Vivian W. Wang

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.

Dysregulated microRNAs and their predicted targets associated with endometrioid endometrial adenocarcinoma in Hong Kong women

The objective of this study, a parallel study to global gene expression profiling, was to identify dysregulated microRNAs (miRNAs) associated with endometrioid endometrial adenocarcinoma (EEC), examine their correlation with clinico‐pathological characteristics and identify predicted target genes of the dysregulated miRNAs. Using real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR), profiling of miRNA expression was performed in 30 EECs and 22 normal counterparts in which genome‐wide gene expression had been previously profiled and reported. Clustering analysis identified 30 miRNAs which were significantly dysregulated in EEC. The expression of a sub‐group of miRNAs was significantly correlated with clinico‐pathological characteristics including stage, myometrial invasion, recurrence and lymph node involvement. By searching for predicted miRNA targets that were linked to the dysregulated genes previously identified, 68 genes were predicted as candidate targets of these 30 dysregulated miRNAs. miR‐205 was significantly overexpressed in EECs compared with normal controls. After transfection of a miR‐205 inhibitor, the expression of miR‐205 in endometrial cancer cell line RL95‐2 cells decreased whereas its predicted target gene, JPH4, showed increased protein expression. JPH4 seems to be a real miR‐205 target in vitro and in vivo, and a candidate tumor suppressor gene in EEC. Based on this study in EEC, miRNAs predicted to be involved in tumorigenesis and tumor progression have been identified and placed in the context of the transcriptome of EEC. This work provides a framework on which further research into novel diagnosis and treatment of EEC can be focused.

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.

Genome-wide gene expression profiling of cervical cancer in Hong Kong women by oligonucleotide microarray.

An analysis of gene expression profiles obtained from cervical cancers was performed to find those genes most aberrantly expressed. Total RNA was prepared from 29 samples of cervical squamous cell carcinoma and 18 control samples, and hybridized to Affymetrix oligonucleotide microarrays with probe sets complementary to over 20,000 transcripts. Unsupervised hierarchical clustering of the expression data readily distinguished normal cervix from cancer. Supervised analysis of gene expression data identified 98 and 139 genes that exhibited >2‐fold upregulation and >2‐fold downregulation, respectively, in cervical cancer compared to normal cervix. Several of the genes that were differentially regulated included SPP1 (Osteopontin), CDKN2A (p16), RPL39L, Clorf1, MAL, p11, ARS and NICE‐1. These were validated by quantitative RT‐PCR on an independent set of cancer and control specimens. Gene Ontology analysis showed that the list of differentially expressed genes included ones that were involved in multiple biological processes, including cell proliferation, cell cycle and protein catabolism. Immunohistochemical staining of cancer specimens further confirmed differential expression of SPP1 in cervical cancer cells vs. nontumor cells. In addition, 2 genes, CTGF and RGS1 were found to be upregulated in late stage cancer compared to early stage cancer, suggesting that they might be involved in cancer progression. The pathway analysis of expression data showed that the SPP1, VEGF, CDC2 and CKS2 genes were coordinately differentially regulated between cancer and normal. The present study is promising and provides potential new insights into the extent of expression differences underlying the development and progression of cervical squamous cell cancer. This study has also revealed several genes that may be highly attractive candidate molecular markers/targets for cervical cancer diagnosis, prognosis and therapy.

Prevalence of human papillomavirus in cervical cancer: A multicenter study in China

A large‐scale epidemiologic survey on the prevalence of different types of human papillomavirus (HPV) in cervical cancer in China is indicated because of the implications for the development of diagnostic probes and vaccines against cervical cancer. A total of 809 cervical cancer specimens were collected from 5 regions in China including Shanghai, Guangzhou, Sichuan, Beijing and Hong Kong. HPV DNA was detected in 83.7% of the specimens. HPV‐16 was present in 79.6%, HPV‐18 in 7.5%, HPV‐52 in 2.6% and HPV‐58 in 3.8% of all HPV‐positive specimens. The prevalences of HPV‐16 and HPV‐18 in Hong Kong were 61.7 and 14.8%, respectively, representing a lower HPV‐16 and a higher HPV‐18 proportion compared with the other regions. HPV‐16 remained the most common HPV infection in both squamous cell carcinoma (SCC) and adenocarcinoma (AC). The proportion of HPV‐18 infection was significantly higher in AC than in SCC.

Dysregulated microRNAs in the pathogenesis and progression of cervical neoplasm.

MicroRNAs (miRNAs) play an important role in a variety of physiological as well as pathophysiological processes, including carcinogenesis. The aim of this study is to identify a distinct miRNA expression signature for cervical intraepithelial neoplasia (CIN) and to unveil individual miRNAs that may be involved in the development of cervical carcinoma. Expression profiling using quantitative real-time RT-PCR of 202 miRNAs was performed on micro-dissected high-grade CIN (CIN 2/3) tissues and compared to normal cervical epithelium. Unsupervised hierarchical clustering of the miRNA expression pattern displayed a distinct separation between the CIN and normal cervical epithelium samples. Supervised analysis identified 12 highly differentially regulated miRNAs, including miR-518a, miR-34b, miR-34c, miR-20b, miR-338, miR-9, miR-512-5p, miR-424, miR-345, miR-10a, miR-193b and miR-203, which distinguished the high-grade CIN specimens from normal cervical epithelium. This miRNA signature was further validated by an independent set of high-grade CIN cases. The same characteristic signature can also be used to distinguish cervical squamous cell carcinoma from normal controls. Target prediction analysis revealed that these dysregulated miRNAs mainly control apoptosis signaling pathways and cell cycle regulation. These findings contribute to understanding the role of microRNAs in the pathogenesis and progression of cervical neoplasm at the molecular level.

