR-HPV in normal cervix (436 cases), CIN1 (88 cases), CIN2+ (47 cases) group were 29.82%, 85.23% and 100%, respectively. The overall prevalence and median viral load increased coordinating with the pathological degree of cervical lesions (P<0.001). The positive rate and viral load of single infection with HR-HPV belongs to α9 species increased coordinating with the pathological degree of cervical lesions (P<0.05). The viral load of single infection with HR-HPV belongs to α7 species in CIN1 group was higher than those of normal group and CIN2+ group, but without statistical significance (P=0.130). The viral load of multiple infections in CIN1 group was 559.13 pg/ml, significantly higher than 37.73 pg/ml of normal histology (P=0.025), but without significant difference of 332.91 pg/ml of CIN2+ group (P=0.790). The median viral load of HPV single infection in CIN1 group was 167.93 pg/ml, significantly lower than 559.73 pg/ml of multiple infections (P=0.044). The incidence of co-infection with HR-HPVs belong to α9 species was 80.56%, dominated in all patterns of multiple infections and their median viral load increased coordinating with the pathological degree of cervical lesions, but without significant difference (P>0.05). The incidence of co-infection with HR-HPVs belong to α7 species was 66.67%, their median viral load in CIN1 group was higher than that of CIN2+ group, but without statistical significance (P>0.05). Conclusions: Viral loads of single/ multiple infections with HR-HPVs belong to different species show different tendencies coordinating with the pathological degree of cervical lesions. Women with high grade of cervical lesion were dominantly infected with high viral load of HR-HPVs belong to α9 species, and the viral load of multiple infections is higher than that of single infection in low grade of cervical lesion.

Deregulation of LIMD1-VHL-HIF-1α-VEGF pathway is associated with different stages of cervical cancer.

To understand the mechanism of cellular stress in basal-parabasal layers of normal cervical epithelium and during different stages of cervical carcinoma, we analyzed the alterations (expression/methylation/copy number variation/mutation) of HIF-1α and its associated genes LIMD1, VHL and VEGF in disease-free normal cervix (n = 9), adjacent normal cervix of tumors (n = 70), cervical intraepithelial neoplasia (CIN; n = 32), cancer of uterine cervix (CACX; n = 174) samples and two CACX cell lines. In basal-parabasal layers of normal cervical epithelium, LIMD1 showed high protein expression, while low protein expression of VHL was concordant with high expression of HIF-1α and VEGF irrespective of HPV-16 (human papillomavirus 16) infection. This was in concordance with the low promoter methylation of LIMD1 and high in VHL in the basal-parabasal layers of normal cervix. LIMD1 expression was significantly reduced while VHL expression was unchanged during different stages of cervical carcinoma. This was in concordance with their frequent methylation during different stages of this tumor. In different stages of cervical carcinoma, the expression pattern of HIF-1α and VEGF was high as seen in basal-parabasal layers and inversely correlated with the expression of LIMD1 and VHL. This was validated by demethylation experiments using 5-aza-2'-deoxycytidine in CACX cell lines. Additional deletion of LIMD1 and VHL in CIN/CACX provided an additional growth advantage during cervical carcinogenesis through reduced expression of genes and associated with poor prognosis of patients. Our data showed that overexpression of HIF-1α and its target gene VEGF in the basal-parabasal layers of normal cervix was due to frequent inactivation of VHL by its promoter methylation. This profile was maintained during different stages of cervical carcinoma with additional methylation/deletion of VHL and LIMD1.

Stromal p16 Overexpression in Gastric-type Mucinous Carcinoma of the Uterine Cervix.

Gastric-type mucinous carcinoma (MC-G) of the uterine cervix displays distinct morphological features and an aggressive clinical course. The expression status of p16 in the stroma has not been investigated in adenocarcinoma of the uterine cervix. Stromal p16 expression was evaluated in endocervical adenocarcinomas, including usual-type endocervical adenocarcinoma (UEA), intestinal-type mucinous carcinoma (MC-I), and MC-G. Whether stromal p16 expression varied significantly according to the histological subtype and whether the expression status is associated with clinicopathological characteristics of MC-G was also investigated. Immunostaining of p16 was performed for 24, 19, and 18 cases of UEA, MC-I, and MC-G, respectively. UEA and MC-I subtypes exhibited horizontally continuous, strong nuclear p16 immunoreactivity in the tumor cells, whereas none of the MC-G cases showed diffuse and strong nuclear immunoreactivity for p16 in the tumor cells. Instead, 10 (55.6%) cases of MC-G displayed moderately to strongly positive p16 expression in the stroma. Stromal p16 expression of MC-G was significantly higher than that of normal cervix, UEA, and MC-I. Metastatic MC-G had significantly higher stromal p16 expression than primary MC-G. Further, stromal p16 overexpression in MC-G was associated with advanced stage, parametrial invasion, and lymphovascular invasion. Stromal p16 expression of MC-G was significantly higher than that of normal cervix and other histological subtypes of adenocarcinoma and was associated with advanced stage, parametrial invasion, and lymphovascular invasion, reflecting the aggressive behavior of MC-G. Our observations suggest that stromal p16 expression is involved in the development and progression of MC-G.

