[Show abstract][Hide abstract] ABSTRACT: Papanicolaou (Pap) triage, with high specificity, has been recommended for primary Human papillomavirus (HPV) testing but is flawed by poor sensitivity and cytologist dependence. We evaluated the potential role of microRNA (miRNA) detection in cervical exfoliated cells in HPV-positive women from a clinic-based population.
Journal of the National Cancer Institute. 09/2014; 106(9).
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to characterize the clinical significance of GMFG, a novel ADF/cofilin superfamily protein, and investigate its role in cell migration and invasion in epithelial ovarian cancer (EOC).
The expression of GMFG in EOC tissues and ovarian cancer cell lines was evaluated by immunohistochemistry and immunoblotting respectively. The data were statistically analyzed for the associations of GMFG expression with clinicopathologic parameters and survival. In vitro cell migration and invasion assays were performed to determine the role of GMFG in cell migratory behaviors. The effect of GMFG on reorganization of actin cytoskeleton was investigated by immunostaining.
GMFG was overexpressed in EOC. Up-regulated GMFG expression was closely correlated with advanced FIGO stage and chemoresistance of the disease. EOC patients with higher GMFG expression showed poorer progression-free survival (PFS) and overall survival (OS). In vitro cellular assays revealed that GMFG promoted cell migration and invasion. GMFG expression altered actin cytoskeleton organization probably by interacting with Arp2/3 complex.
GMFG expression independently predicts poorer prognosis in patients with EOC. Ectopic overexpression of GMFG contributes to the malignant biological behavior of ovarian cancer cells.
[Show abstract][Hide abstract] ABSTRACT: Glycogen synthase kinase-3 (GSK-3) plays an important role in human cancer. The aim of this study is to evaluate the clinicopathological significance of expression of GSK-3α/β and pGSK-3α/β(Tyr279/216) in patients with epithelial ovarian cancer and to investigate whether GSK-3 inhibition can influence cell viability and tumor growth of ovarian cancer.
OncoTargets and Therapy 01/2014; 7:1159-68. · 2.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acquired chemo-resistance is one of the key causal factors in cancer death. Emerging evidences suggest that miRNA and epithelial-mesenchymal transition play critical roles in the chemo-resistance in cancers. Here, we showed the association of paclitaxel-resistance with miR-375 over-expression and epithelial-mesenchymal transition inducement in cervical cancer. Using different cervical cancer cell models, we found that paclitaxel transiently induced up-regulation of miR-375 expression, proliferation inhibition, transition from epithelial to mesenchymal phenotype, and consequently impaired paclitaxel sensitivity. Forced over-expression of miR-375 may suppress Ecadherin expression by a directly targeting pathway, which led to paclitaxel resistance. Contrarily, re-expression of Ecadherin partly reversed epithelial-mesenchymal transition phenotype and miR-375 induced paclitaxel-resistance. Our findings suggest that paclitaxel-induced miR-375 over-expression facilitates epithelial-mesenchymal transition process via directly targeting Ecadherin, proliferation inhibition, and consequently results in chemo-resistance in cervical cancer cells. A reversion of miR-375 or Ecadherin expression may be a novel therapeutic approach for overcoming chemo-resistance in cervical cancer.
