Yinghua He

The DNA methylome of human peripheral blood mononuclear cells

Analysis across the genome of patterns of DNA methylation reveals a rich landscape of allele-specific epigenetic modification and consequent effects on allele-specific gene expression.

Single base-resolution methylome of the silkworm reveals a sparse epigenomic map

Epigenetic regulation in insects may have effects on diverse biological processes. Here we survey the methylome of a model insect, the silkworm Bombyx mori, at single-base resolution using Illumina high-throughput bisulfite sequencing (MethylC-Seq). We conservatively estimate that 0.11% of genomic cytosines are methylcytosines, all of which probably occur in CG dinucleotides. CG methylation is substantially enriched in gene bodies and is positively correlated with gene expression levels, suggesting it has a positive role in gene transcription. We find that transposable elements, promoters and ribosomal DNAs are hypomethylated, but in contrast, genomic loci matching small RNAs in gene bodies are densely methylated. This work contributes to our understanding of epigenetics in insects, and in contrast to previous studies of the highly methylated genomes of Arabidopsis and human, demonstrates a strategy for sequencing the epigenomes of organisms such as insects that have low levels of methylation.

DNA methylation mediated repression of miR-886-3p predicts poor outcome of human small cell lung cancer

Small cell lung cancer (SCLC) is one of the most aggressive types of cancer, yet the pathologic mechanisms underlying its devastating clinical outcome remain elusive. In this report, we surveyed 924 miRNA (miR) for their expressions in the formalin-fixed paraffin-embedded specimens from 42 patients with SCLC, and found that the downregulated miR-886-3p is closely correlated with the shorter survival of SCLC. This correlation was validated with another 40 cases. It was further discovered that loss of miR-886-3p expression was mediated by DNA hypermethylation of its promoter in both cultured SCLC cells and tumor samples. Moreover, miR-886-3p potently repressed cell proliferation, migration, and invasion of NCI-H446 cell in cell culture via suppression of the expression of its target genes: PLK1 and TGF-β1 at posttranscription levels. Forced upregulation of miR-886-3p greatly inhibited in vivo tumor growth, bone/muscle invasion, and lung metastasis of NCI-H446 cells. This newly identified miR-886-3p-PLK1/TGF-β1 nexus that modulates SCLC aggression suggests that both loss of miR-886-3p expression and hypermethylation of the miR-886 promoter are the promising indicators for poor outcome of as well as new therapeutic targets for SCLC.

Methylcap-Seq Reveals Novel DNA Methylation Markers for the Diagnosis and Recurrence Prediction of Bladder Cancer in a Chinese Population

Purpose There is a need to supplement or supplant the conventional diagnostic tools, namely, cystoscopy and B-type ultrasound, for bladder cancer (BC). We aimed to identify novel DNA methylation markers for BC through genome-wide profiling of BC cell lines and subsequent methylation-specific PCR (MSP) screening of clinical urine samples. Experimental Design The methyl-DNA binding domain (MBD) capture technique, methylCap/seq, was performed to screen for specific hypermethylated CpG islands in two BC cell lines (5637 and T24). The top one hundred hypermethylated targets were sequentially screened by MSP in urine samples to gradually narrow the target number and optimize the composition of the diagnostic panel. The diagnostic performance of the obtained panel was evaluated in different clinical scenarios. Results A total of 1,627 hypermethylated promoter targets in the BC cell lines was identified by Illumina sequencing. The top 104 hypermethylated targets were reduced to eight genes (VAX1, KCNV1, ECEL1, TMEM26, TAL1, PROX1, SLC6A20, and LMX1A) after the urine DNA screening in a small sample size of 8 normal control and 18 BC subjects. Validation in an independent sample of 212 BC patients enabled the optimization of five methylation targets, including VAX1, KCNV1, TAL1, PPOX1, and CFTR, which was obtained in our previous study, for BC diagnosis with a sensitivity and specificity of 88.68% and 87.25%, respectively. In addition, the methylation of VAX1 and LMX1A was found to be associated with BC recurrence. Conclusions We identified a promising diagnostic marker panel for early non-invasive detection and subsequent BC surveillance.