Clinical and Prognostic Significance of Human Papillomavirus in a Chinese Population of Cervical Cancers

Objective: To investigate the clinical and prognostic significance of human papillomavirus (HPV) in a Chinese population of cervical cancers. Methods: We studied 121 cervical cancer tissue samples from patients treated at our hospital. Identification and typing of HPV were done by polymerase chain reaction (PCR) using consensus primers MY11 and MY09 followed by direct DNA sequencing. The results were correlated with various clinical and prognostic parameters. Results: We found HPV DNA in 95 (78.5%) cases, including HPV-16 in 59 (48.8%) and HPV-18 in 14 (11.6%) cases. χ2 analysis revealed no significant correlation between the presence of HPV DNA and age at diagnosis, clinical stage, histologic type, tumor grading, 2-year and 5-year survival rate. Of the factors evaluated, age at diagnosis and histologic type were found to have a statistically significant relationship with HPV type. The mean age of the HPV-18 group was 48.6 years compared to 57.1 years for the HPV-16 group (p = 0.045) and 58.2 years for the HPV-negative group (p = 0.04). HPV-18 was detected more often in adenocarcinomas (AC) than in squamous cell carcinomas (SCC). Conversely HPV-16 was detected significantly more often in SCC (p < 0.0001). The HPV-negative group also had a higher incidence of SCC (p = 0.007). HPV-18-positive patients seemed to have more nodal involvement than both HPV-16-positive patients (45.5 vs. 20.8%) and HPV-negative patients (45.5 vs. 18.2%); however, it did not reach statistical significance. Conclusions: These observations suggest that the presence of HPV DNA does not bear any clinical or prognostic significance in a Chinese population of cervical cancers. HPV-18 is found more often in younger patients and is associated with AC.

Allelic loss on chromosome 1 is associated with tumor progression of cervical carcinoma

Alterations in chromosome 1 are common in human malignancies. The frequency of loss of heterozygosity (LOH) on chromosome 1 in cervical carcinoma and its clinical significance are not clearly understood.

p53 Polymorphism and Human Papillomavirus Infection in Hong Kong Women with Cervical Cancer

In this study we investigated the involvement of p53 polymorphism at codon 72, the infection and typing of human papillomavirus (HPV) and the correlation of p53 polymorphism with HPV type and other clinicopathologic characteristics in 72 Hong Kong women with cervical cancer. We developed a simple and nonradioactive method for determining polymorphism at codon 72 of the p53 gene. The homozygous p53 arginine allele (Arg/Arg) was detected in 22 (31%), the homozygous p53 proline allele (Pro/Pro) in 14 (19%) and the heterozygous allele (Arg/Pro) in 36 (50%) cases, respectively. Using the consensus primers MY11 and MY09, HPV infection was detected in 55 of 72 (76%) cases. The prevalent types were HPV-16 (55%), HPV-18 (16%) and HPV-58 (9%). The number of HPV-positive cases with Arg/Arg, Pro/Pro and Arg/Pro were 17 (31%), 12 (22%) and 26 (47%), respectively. The p53 polymorphism at codon 72 was not significantly correlated with any of the HPV types (p > 0.05). No striking overrepresentation of homozygous arginine-72 p53 was observed in HPV-associated cervical cancer. The results in this study did not show that any p53 polymorphic form has a prognostic significance for cervical cancer.

Genome-Wide Characterization of Transcriptional Patterns in High and Low Antibody Responders to Rubella Vaccination

Immune responses to current rubella vaccines demonstrate significant inter-individual variability. We performed mRNA-Seq profiling on PBMCs from high and low antibody responders to rubella vaccination to delineate transcriptional differences upon viral stimulation. Generalized linear models were used to assess the per gene fold change (FC) for stimulated versus unstimulated samples or the interaction between outcome and stimulation. Model results were evaluated by both FC and p-value. Pathway analysis and self-contained gene set tests were performed for assessment of gene group effects. Of 17,566 detected genes, we identified 1,080 highly significant differentially expressed genes upon viral stimulation (p<1.00E−15, FDR<1.00E−14), including various immune function and inflammation-related genes, genes involved in cell signaling, cell regulation and transcription, and genes with unknown function. Analysis by immune outcome and stimulation status identified 27 genes (p≤0.0006 and FDR≤0.30) that responded differently to viral stimulation in high vs. low antibody responders, including major histocompatibility complex (MHC) class I genes (HLA-A, HLA-B and B2M with p = 0.0001, p = 0.0005 and p = 0.0002, respectively), and two genes related to innate immunity and inflammation (EMR3 and MEFV with p = 1.46E−08 and p = 0.0004, respectively). Pathway and gene set analysis also revealed transcriptional differences in antigen presentation and innate/inflammatory gene sets and pathways between high and low responders. Using mRNA-Seq genome-wide transcriptional profiling, we identified antigen presentation and innate/inflammatory genes that may assist in explaining rubella vaccine-induced immune response variations. Such information may provide new scientific insights into vaccine-induced immunity useful in rational vaccine development and immune response monitoring.