PLoS ONE 01/2014; 9(10):e109299. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: HPV58 is relatively prevalent in China and other Asian countries. In this study, the HPV58 genome in cervical lesions was decoded from five CIN2/3 and five cervical cancer tissues using rolling-circle amplification of total cell DNA and deep sequencing and verified by whole genome cloning and sequencing. HPV58 isolates from China feature a total of 52 nucleotide substitutions (0.66%) from reference HPV58, which appear mainly in two regions, with 12 from nt 3430 to 4136 covering E2/E4/E5 ORFs and 13 from nt 4621 to 5540 covering L2 ORF, and could be grouped as HPV58 Chinese Zhejiang-1, -2, and -3 (CNZJ-1, -2, and -3) according to their sequence similarities and restriction enzyme digestions. Phylogenetically, CNZJ-3 is similar to the reference HPV58 close to sublineage A1. The other two are close to sublineage A2. Analysis of cervical lesion-derived RNA revealed abundant HPV58 early transcripts spliced at the E6 and E1/E2 ORFs, where two 5' splice sites at nt 232 and nt 898 and two 3' splice sites at nt 510 and nt 3355 can be identified. Thus, our study represents the first genome-wide analysis of HPV58 and its expression in cervical lesions.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: To assess the feasibility of validating microRNA (miRNA) profile related to paclitaxel-sensitivity in formalin-fixed paraffin-embedded (FFPE) samples of serous ovarian carcinoma (OC) patients. METHODS: Deregulated miRNAs identified by miRNA microarray were further detected in 45 FFPE OC samples using Realtime PCR. Correlations between paired FFPE and frozen tumor samples were analyzed. Survival times were compared between 6 high and low miRNAs groups. Western blot and luciferase reporter assay were used for validating the target of miRNA. RESULTS: Sixteen up-regulated miRNAs and twenty-three down-regulated miRNAs were revealed in pacilitaxel-resistant ST30 cells. The up-regulated miRNAs (miR-320a, 22 and 129-5p) and down-regulated miRNAs (miR-9, 155 and 640) were confirmed in paclitaxel-resistant FFPE tumor samples, compared with paclitaxel-sensitive samples. Higher miR-9 and miR-640 showed better survival time in OC patients. Expressions of miR-9, 155 and 22 in FFPE samples were closely mimicked by those in frozen tissues. RAB34 was validated as a direct target of miR-9. CONCLUSIONS: We validated miRNA profile in pacilitaxel-resistant OC using FFPE samples, which might enable treatment stratification and help us to predict outcomes in OC patients. FFPE samples are feasible materials for miRNA research.
BMC Cancer 04/2013; 13(1):216. · 3.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVE: Expression of the mitochondrial protein, nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) Fe-S protein 3 (NDUFS3), down-regulates in some cancers including breast and kidney. This study evaluates NDUFS3 expression and its clinical significance in human serous ovarian adenocarcinoma. METHODS: 30 ovarian normal epithelium, 30 benign serous adenoma, and 100 serous carcinoma tissues were collected, and the expression of NDUFS3 protein and messenger RNA was evaluated by immunohistochemistry, Western blot, and real-time reverse transcription-polymerase chain reaction, respectively. Relationships between NDUFS3 protein expression and clinicopathologic parameters and disease-free and overall survival were also studied. RESULTS: The expression of NDUFS3 messenger RNA and protein was significantly decreased in serous ovarian adenocarcinoma compared with normal ovarian epithelium and benign serous adenoma (both P < 0.001). Reduced NDUFS3 immunostaining correlated with advanced Federation of Gynecology and Obstetrics stage (P = 0.002) and suboptimal residual disease after primary surgery (P = 0.021). Reduced NDUFS3 expression also correlated with shorter disease-free (P = 0.002) and overall survival (P = 0.004). CONCLUSIONS: NDUFS3 expression is down-regulated in serous ovarian adenocarcinoma, suggesting that NDUFS3 may contribute to the development of serous ovarian cancer.
International Journal of Gynecological Cancer 02/2013; · 1.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Angiogenesis is a vital process for proper embryonic development, wound healing, malignant tumor growth and metastasis. Two glia maturation factor genes, glia maturation factor-β (GMFB) and glia maturation factor-γ (GMFG), presenting different expression patterns and distinct biological functions are found in vertebrates. But, the role of GMFB and GMFG in vascular development remains largely unknown. Here, we showed that both GMFB and GMFG are highly conserved in vertebrates. Whole-mount in situ hybridization and quantitative RT-PCR results revealed that GMFB and GMFG were differently expressed during zebrafish embryogenesis. GMFB was highly enriched in brain and GMFG was predominantly expressed in endothelial cells. By gene specific MO, knockdown of GMFG, but not GMFB, severely disrupted angiogenic sprouting of intersegmental vessels (ISVs), but this angiogenic defects were prevented by overexpression of a MO-resistant form of zebrafish GMFG mRNA. In addition, the expressions of angiogenic factors VEGF-A, STAT3, MMP2, MMP9, and MMP13 were significantly decreased in endothelial cells of GMFG morphants. Our findings provide the first in vivo evidence that GMFG is an important regulator for angiogenic sprouting during angiogenesis in zebrafish and suggest that GMFG may act as a novel potential target for anti-angiogenesis therapy in clinical settings.
