Mei-ng Yu

Dysregulation of microRNA-204 mediates migration and invasion of endometrial cancer by regulating FOXC1

MicroRNAs (miRNAs) regulate mRNA stability and protein expression, and certain miRNAs have been demonstrated to act either as oncogenes or tumor suppressors. Differential miRNA expression signatures have been documented in many human cancers but the role of miRNAs in endometrioid endometrial cancer (EEC) remains poorly understood. This study identifies significantly dysregulated miRNAs of EEC cells, and characterizes their impact on the malignant phenotype. We studied the expression of 365 human miRNAs using Taqman low density arrays in EECs and normal endometriums. Candidate differentially expressed miRNAs were validated by quantitative real‐time PCR. Expression of highly dysregulated miRNAs was examined in vitro through the effect of anti‐/pre‐miRNA transfection on the malignant phenotype. We identified 16 significantly dysregulated miRNAs in EEC and 7 of these are novel findings with respect to EEC. Antagonizing the function of miR‐7, miR‐194 and miR‐449b, or overexpressing miR‐204, repressed migration, invasion and extracellular matrix‐adhesion in HEC1A endometrial cancer cells. FOXC1 was determined as a target gene of miR‐204, and two binding sites in the 3′‐untranslated region were validated by dual luciferase reporter assay. FOXC1 expression was inversely related to miR‐204 expression in EEC. Functional analysis revealed the involvement of FOXC1 in migration and invasion of HEC1A cells. Our results present dysfunctional miRNAs in endometrial cancer and identify a crucial role for miR‐204‐FOXC1 interaction in endometrial cancer progression. This miRNA signature offers a potential biomarker for predicting EEC outcomes, and targeting of these cancer progression‐ and metastasis‐related miRNAs offers a novel potential therapeutic strategy for the disease.

Evaluation of extraction methods from paraffin wax embedded tissues for PCR amplification of human and viral DNA

Aim—To evaluate the efficiency of phenol/chloroform, microwave, and Qiagen spin column based DNA extractions from paraffin wax embedded tissue for use in the polymerase chain reaction (PCR). In addition, to assess the reliability of amplifying a housekeeping gene to indicate successful viral DNA extraction. Methods—DNA samples extracted from 20 blocks of cervical carcinoma tissues using the three methods were subjected to PCRs targeting 509 bp and 355 bp of the β globin gene, and 450 bp and 150 bp of human papillomavirus (HPV) DNA. Results—Microwave extraction showed the highest positive rate for β globin PCR, whereas the spin column method was the most efficient for HPV DNA extraction. When the 509 bp β globin and 450 bp HPV PCR results were correlated, two of 10, eight of 12, and nine of 10 β globin positive extractions prepared by means of the phenol/chloroform, microwave, and spin column methods, respectively, yielded HPV DNA of the expected size. For the β globin negative samples, HPV was detected in three of 10, two of eight, and four of 10 samples. Conclusions—HPV DNA extraction was most efficient using the Qiagen spin column and had the highest positive predictive value when a housekeeping gene was used as an indicator of successful viral DNA extraction; the phenol/chloroform method was the least efficient. The potential drawbacks of some extraction methods when using a human housekeeping gene to assess the quality of viral DNA extraction need to be considered.

Identification of molecular markers and signaling pathway in endometrial cancer in Hong Kong Chinese women by genome-wide gene expression profiling