Apatinib, a novel tyrosine kinase inhibitor, suppresses tumor growth in cervical cancer and synergizes with Paclitaxel

ABSTRACT Apatinib is a novel tyrosine kinase inhibitor that targets VEGFR2 signal and exhibits potent anti-tumor effects in human cancers. In this study, we aim to investigate the efficacy of Apatinib in cervical cancer. The protein expression of VEGFR2 and its relationships with clinical parameters were investigated in a panel of cervical cancer patients. In vitro, a series of experiments were performed to detect the effects of Apatinib on the proliferation, apoptosis and cell cycle in cervical cancer cells. Both the immortalized cell lines and primary cultured tissues were used to investigate the synergy between Apatinib and chemotherapeutic drugs. The in vivo effects of Apatinib were validated in a nude mouse model. Compared to that in normal cervix, VEGFR2 protein was significantly upregulated in cervical cancer tissues (P < 0.001); this was positively correlated with advanced tumor stage, lymph node metastasis, and a poor prognosis. In vitro, Apatinib markedly induced apoptosis and G1-phase arrest, suppressed cell growth, and decreased colony formation ability. We also found that primary cancer tissues with higher level of VEGFR2 were much more sensitive to Apatinib. Further, we proved that Apatinib significantly increased the sensitivity to Paclitaxel in cervical cancer cells and the mouse model. Collectively, we firstly report the anti-tumor efficacy of Apatinib in cervical cancer. Moreover, Apatinib synergized with Paclitaxel to achieve more significant suppression on tumor growth, proposing that Apatinib might be a potent drug for cervical cancer.

Interaction between abnormal expression of fragile histidine triad and methyl-CpG-binding protein 2 on cervical cancerization

Objective: To explore the relationship between abnormal expression of fragile histidine triad (FHIT) gene and methyl-CpG-binding protein 2 (MeCP2) as well as their interaction on cervical cancerization. Methods: A total of 73 patients with cervical squamous cell carcinoma (SCC), 113 patients with cervical intraepithelial neoplasia (CIN Ⅰ, n=45; CINⅡ/Ⅲ, n=68) and 60 women with normal cervix (NC) were included in the study. Real time PCR and Western blot were performed to detect the expression levels of mRNA and protein about FHIT and MeCP2, respectively. The methylation status of FHIT gene CpG island was tested by methylation-specifc PCR (MSP). Kruskal-Wallis H test, χ(2) test, trend χ(2) test and Spearman correlation analysis were conducted with software SPSS 20.0. The interaction was evaluated by generalized multifactor dimensionality reduction (GMDR) model. Results: With the deterioration of cervical lesion, the methylation rates of FHIT gene CpG island (χ(2)=18.64, P<0.001; trend χ(2)=18.08, P<0.001) increased gradually, while the expression levels of FHIT mRNA (H=27.32, P<0.001; trend χ(2)=12.65, P<0.001) and protein (H=47.10, P<0.001; trend χ(2)=29.79, P<0.001) decreased gradually. There was a negative correlation between the methylation rates of FHIT gene CpG island and the expression level of FHIT protein (r=-0.226, P<0.001). The levels of MeCP2 mRNA (H=26.19, P<0.001; trend χ(2)=11.81, P=0.001) and protein (H=69.02, P<0.001; trend χ(2)=47.44, P<0.001) increased gradually with the aggravation of cervical lesions. There was a positive correlation between the expression level of MeCP2 protein and the FHIT mRNA Ct ratio (r=0.254, P<0.001). Expression of proteins were negatively correlated between MeCP2 and FHIT (r=-0.213, P=0.001). The results analyzed by GMDR model showed that there were interactions among high MeCP2 protein expression, the CpG island methylation of FHIT and mRNA and protein expression in CINⅡ/Ⅲ group, and among high MeCP2 mRNA and protein expression, the CpG island methylation of FHIT and low mRNA and protein expression in SCC group. Conclusion: High expression of MeCP2 mRNA and protein, the CpG island methylation and low mRNA and protein expression of FHIT could increase the risk of cervical carcinogenesis, and there might be a synergistic effect on cervical carcinogenesis.