Experimental Cell Research 01/2013; · 3.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to evaluate the effectiveness of conization in the diagnosis and treatment of high-grade cervical intraepithelial neoplasia (CIN) in post-menopausal women. A total of 101 post-menopausal patients who were diagnosed with high-grade lesion CIN by biopsy and in whom conization was used as the primary treatment were examined and 202 pre-menopausal patients were studied as the controls. Clinical and pathological data including symptoms, cytological examination and HPV DNA test results before and after conization treatment were analyzed. Both the cytological abnormalities (57.9 vs. 58.5%, P=0.260) and the positive rate of the HPV DNA test (89.5 vs. 86.4%, P=0.812) did not show a significant difference between the post- and pre-menopausal group. The rate of satisfactory colposcopy was significantly lower in post-menopausal patients compared with pre-menosausal patients (23.2 vs. 68.9%, P<0.001). Post-menopausal patients presented a significantly lower diagnostic consistency between colpscopy-directed biopsy and conization (46.4 vs. 68.9%, P=0.004), and a significantly higher positive margin rate of conization (20.8 vs. 10.9%, P=0.020). A total of 10 of the 101 post-menopausal and 2 of the 202 pre-menopausal women were diagnosed with invasive cancer by conization and underwent further treatment. In conclusion, these data suggest that conization, as a conservative primary treatment, is not suitable for post-menopausal women with high-grade lesion CIN due to the lower rate of satisfactory colposcopy, lower consistency of diagnosis between colposcopy-directed biopsy and conization, and a higher positive margin of conization.
Experimental and therapeutic medicine 01/2013; 5(1):185-188. · 0.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The association of cancer stem cells with epithelial-mesenchymal transition (EMT) is receiving attention. We found in our previous study that EMT existed from CD24- phenotype cells to their differentiated cells. It was shown that cyclin D1 functioned in sustaining self-renewal independent of CDK4/CDK6 activation, but its effect on the EMT mechanism in ovarian cancer stem cells is unclear.
The anchorage-independent spheroids from ovarian adenocarcinoma cell line 3AO were formed in a serum-free medium. CD24- and CD24+ cells were isolated by fluorescence-activated cell sorting. Cell morphology, viability, apoptosis, and migratory ability were observed. Stem-related molecule Bmi-1, Oct-4 and EMT-related marker E-cadherin, and vimentin expressions were analyzed. Cyclin D1 expression in CD24- phenotype enriched spheroids was knocked down with small interfering RNA, and its effects on cell proliferation, apoptosis, migration ability, and EMT-related phenotype after transfection were observed.
In our study, CD24- cells presented stronger proliferative, anti-apoptosis capacity, and migratory ability, than CD24+ cells or parental cells. CD24- cells grew with a scattered spindle-shape within 3 days of culture and transformed into a cobblestone-like shape, identical to CD24+ cells or parental cells at 7 days of culture. CD24- cells or spheroids highly expressed cyclin D1, Bmi-1, and vimentin, and seldom expressed E-cadherin, while CD24+ or parental cells showed the opposite expression. Furthermore, cyclin D1-targeted small interfering RNA resulted in decreased vimentin expression in spheroids. Transfected cells also exhibited an obvious decrease in cell viability and migration, but an increase in cell apoptosis.
Cancer stem cell-like cells possess mesenchymal characteristics and EMT ability, and cyclin D1 involves in EMT mechanism, suggesting that EMT of cancer stem cell-like cells may play a key role in invasion and metastasis of ovarian cancer.
