Kwong Wai Choy

MiR-222 Overexpression Confers Cell Migratory Advantages in Hepatocellular Carcinoma through Enhancing AKT Signaling

Purpose: This study aims to profile the expressions of 156 microRNAs (miRNA) in hepatocellular carcinoma (HCC) and to characterize the functions of miR-222, the most significantly upregulated candidate identified. Experimental Design: miRNA expression profile in HCC tumors, matching adjacent cirrhotic livers, and cell lines was conducted using quantitative PCR. Common miR-222 upregulations were further validated in a larger cohort of tumors. The functional effects of miR-222 inhibition on HCC cell lines were examined. The downstream modulated pathways and target of miR-222 were investigated by coupling gene expression profiling and pathway analysis, and by in silico prediction, respectively. Luciferase reporter assay was done to confirm target interaction. Results: We identified a 40-miRNA signature that could discriminate tumors from adjacent cirrhotic liver tissue, and further corroborated common miR-222 overexpression in tumors relative to its premalignant counterpart (55.3%; P < 0.0001). Increased miR-222 expression correlated significantly with advanced stage HCC and with the shorter disease-free survival of patients (P ≤ 0.01). Inhibition of miR-222 in Hep3B and HKCI-9 significantly retarded cell motility (P < 0.05). Further investigations suggested that AKT signaling was the major pathway influenced by miR-222. A consistent reduction of AKT phosphorylation in Hep3B and HKCI-9 was shown following miR-222 suppression. The protein phosphatase 2A subunit B (PPP2R2A) was predicted as a putative miR-222 target in silico. We found that miR-222 inhibition could augment the tumor protein level and restore luciferase activity in reporter construct containing the PPP2R2A 3′ untranslated region (P = 0.0066). Conclusions: Our study showed that miR-222 overexpression is common in HCC and could confer metastatic potentials in HCC cells, possibly through activating AKT signaling. Clin Cancer Res; 16(3); 867–75

Therapeutic potentials of gene silencing by RNA interference: principles, challenges, and new strategies.

During recent decades there have been remarkable advances in biology, in which one of the most important discoveries is RNA interference (RNAi). RNAi is a specific post-transcriptional regulatory pathway that can result in silencing gene functions. Efforts have been done to translate this new discovery into clinical applications for disease treatment. However, technical difficulties restrict the development of RNAi, including stability, off-target effects, immunostimulation and delivery problems. Researchers have attempted to surmount these barriers and improve the bioavailability and safety of RNAi-based therapeutics by optimizing the chemistry and structure of these molecules. This paper aimed to describe the principles of RNA interference, review the therapeutic potential in various diseases and discuss the new strategies for in vivo delivery of RNAi to overcome the challenges.

EZH2-Mediated Concordant Repression of Wnt Antagonists Promotes β-Catenin–Dependent Hepatocarcinogenesis

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the Polycomb-repressive complex 2 (PRC2) that represses gene transcription through histone H3 lysine 27 trimethylation (H3K27me3). Although EZH2 is abundantly present in various cancers, the molecular consequences leading to oncogenesis remain unclear. Here, we show that EZH2 concordantly silences the Wnt pathway antagonists operating at several subcellular compartments, which in turn activate Wnt/β-catenin signaling in hepatocellular carcinomas (HCC). Chromatin immunoprecipitation promoter array and gene expression analyses in HCCs revealed EZH2 occupancy and reduced expression of Wnt antagonists, including the growth-suppressive AXIN2, NKD1, PPP2R2B, PRICKLE1, and SFRP5. Knockdown of EZH2 reduced the promoter occupancy of PRC2, histone deacetylase 1 (HDAC1), and H3K27me3, whereas the activating histone marks were increased, leading to the transcriptional upregulation of the Wnt antagonists. Combinatorial EZH2 and HDAC inhibition dramatically reduced the levels of nuclear β-catenin, T-cell factor-dependent transcriptional activity, and downstream pro-proliferative targets CCND1 and EGFR. Functional analysis revealed that downregulation of EZH2 reduced HCC cell growth, partially through the inhibition of β-catenin signaling. Conversely, ectopic overexpression of EZH2 in immortalized hepatocytes activated Wnt/β-catenin signaling to promote cellular proliferation. In human HCCs, concomitant overexpression of EZH2 and β-catenin was observed in one-third (61/179) of cases and significantly correlated with tumor progression. Our data indicate that EZH2-mediated epigenetic silencing contributes to constitutive activation of Wnt/β-catenin signaling and consequential proliferation of HCC cells, thus representing a novel therapeutic target for this highly malignant tumor.