Loss of miR-638 in vitro promotes cell invasion and a mesenchymal-like transition by influencing SOX2 expression in colorectal carcinoma cells

BackgroundColorectal carcinoma (CRC) is a major cause of cancer mortality. The aberrant expression of several microRNAs is associated with CRC progression; however, the molecular mechanisms underlying this phenomenon are unclear.MethodsmiR-638 and SRY-box 2 (SOX2) expression levels were detected in 36 tumor samples and their adjacent, non-tumor tissues from patients with CRC, as well as in 4 CRC cell lines, using real-time quantitative RT-PCR (qRT-PCR). SOX2 expression levels were detected in 90 tumor samples and their adjacent tissue using immunohistochemistry. Luciferase reporter and Western blot assays were used to validate SOX2 as a target gene of miR-638. The regulation of SOX2 expression by miR-638 was assessed using qRT-PCR and Western blot assays, and the effects of exogenous miR-638 and SOX2 on cell invasion and migration were evaluated in vitro using the HCT-116 and SW1116 CRC cell lines.ResultsWe found that miR-638 expression was differentially impaired in CRC specimens and dependent on tumor grade. The inhibition of miR-638 by an antagomiR promoted cell invasion and a mesenchymal-like transition (lamellipodium stretching increased and cell-cell contacts decreased, which was accompanied by the suppression of the epithelial cell marker ZO-1/E-cadherin and the upregulation of the mesenchymal cell marker vimentin). A reporter assay revealed that miR-638 repressed the luciferase activity of a reporter gene coupled to the 3′-untranslated region of SOX2. miR-638 overexpression downregulated SOX2 expression, and miR-638 inhibition upregulated SOX2 expression. Moreover, miR-638 expression levels were correlated inversely with SOX2 mRNA levels in human CRC tissues. The RNAi-mediated knockdown of SOX2 phenocopied the invasion-inhibiting effect of miR-638; furthermore, SOX2 overexpression blocked the miR-638-induced CRC cell transition to epithelial-like cells.ConclusionsThese results demonstrate that the loss of miR-638 promotes invasion and a mesenchymal-like transition by directly targeting SOX2 in vitro. These findings define miR-638 as a new, invasion-associated tumor suppressor of CRC.

miR-193a-3p regulates the multi-drug resistance of bladder cancer by targeting the LOXL4 gene and the Oxidative Stress pathway

BackgroundChemoresistance is a major obstacle to the curative cancer chemotherapy and presents one of the most formidable challenges in both research and management of cancer.ResultsFrom the detailed studies of a multi-chemosensitive (5637) versus a chemoresistant (H-bc) bladder cancer cell lines, we showed that miR-193a-3p [GenBank: NR_029710.1] promotes the multi-chemoresistance of bladder cancer cells. We further demonstrated that lysyl oxidase-like 4 (LOXL4) gene [GenBank: NM_032211.6] is a direct target of miR-193a-3p and executes the former’s impact on bladder cancer chemoresistance. The Oxidative Stress pathway activity is drastically affected by a forced reversal of miR-193a-3p or LOXL4 levels in cell and may act at the downstream of LOXL4 gene to relay the miR-193a-3p’s impact on the multi-chemoresistance in both cultured cells and the tumor xenografts in nude mice.ConclusionsIn addition to a new mechanistic insight, our results provide a set of the essential genes in this newly identified miR-193a-3p/LOXL4/Oxidative Stress axis as the diagnostic targets for a guided anti-bladder cancer chemotherapy.

Abnormal methylation of seven genes and their associations with clinical characteristics in early stage non-small cell lung cancer

To identify novel abnormally methylated genes in early stage non-small cell lung cancer (NSCLC), we analyzed the methylation status of 13 genes (ALX1, BCL2, FOXL2, HPP1, MYF6, OC2, PDGFRA, PHOX2A, PITX2, RARB, SIX6, SMPD3 and SOX1) in cancer tissues from 101 cases of stage I NSCLC patients and lung tissues from 30 cases of non-cancerous lung disease controls, using methylation-specific PCR (MSP). The methylation frequencies (29.70–64.36%) of 7 genes (MYF6, SIX6, SOX1, RARB, BCL2, PHOX2A and FOLX2) in stage I NSCLC were significantly higher compared with those in non-cancerous lung disease controls (P<0.05). The co-methylation of SIX6 and SOX1, or the co-methyaltion of SIX6, RARB and SOX1 was associated with adenosquamous carcinoma (ADC), and the co-methylation of BCL2, RARB and SIX6 was associated with smoking. A panel of 4 genes (MYF6, SIX6, BCL2 and RARB) may offer a sensitivity of 93.07% and a specificity of 83.33% in the diagnosis of stage I NSCLC. Furthermore, we also detected the expression of 8 pathological markers (VEGF, HER-2, P53, P21, EGFR, CHGA, SYN and EMA) in cancer tissues of stage I NSCLC by immunohistochemistry, and found that high expression levels of p53 and CHGA were associated with the methylation of BCL2 (P=0.025) and PHOX2A (P=0.023), respectively. In this study, among the 7 genes which demonstrated hypermethylation in stage I NSCLC compared with non-cancerous lung diseases, 5 genes (MYF6, SIX6, PHOX2A, FOLX2 and SOX1) were found for the first time to be abonormally methylated in NSCLC. Further study of these genes shed light on the carcinogenesis of NSCLC.