OncoTargets and Therapy 01/2013; 6:667-77. · 2.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to investigate the role of the cell surface expression levels of HLA class I and II molecules, the costimulatory molecules CD80/B7-1 and CD86/B7-2, and the adhesion molecules CD54 and CD58 during cervical carcinogenesis. The expression levels of MHC class I and II molecules, the costimulatory molecules CD80/B7-1 and CD86/B7-2 and the adhesion molecules CD54 and CD58 on CD14+ peripheral blood monocytes (PBMs) from 21 cases of cervical intraepithelial neoplasia (CIN) II-III, 51 squamous cervical carcinomas (SCCs) and 18 healthy controls were analyzed using flow cytometry analysis. We found increased expression levels of HLA-DR (p=0.000), HLA-DQ (p=0.000), CD86/B7-2 (p=0.002) and CD58 (p=0.000) on PBMs from patients with SCC and CIN II-III, compared with healthy control subjects, whereas no significant difference existed in the expression levels of HLA class I antigens, HLA-DP CD80/B7-1 and CD54. Upregulated expression levels of HLA-DR, HLA-DQ, CD86/B7-2 and CD58 were associated with disease progression, indicating that an increased expression of HLA-DR, HLA-DQ, CD86/B7-2 and CD58 on PBMs may be correlated with the evolution of cervical cancer.
Molecular Medicine Reports 09/2012; 6(6):1301-4. · 1.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of the present study was to investigate the correlation between fibrinogen level, platelet count and prognosis in patients with epithelial ovarian cancer (EOC).
Preoperative fibrinogen level and platelet count in 136 EOC patients and 146 patients with benign ovarian tumor, and their associations with clinicopathologic parameters and survival in EOC patients, were retrospectively analyzed.
The fibrinogen level in EOC was higher than that in benign patients (3.95 ± 1.37 g/L versus 2.88 ± 0.6 g/L, P < 0.001), and 36.0% (49/136) of EOC patients had hyperfibrinogenemia (fibrinogen >4.0 g/L). The platelet count in EOC was higher than that in benign patients (251.5 ± 89.4 × 10(9) /L versus 206.7 ± 49.0 × 10(9) /L P < 0.001), and 7.4% (10/136) of EOC patients had thrombocytosis (platelet count >400 × 10(9) /L). Hyperfibrinogenemia was associated with International Federation of Gynecologists and Obstetricians (FIGO) stage, non-optimal cytoreduction and poor chemo-response, but not with histologic type and grade, CA-125 level, chemotherapy method, and age. EOC patients with advanced disease showed higher rate of elevated thrombocyte count than patients with early disease (30.7% versus 8.3%, P = 0.002). The rate of thrombocytosis was higher in patients with hyperfibrinogenemia than in those with normal fibrinogen (9/10 versus 1/10, P < 0.001). A significant correlation between platelet count and fibrinogen level was observed in EOC patients (P < 0.001). In multivariate analysis, overall survival was influenced by tumor stage (P < 0.001), chemotherapy with taxane (P < 0.001) and fibrinogen level (P = 0.004), and disease-free survival was only influenced by tumor stage (P < 0.001).
Our findings suggest that hyperfibrinogenemia may be a predictor for poor chemo-response and have a potential role as independent prognostic factors in EOC patients.
Journal of Obstetrics and Gynaecology Research 03/2012; 38(4):651-7. · 0.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pelvic lymph node metastases are regarded as the most important risk factor and a predictor of poor prognosis for patients with cervical cancer. Exploration of metastasis-related molecules is helpful toward improving the prognosis in cervical cancer. To identify the role of miR-375 in metastasis and progression of cervical cancer, we examined the expression of miR-375 in 170 cervical cancer tissues and 68 normal cervical tissues, using stem-loop quantitative PCR, and found that the expression of miR-375 in cervical cancer tissues was significantly decreased by 4.45-fold, compared with 68 normal tissues. A significant correlation existed between miR-375 expression and clinicopathologic parameters, including lymph node metastasis of cervical cancer. Overexpressed miR-375 suppressed cell proliferation, blocked G1-to-S cell-cycle transition, and inhibited cell migration and invasion in human cervical SiHa and CaSki cells. SP1, a potential target gene of miR-375, was inversely correlated with miR-375 expression in cervical cancer tissues. Moreover, SP1 was negatively regulated by miR-375, and knockdown of SP1 by siRNA inhibited cell malignant behaviors. Thus, our findings suggest that down-regulated miR-375 promotes cell malignant behaviors via the target gene SP1 and may consequently contribute to the progression of cervical cancer.