Non-invasive prenatal testing for fetal chromosomal abnormalities by low-coverage whole-genome sequencing of maternal plasma DNA: review of 1982 consecutive cases in a single center

To review the performance of non‐invasive prenatal testing (NIPT) by low‐coverage whole‐genome sequencing of maternal plasma DNA at a single center.

Secondary findings from non‐invasive prenatal testing for common fetal aneuploidies by whole genome sequencing as a clinical service

To report secondary or additional findings arising from introduction of non‐invasive prenatal testing (NIPT) for aneuploidy by whole genome sequencing as a clinical service.

Prevalence of recurrent pathogenic microdeletions and microduplications in over 9500 pregnancies.

The implementation of chromosomal microarray analysis (CMA) in prenatal testing for all patients has not achieved a consensus. Technical alternatives such as Prenatal BACs‐on‐BeadsTM (PNBoBsTM) have thus been applied. The aim of this study was to provide the frequencies of the submicroscopic defects detectable by PNBoBsTM under different prenatal indications.

TBX6 Null Variants and a Common Hypomorphic Allele in Congenital Scoliosis

BACKGROUND Congenital scoliosis is a common type of vertebral malformation. Genetic susceptibility has been implicated in congenital scoliosis. METHODS We evaluated 161 Han Chinese persons with sporadic congenital scoliosis, 166 Han Chinese controls, and 2 pedigrees, family members of which had a 16p11.2 deletion, using comparative genomic hybridization, quantitative polymerase-chain-reaction analysis, and DNA sequencing. We carried out tests of replication using an additional series of 76 Han Chinese persons with congenital scoliosis and a multicenter series of 42 persons with 16p11.2 deletions. RESULTS We identified a total of 17 heterozygous TBX6 null mutations in the 161 persons with sporadic congenital scoliosis (11%); we did not observe any null mutations in TBX6 in 166 controls (P<3.8×10(-6)). These null alleles include copy-number variants (12 instances of a 16p11.2 deletion affecting TBX6) and single-nucleotide variants (1 nonsense and 4 frame-shift mutations). However, the discordant intrafamilial phenotypes of 16p11.2 deletion carriers suggest that heterozygous TBX6 null mutation is insufficient to cause congenital scoliosis. We went on to identify a common TBX6 haplotype as the second risk allele in all 17 carriers of TBX6 null mutations (P<1.1×10(-6)). Replication studies involving additional persons with congenital scoliosis who carried a deletion affecting TBX6 confirmed this compound inheritance model. In vitro functional assays suggested that the risk haplotype is a hypomorphic allele. Hemivertebrae are characteristic of TBX6-associated congenital scoliosis. CONCLUSIONS Compound inheritance of a rare null mutation and a hypomorphic allele of TBX6 accounted for up to 11% of congenital scoliosis cases in the series that we analyzed. (Funded by the National Basic Research Program of China and others.).

Detection of cryptic pathogenic copy number variations and constitutional loss of heterozygosity using high resolution SNP microarray analysis in 117 patients referred for cytogenetic analysis and impact on clinical practice

Background: Microarray genome analysis is realising its promise for improving detection of genetic abnormalities in individuals with mental retardation and congenital abnormality. Copy number variations (CNVs) are now readily detectable using a variety of platforms and a major challenge is the distinction of pathogenic from ubiquitous, benign polymorphic CNVs. The aim of this study was to investigate replacement of time consuming, locus specific testing for specific microdeletion and microduplication syndromes with microarray analysis, which theoretically should detect all known syndromes with CNV aetiologies as well as new ones. Methods: Genome wide copy number analysis was performed on 117 patients using Affymetrix 250K microarrays. Results: 434 CNVs (195 losses and 239 gains) were found, including 18 pathogenic CNVs and 9 identified as “potentially pathogenic”. Almost all pathogenic CNVs were larger than 500 kb, significantly larger than the median size of all CNVs detected. Segmental regions of loss of heterozygosity larger than 5 Mb were found in 5 patients. Conclusions: Genome microarray analysis has improved diagnostic success in this group of patients. Several examples of recently discovered “new syndromes” were found suggesting they are more common than previously suspected and collectively are likely to be a major cause of mental retardation. The findings have several implications for clinical practice. The study revealed the potential to make genetic diagnoses that were not evident in the clinical presentation, with implications for pretest counselling and the consent process. The importance of contributing novel CNVs to high quality databases for genotype–phenotype analysis and review of guidelines for selection of individuals for microarray analysis is emphasised.

Prevalence of levator ani muscle injury in Chinese women after first delivery.

To assess the prevalence of levator ani muscle injury in Chinese women after their first delivery and investigate associated factors.

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.