Endometrial cancer is the third most common gynecologic malignancy and the ninth most common malignancy for females overall in Hong Kong. Approximately 80% or more of these cancers are endometrioid endometrial adenocarcinomas. The aim of this study was to reveal genes contributing to the development of endometrioid endometrial cancer, which may impact diagnosis, prognosis and treatment of the disease. Whole-genome gene expression analysis was completed for a set of 55 microdissected sporadic endometrioid endometrial adenocarcinomas and 29 microdissected normal endometrium specimens using the Affymetrix Human U133 Plus 2.0 oligonucleotide microarray. Selected genes of interest were validated by quantitative real-time-polymerase chain reaction (qRT-PCR). Pathway analysis was performed to reveal gene interactions involved in endometrial tumorigenesis. Unsupervised hierarchical clustering displayed a distinct separation between the endometrioid adenocarcinomas and normal endometrium samples. Supervised analysis identified 117 highly differentially regulated genes (⩾4.0-fold change), which distinguished the endometrial cancer specimens from normal endometrium. Twelve novel genes including DKK4, ZIC1, KIF1A, SAA2, LOC16378, ALPP2, CCL20, CXCL5, BST2, OLFM1, KLRC1 and MBC45780 were deregulated in the endometrial cancer, and further validated in an independent set of 56 cancer and 29 normal samples using qRT-PCR. In addition, 10 genes were differentially regulated in late-stage cancer, as compared to early-stage disease, and may be involved in tumor progression. Pathway analysis of the expression data from this tumor revealed an interconnected network consisting of 21 aberrantly regulated genes involved in angiogenesis, cell proliferation and chromosomal instability. The results of this study highlight the molecular features of endometrioid endometrial cancer and provide insight into the events underlying the development and progression of endometrioid endometrial cancer.

MicroRNA-182 plays an onco-miRNA role in cervical cancer.

OBJECTIVES The purposes of this study were to identify aberrantly expressed miRNAs and investigate their pathogenic roles in cervical cancer. METHODS miRNA expression was assessed in cervical cancer cell lines, micro-dissected normal cervical epithelium cells and primary cervical carcinoma by TaqMan RT-PCR. Spatial expression of miR-182 in cervical carcinoma and normal cervix was explored by in situ hybridization. HeLa xenograft mice model was used for evaluation of the effect on tumor growth of miR-182 inhibitor. Western blot, flow cytometry and gene expression analysis were used for identification of the functional role of miR-182 in HeLa cells. RESULTS Two up-regulated (miR-182 and -183) and nine down-regulated (miR-211, 145, 223, 150, 142-5p, 328, 195, 199b, 142-3p) microRNAs were consistently identified in cervical cancer cell lines. Further investigation confirmed the most up-regulated miRNA (miR-182) was significantly elevated in primary cervical carcinoma and discovered a significant correlation between the increased expression of miR-182 and advanced stages of cervical cancer. In HeLa xenograft mouse model, we demonstrated that inhibition of the miR-182 could exert the effect of tumor growth regression. Western blot, flow cytometry and pathway analysis for the HeLa cells with miR-182 over/down-expression in vitro showed that miR-182 was involved in apoptosis and cell cycle pathways, it also associated with the regulation of FOXO1. CONCLUSIONS Our findings indicated that miR-182 plays an onco-miRNA role in cervical cancer and its alteration is associated with cervical cancer pathogenesis by disrupting cell proliferation.

Depth-resolved fluorescence spectroscopy of normal and dysplastic cervical tissue

A portable confocal system with the excitations at 355nm and 457nm was instrumented to investigate the depth-resolved fluorescence of cervical tissue. The study focused on extracting biochemical and morphological information carried in the depth-resolved signals measured from the normal squamous epithelial tissue and squamous intraepithelial lesions. Strong keratin fluorescence with the spectral characteristics similar to collagen were observed from the topmost keratinizing layer of all tissue samples. It was found that NADH and FAD fluorescence measured from the underlying non-keratinizing epithelial layer were strongly correlated to the tissue pathology. This study demonstrates that the depth-resolved fluorescence spectroscopy can potentially provide more accurate diagnostic information for determining tissue pathology.

Depth-resolved fluorescence spectroscopy reveals layered structure of tissue.

A confocal fluorescence spectroscopy system is instrumented to study depth-resolved autofluorescence in biological tissue. The system provides the capability of optical sectioning with the maximal detectiondepth up to 120 m in the examined tissue samples. It was found that the topmost keratinizing epithelial layer produces strong fluorescence similar to collagen. The fluorescence signal from epithelial tissue between the keratinizing layer and stroma can be well resolved. The study results show that depth-resolved fluorescence spectroscopy has the potential to provide more accurate information for the diagnosis of tissue pathology.