American Journal Of Pathology 09/2011; 179(5):2580-8. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Quantitative real-time RT-PCR (RT-qPCR) is being widely used in microRNA expression research. However, few reports detailed a robust identification and validation strategy for suitable reference genes for normalisation in microRNA RT-qPCR studies. The aim of this study was to identify the most stable reference gene(s) for quantification of microRNA expression analysis in uterine cervical tissues. A microarray was performed on 6 pairs of uterine cervical tissues to identify the candidate reference genes. The stability of candidate reference genes was assessed by RT-qPCR in 23 pairs of uterine cervical tissues. The identified most stable reference genes were further validated in other cohort of 108 clinical uterine cervical samples: (HR-HPV- normal, n=21; HR-HPV+ normal, n=19; cervical intraepithelial neoplasia [CIN], n=47; cancer, n=21), and the effects of normalizers on the relative quantity of target miR-424 were assessed. In the array experiment, miR-26a, miR-23a, miR-200c, let-7a, and miR-1979 were identified as candidate reference genes for subsequent validation. MiR-23a was identified as the most reliable reference gene followed by miR-191. The use of miR-23a and miR-191 to normalize expression data enabled detection of a significant dereg-ulation of miR-424 between normal, CIN and cancer tissue. Our results suggested that miR-23a and miR-191 are the optimal reference microRNAs that can be used for normalization in profiling studies of cervical tissues; miR-23a is a novel microRNA normalizer.
Experimental and Molecular Medicine 04/2011; 43(6):358-66. · 2.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: miRNAs have the potential to act on diverse downstream genes, and miRNA signatures of HPV-infected tissues may provide insight into HPV-related carcinogenesis. We set out to profile miRNA expression in HPV-infected samples and relate this to histological and grade-specific alterations in the spectrum of cervical carcinogenesis in vivo. A total of 31 miRNAs showed significant and continuous expression along with the progression from normal cervical tissue to cancer, and six of them were validated in 133 samples. By bioinformatics analyses, we established a putative HPV-associated miRNA-mRNA regulatory network, showing that miR-29 is the most highly enriched. We also found that YY1 and CDK6 were both positively correlated with E6/E7 RNA expression and targeted by tumour-suppressive miR-29. Evidence of miR-29 involvement in HPV infection was further verified in patient samples and by various experimental approaches. Taken together, our results suggest that HPVs have oncogenic properties at least in part by reshaping the milieu of cellular miRNAs. miR-29 restrains cell cycle progression and induces apoptosis via YY1 and CDK6 promoting malignant transformation induced by HPV, although the abnormality of miR-29 in HPV-infected cells might be regulated in an indirect way.
The Journal of Pathology 02/2011; 224(4):484-95. · 7.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The specific intratype HPV genome variations may be associated with the development of cervical cancer in specific geographic regions and a given population. Human papillomavirus (HPV) 58 and 52 have been found to be relatively prevalent among Asian women including Chinese women. This study aimed to assess the risk of HPV 58 and 52 variants for cervical cancer and its precursors in Chinese women.
A total of 2021cervical samples were collected. After DNA extraction and genotyping, a total of 298 (177 HPV58-single positive and 121 HPV52-single positive) DNA samples were analyzed for E6 and E7 sequence variations by direct sequencing.
A total of 29 new reported variations of HPV 58 and 52 were found. For HPV58, the presence of C632T (T20I) and G760A (G63S) variants in E7 showed a positive trend of the association with the severity of neoplasia (P(trend)<0.05, χ² test for trend).
These findings suggest that C632T (T20I) and G760A (G63S) variants in HPV58 E7 are probably risk factors associated with the development of cervical cancer in Chinese women. The presence of HPV58/52 E6 and E7 variants may be different in Chinese women.