Identification of miRNAs in cervical mucus as a novel diagnostic marker for cervical neoplasia

microRNAs (miRNAs) play important roles in regulation of gene expression during cervical carcinogenesis. We investigated expression profiles of miRNAs in cervical cancer and its precursor lesions by utilizing cervical mucus. Cervical mucus was collected from 230 patients with a normal cervix, cervical intraepithelial neoplasia (CIN), squamous cell carcinoma (SCC), or adenocarcinoma (AD). The levels of miRNA in the mucus were quantified by miRNA array and real-time reverse-transcriptase polymerase chain reaction (RT-PCR). The performance for detecting diseases was statistically analysed. The expression of miRNAs was further validated in the surgical tissues of enrolled patients. Four miRNAs (miR-126-3p, -20b-5p, -451a, and -144-3p) were significantly up-regulated in SCC and AD compared with normal, and their expression levels correlated with disease severity and high-risk human papillomavirus infection. Receiver operating characteristic curve analyses revealed that the area under the curve values for miR-126-3p, -20b-5p, -451a, and -144-3p were 0.89, 0.90, 0.94, and 0.93, respectively, for SCC plus AD compared with normal, showing high accuracy of cancer detection. Real-time RT-PCR analyses confirmed the expression of these four miRNAs in frozen tissues from cervical cancer. miR-126-3p, -20b-5p, -451a, and -144-3p in cervical mucus are promising biomarkers for cervical cancer and high-grade CINs.

NK-Cell Count and its Function in Producing Interferon Gamma Associated with the Cervical Cancer Natural History

Natural killer cell (NK-cell) has an important role in tumor immune surveillance. We investigate the NK-cell count and its function in producing IFN-I³ on normal cervix, subclinical high risk-HPV (hr-HPV) infection, precancerous lesion, and cervical cancer in order to understand the natural history of uterine cervical cancer.

Molecular Signatures of High-Grade Cervical Lesions.

Cervical cancer is the fourth most common neoplasia in women and the infection with human papilloma virus (HPV) is its necessary cause. Screening methods, currently based on cytology and HPV DNA tests, display low specificity/sensitivity, reducing the efficacy of cervical cancer screening programs. Herein, molecular signatures of cervical cytologic specimens revealed by liquid chromatography-mass spectrometry (LC-MS), were tested in their ability to provide a metabolomic screening for cervical cancer. These molecules were tested whether they could clinically differentiate insignificant HPV infections from precancerous lesions. For that, high-grade squamous intraepithelial lesions (HSIL)-related metabolites were compared to those of no cervical lesions in women with and without HPV infection. Samples were collected from women diagnosed with normal cervix (N = 40) and from those detected with HSIL from cytology and colposcopy (N = 40). Liquid-based cytology diagnosis, DNA HPV-detection test, and LC-MS analysis were carried out for all the samples. The same sample, in a customized collection medium, could be used for all the diagnostic techniques employed here. The metabolomic profile of cervical cancer provided by LC-MS was found to indicate unique molecular signatures for HSIL, being two ceramides and a sphingosine metabolite. These molecules occurred independently of women’s HPV status and could be related to the pre-neoplastic phenotype. Statistical models based on such findings could correctly discriminate and classify HSIL and no cervical lesion women. The results showcase the potential of LC-MS as an emerging technology for clinical use in cervical cancer screening, although further validation with a larger sample set is still necessary.

GDF15 promotes the proliferation of cervical cancer cells by phosphorylating AKT1 and Erk1/2 through the receptor ErbB2