MiR-193a-3p promotes the multi-chemoresistance of bladder cancer by targeting the HOXC9 gene

Chemoresistance prevents the curative cancer chemotherapy and presents a formidable challenge for both cancer researchers and clinicians. We have previously shown that miR-193a-3p promotes the multi-chemoresistance of bladder cancer cells via repressing its three target genes: SRSF2, PLAU and HIC2. Here, we showed that as a new direct target, the homeobox C9 (HOXC9) gene also executes the promoting effect of miR-193a-3p on the bladder cancer chemoresistance from a systematic study of multi-chemosensitive (5637) and resistant (H-bc) bladder cancer cell lines in both cell culture and tumor-xenograft/nude mice system. Paralleled with the changes in the drug-triggered cell death, the activities of both DNA damage response and oxidative stress pathways were drastically altered by a forced reversal of miR-193a-3p or HOXC9 levels in bladder cancer cells. In addition to a new mechanistic insight, our results provide a set of the essential genes in the miR-193a-3p/HOXC9/DNA damage response/oxidative stress pathway axis as the diagnostic targets for the guided anti-bladder cancer chemotherapy.

Genome-wide methylation profiling of the different stages of hepatitis B virus-related hepatocellular carcinoma development in plasma cell-free DNA reveals potential biomarkers for early detection and high-risk monitoring of hepatocellular carcinoma

BackgroundAn important model of hepatocellular carcinoma (HCC) that has been described in southeast Asia includes the transition from chronic hepatitis B infection (CHB) to liver cirrhosis (LC) and, finally, to HCC. The genome-wide methylation profiling of plasma cell-free DNA (cfDNA) has not previously been used to assess HCC development. Using MethylCap-seq, we analyzed the genome-wide cfDNA methylation profiles by separately pooling healthy control (HC), CHB, LC and HCC samples and independently validating the library data for the tissue DNA and cfDNA by MSP, qMSP and Multiplex-BSP-seq.ResultsThe dynamic features of cfDNA methylation coincided with the natural course of HCC development. Data mining revealed the presence of 240, 272 and 286 differentially methylated genes (DMGs) corresponding to the early, middle and late stages of HCC progression, respectively. The validation of the DNA and cfDNA results in independent tissues identified three DMGs, including ZNF300, SLC22A20 and SHISA7, with the potential for distinguishing between CHB and LC as well as between LC and HCC. The area under the curve (AUC) ranged from 0.65 to 0.80, and the odds ratio (OR) values ranged from 5.18 to 14.2.ConclusionsOur data revealed highly dynamic cfDNA methylation profiles in support of HBV-related HCC development. We have identified a panel of DMGs that are predictive for the early, middle and late stages of HCC development, and these are potential markers for the early detection of HCC as well as the screening of high-risk populations.

The miR-193a-3p regulated PSEN1 gene suppresses the multi-chemoresistance of bladder cancer

Chemoresistance prevents the curative cancer therapy, our understanding of which remains inadequate. Among the differentially expressed genes between the chemosensitive (5637) and chemoresistant (H-bc) bladder cancer cell lines, the expression level of the PSEN1 gene (presenilin 1), a key component of the γ-secretase, is negatively correlated with chemoresistance. A small interfering RNA mediated repression of the PSEN1 gene suppresses cell apoptosis and de-sensitizes 5637 cells, while overexpression of the presenilin 1 sensitizes H-bc cells to the drug-triggered cell death. As a direct target of microRNA-193a-3p that promotes the multi-chemoresistance of the bladder cancer cell, PSEN1 acts as an important executor for the microRNA-193a-3ps positive impact on the multi-chemoresistance of bladder cancer, probably via its activating effect on DNA damage response pathway. In addition to the mechanistic insights, the key players in this microRNA-193a-3p/PSEN1 axis are likely the diagnostic and/or therapeutic targets for an effective chemotherapy of bladder cancer.