[Show abstract][Hide abstract] ABSTRACT: Human papillomavirus (HPV) type 16 is the major etiological agents of cervical cancer. Recently, the studies have demonstrated that HPV intratypic variations could affect oncogenic potential to cervical cancer development. The objectives of this study were to identify HPV-16 E6 and E7 variants prevalent in Chinese women and to assess the risk of them for invasive cervical carcinoma and cervical intraepithelial neoplasia (CIN).
DNA samples were genotyped by flow-through hybridization (HybriMax) and amplified by using primers specific for E6 and E7. Products were directly sequenced and analyzed using BLAST on PubMed.
A total of 170 cervical samples (33 cases of normal control, 11 of CIN 1, 72 of CIN 2-3, and 54 of invasive cervical carcinoma) were HPV-16-positive and were analyzed for E6 and E7 sequence variation. The results showed that HPV-16 Asian lineage was the most frequently detected (77%) and that the Asian variant presented a significantly higher disease risk for cervical cancer and CIN. In addition, 3 novel variants at E6 (Q20P, H118Q, and Q123K) and 2 at E7 (D75N and T86P) were found. The substitution G368T at E6 leading to a premature stop codon occurred in an isolate of normal control sample.
This study reported the distribution of HPV-16 E6 and E7 gene variations in women from southeastern China, which are different from that showed in previous studies and may be important when developing an effective vaccine for this area.
International Journal of Gynecological Cancer 11/2010; 20(8):1391-8. · 1.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: For quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR), the most commonly used normalization strategy is to select a stable reference gene. However, no suitable reference genes have been identified in cervical tissues to date. The aim of this study was to identify the most stable gene or a set of genes as reference genes for RT-qPCR analysis in cervical tissues from a panel of 12 candidates (ALAS1, PPIA, GAPDH, HBB, TBP, ACTIN, B2M, MBNL2, PGKL, RPLP0, RPL-4, and EEF1A1). In total, 20 normal and 20 cervical cancer specimens were examined. Gene expression data were analyzed using two different statistical models (geNorm and NormFinder). EEF1A1 was identified as the most stable and reliable reference gene, followed by GAPDH and RPLP0, whereas EEF1A1 and GAPDH were the best two-gene combination by NormFinder. The expression validity of EEF1A1 was further determined in 21 normal, 22 cervical intraepithelial neoplasia (CIN(2-3)), and 18 cancer tissues; no expression differences were found among normal, CIN(2-3), and cancer tissues (P>0.05). Our results suggested that EEF1A1 can be used as a reference gene for normalization in gene profiling studies in clinic cervical samples, and the combination of EEF1A1 and GAPDH could be recommended as a much more reliable normalization strategy.
[Show abstract][Hide abstract] ABSTRACT: To study short-term type-specific human papillomavirus (HPV) persistence and associated predictors in an asymptomatic general female population in Zhejiang, China.
Persistence was evaluated in women who were HPV positive at enrollment and who returned within 6-14 months. Liquid-based cytology screening was encouraged for returning women.
Persistence was evaluated in 548 women. Overall persistence was 49.1%, and established high-risk HPV persistence was 54.5%. The types associated with the highest level of persistence were HPV-52, HPV-58, HPV-56, and HPV-16-most of which belong to alpha9 species. In total, 252 women refused cytology screening. No differences were found regarding HPV persistence and other variables among women who returned for follow-up, women who accepted cytology screening, and women who refused cytology screening. Among women aged 35 years or older, there were no differences between those with normal cytology and those with abnormal cytology in the short-term persistence of HPV types, except for HPV-58 (P<0.01) and HPV-81 (P=0.04). Established high-risk HPV persistence increased with age, low income, and early sexual experience.
The data support close surveillance of older women with established high-risk HPV infections, and conservative management of women with non-alpha9 HPV and no risk factors.
International journal of gynaecology and obstetrics: the official organ of the International Federation of Gynaecology and Obstetrics 09/2010; 110(3):217-22. · 1.41 Impact Factor