BackgroundGrowth differentiation factor 15 (GDF15) is a member of the TGF-β superfamily, and evidence suggests that a substantial amount of GDF15 is secreted in various human cancers, such as ovarian cancer, prostate cancer, and breast cancer, among others. However, the function of GDF15 in cervical cancer has not yet been reported.MethodsImmunohistochemistry was used to detect GDF15 expression in normal cervix and in different cervical cancer lesions. Cell growth curves, MTT, tumor formation assays and flow cytometry were utilized to observe the effects of ectopic GDF15 expression on the proliferation and cell cycle of cervical cancer cells. Real-time PCR, western blotting and immunoprecipitation assays were conducted to measure the expression of genes related to the cell cycle and the PI3K/AKT and MAPK/ERK signaling pathways. A chromatin immunoprecipitation assay was performed to confirm whether C-myc bound to a specific region of the GDF15 promoter. Inhibitor treatment and immunoprecipitation assays were employed to identify the association between GDF15 and ErbB2.ResultsGDF15 expression gradually increased during the progression of cervical carcinogenesis. GDF15 promoted cervical cancer cell proliferation via exogenous rhGDF15 treatment or the use of gene editing technology in vitro and in vivo and significantly accelerated the cell cycle transition from G0/G1 to S phase. The expression of p-ErbB2, p-AKT1, p-Erk1/2, CyclinD1 and CyclinE1 was up-regulated and the expression of p21 was down-regulated in GDF15-overexpressing and rhGDF15-treated cervical cancer cells. C-myc trans-activated GDF15 expression by binding to the E-box motifs in the promoter of GDF15 and contributed to the positive feedback of GDF15/C-myc/GDF15. Furthermore, GDF15 bound to ErbB2 in a protein complex in cervical cancer cells.ConclusionsOur data demonstrated that GDF15 promoted the proliferation of cervical cancer cells via the up-regulation of CyclinD1 and CyclinE1 and the down-regulation of p21 through both the PI3K/AKT and MAPK/ERK signaling pathways in a complex with ErbB2.

Clinico-histological analysis of non-neoplastic lesions of cervix

Background: Cervix from hysterectomy constitutes the majority of gynecological specimens and includes genital infections to invasive carcinoma. These cervical lesions are the source of morbidity and mortality in women. But, there are many benign lesions which appears malignant on gross and microscopy so, misdiagnosed. Hence, clinical and histopathological examination of cervix is required for proper diagnosis and to exclude misdiagnosis.Materials and Methods: This is a retrospective study conducted in Histopathology section of Pathology Department of Rural Institute of Medical Science and Research, Saifai, Etawah (U.P) India from 1 January 2008 to 31 December 2015 on 1544 hysterectomy specimens. Histopathology examination was the diagnostic tool. Cervical pathologies in hysterectomy specimens were evaluated grossly and microscopically and correlated with clinical findings. Results: Vaginal discharge was the most common clinical complaint (43.00%), followed by bleeding complaints (23.38 %). Grossly normal cervix was seen in (32.51 %) and nabothian follicles were found in (20.07%). On histopathology examination, chronic cervicitis was the most common pathology (79.66 %).Conclusion: Cervix is site of shades of grey lesions that include cervical dysplasia to malignancy. But majority of cervical pathologies are benign lesions. However, many times benign lesions are misdiagnosed as malignant. Histopathological examination along with clinical findings is mandatory for the early and accurate diagnosis. Health camps along with cervix screening and educational awareness program should be carried out.

Differential gene expression of extracellular-matrix-related proteins in the vaginal apical compartment of women with pelvic organ prolapse

Pelvic organ prolapse (POP) is a multifactorial disorder that impairs the quality of life (QoL) of older women in particular. The purpose of this study was to elucidate the pathogenesis of POP by focusing on the extracellular matrix (ECM). Patients were classified into two groups-with or without cervical elongation-using the POP quantification system. Specimens were obtained from 29 women with POP during hysterectomy. The expression of fibulin-5, elastin, integrin β1 (ITGβ1), lysyl oxidase-like protein-1 (LOXL1) and collagen in the vagina, uterosacral ligament, and uterine cervix was investigated by quantitative real-time polymerase chain reaction (RT-PCR) and correlation between gene levels and severity of POP examined. The location of proteins was analyzed using immunohistochemical staining and expression of fibulin-5 protein analyzed by Western blotting. Fibulin-5 and elastin were mainly expressed in lamina propria and fibromuscular layers of the vagina and uterosacral ligament. Gene levels of fibulin-5 and ITGβ1 in uterosacral ligaments increased with severity of POP in women with cervical elongation, while no correlation was observed in women with a normal cervix. In women with uterine cervical elongation, each ECM-related gene significantly increased with POP staging. Furthermore, fibulin-5 protein also increased in the uterosacral ligament and uterine cervix. The severity of POP and gene expression of ECM-related proteins were inversely correlated in vaginal tissue in a normal and elongated cervix. These results suggested that the differing progression of the two types of POP have a relationship with ECM-related protein.