Expression of apoptotic regulators and their significance in cervical cancer

Insufficient apoptosis is implicated in many human cancers, including cervical carcinoma. The objectives of this study were to explore changes of apoptosis-regulating gene expression and their clinical significance in cervical cancer. The expression of apoptosis-regulating genes, including five Bcl-2 family and two caspase family members, was evaluated in 43 cervical invasive squamous cell carcinomas, using immunohistochemistry. Specimens in which >or=10% of the neoplastic cells showed cytosolic immunoreactivity were considered to be immunopositive. Results were correlated with clinico-pathologic characteristics of the subjects. All seven apoptotic regulators examined were positive in a proportion of the tumors. The percentage of cases expressing Bax was higher in the patients without evidence of disease after treatment than in the patients alive with disease or who died of disease (P<0.05). A significant difference in disease-free survival was detected between Bax-positive and -negative groups (P<0.05), and in overall survival between Mcl-1-positive and -negative groups (P<0.05). Significant association between the seven markers tested was only found for caspase 3 and Bak immunoreactivity in cervical carcinoma (P<0.05). The results demonstrate expression of multiple apoptosis-modulating proteins in cervical cancer. There appears to be complex regulation of apoptosis protein levels in association with clinical behavior of cervical squamous cell carcinoma.

Risk factors for squamous intraepithelial lesions in systemic lupus erythematosus: A prospective cohort study

We undertook a prospective cohort study to ascertain the risk factors for the development of squamous intraepithelial lesions (SIL) in patients with systemic lupus erythematosus (SLE).

Epidemiologic Risk Profile of Infection With Different Groups of Human Papillomaviruses

This study identified the age‐specific prevalence and epidemiologic risk profile for infection with different groups and species of human papillomaviruses (HPV). Structured interview and HPV testing were conducted for 2,604 Chinese women self‐referred for cervical screening. Independent risk factors for infection were identified by multiple logistic regressions. Overall, a major peak of HPV infection was observed at women aged 26–30 years, and a minor peak at 46–55 years. This pattern was observed for high‐risk, low‐risk, and alpha‐5/7/9 HPVs; but alpha‐3/6 HPVs showed peaks of similar magnitudes in young and older women. Independent risk factors for HPV infection (all types combined) included younger age (OR [95% CI] for >55 vs. ≤30 years = 0.22 [0.09–0.55]; 31‐45 vs. ≤ 30 years = 0.57 [0.33–0.99]), having ≥4 lifetime sexual partners (2.28 [1.06–4.88]), and smoking (2.24 [1.22–4.10]). Young age and smoking were the most consistent independent risk factors observed across different HPV groups. The risk profile for high‐risk HPV was similar to alpha‐5/7/9. Single‐type infection was associated with having more sexual partners, higher education level and oral contraception; whereas multiple‐type infection was associated with smoking. In conclusion, a U‐shaped age‐specific prevalence curve was observed for HPV infection overall, but with a different pattern for different HPV species. Different HPV groups showed variations in their risk profiles. These data are useful for formulating preventative strategy for HPV‐related diseases. Catch‐up vaccination program in Hong Kong should cover a wider age group as the first peak of infection occurred relatively late. J. Med. Virol. 81:1635–1644, 2009.

CHD5 Downregulation Associated with Poor Prognosis in Epithelial Ovarian Cancer

Background: The CHD5 gene located on 1p36 encodes a protein – chromodomain helicase DNA-binding protein 5. CHD5 has been shown to be a tumor suppressor gene candidate. This study investigated the involvement of CHD5 in ovarian cancer and its clinicopathological significance. Methods: CHD5 expression in ovarian cancer and its counterpart were determined by quantitative RT-PCR. The correlation of CHD5 expression to clinicopathological features of the tumor was analyzed. Results: CHD5 expression was downregulated by at least twofold in 32 of 72 (41%) invasive epithelial ovarian carcinomas when compared to 12 controls in Hong Kong Chinese women. CHD5 downregulation was correlated to clinical status (p < 0.05), but not to patient age, tumor type and grade, recurrence and clinical stage (p > 0.05). Survival analysis showed that patients with CHD5 downregulation in their tumors were associated with shorter disease-free and total survival times compared to those without CHD5 downregulation (p < 0.05). Cox proportional-hazards regression analysis indicated that downregulation of CHD5 is an independent adverse prognostic factor in ovarian cancer. Conclusion: This study shows that CHD5 is downregulated in a certain number of ovarian cancers and appears to be an adverse predictor candidate of ovarian cancer disease-free and total survival.