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author:("He, xinghua")
1.  miR-193a-3p regulates the multi-drug resistance of bladder cancer by targeting the LOXL4 gene and the Oxidative Stress pathway 
Molecular Cancer  2014;13(1):234.
Background
Chemoresistance is a major obstacle to the curative cancer chemotherapy and presents one of the most formidable challenges in both research and management of cancer.
Results
From 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.
Conclusions
In 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.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-4598-13-234) contains supplementary material, which is available to authorized users.
doi:10.1186/1476-4598-13-234
PMCID: PMC4200202  PMID: 25311867
LOXL4; miR-193a-3p; Chemoresistance; Bladder cancer; Oxidative stress pathway
2.  Loss of miR-638 in vitro promotes cell invasion and a mesenchymal-like transition by influencing SOX2 expression in colorectal carcinoma cells 
Molecular Cancer  2014;13:118.
Background
Colorectal 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.
Methods
miR-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.
Results
We 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.
Conclusions
These 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.
doi:10.1186/1476-4598-13-118
PMCID: PMC4039649  PMID: 24885288
miR-638; SOX2; CRC; Invasion
3.  Hypermethylation Leads to Bone Morphogenetic Protein 6 Downregulation in Hepatocellular Carcinoma 
PLoS ONE  2014;9(1):e87994.
Background
In the liver, bone morphogenetic protein 6 (BMP-6) maintains balanced iron metabolism. However, the mechanism that underlies greater BMP-6 expression in hepatocellular carcinoma (HCC) tissue than adjacent non-cancerous tissue is unclear. This study sought to investigate the epigenetic mechanisms of BMP-6 expression by analysing the relationship between the DNA methylation status of BMP-6 and the expression of BMP-6.
Methods
Methylation-specific polymerase chain reaction (PCR), bisulphite sequencing PCR, the MethyLight assay, and quantitative real-time PCR were performed to examine BMP-6 methylation and mRNA expression levels. Immunohistochemistry (IHC) was performed on tissue arrays to evaluate the BMP-6 protein level.
Results
BMP-6 mRNA expression was approximately 84.09% lower in HCC tissues than in adjacent non-cancerous tissues, and this low level of expression was associated with a poor prognosis. Moreover, the hypermethylation observed in HCC cell lines and HCC tissues was correlated with the BMP-6 mRNA expression level, and this correlation was validated following treatment with 5-aza-CdR, a demethylation agent. In addition, BMP-6 DNA methylation was upregulated by 68.42% in 114 clinical HCC tissue samples compared to adjacent normal tissues, whereas the BMP-6 staining intensity was downregulated by 77.03% in 75 clinical HCC tissue samples in comparison to adjacent normal tissues. Furthermore, elevated expression of BMP-6 in HCC cell lines inhibited cell colony formation.
Conclusions
Our results suggest that BMP-6 CpG island hypermethylation leads to decreased BMP-6 expression in HCC tissues.
doi:10.1371/journal.pone.0087994
PMCID: PMC3907571  PMID: 24498236
4.  Abnormal methylation of seven genes and their associations with clinical characteristics in early stage non-small cell lung cancer 
Oncology Letters  2013;5(4):1211-1218.
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.
doi:10.3892/ol.2013.1161
PMCID: PMC3629069  PMID: 23599765
DNA methylation; non-small cell lung cancer; stage I; smoking
5.  Methylcap-Seq Reveals Novel DNA Methylation Markers for the Diagnosis and Recurrence Prediction of Bladder Cancer in a Chinese Population 
PLoS ONE  2012;7(4):e35175.
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.
doi:10.1371/journal.pone.0035175
PMCID: PMC3328468  PMID: 22529986
6.  Methylation status of NEUROG2 and NID2 improves the diagnosis of stage I NSCLC 
Oncology Letters  2012;3(4):901-906.
In our previous study, we attempted to develop a tool for the early diagnosis of non-small cell lung cancer (NSCLC) using DNA methylation biomarkers. With the aim of improving the diagnostic potential by optimizing the composition of the target set, in this study, 13 candidate genes (ACTA1, AIDH1A2, CBX8, CDH8, EVX1, MGC16275, NEUROG1, NEUROG2, NID2, OTX2OS1, PGAM2, PHOX2B and TOX) were analyzed by methylation-specific PCR to determine the methylation status of each gene in 5 NSCLC cell lines and in lung tissue samples from 15 healthy volunteers, 103 stage I NSCLC patients and 26 non-cancerous control patients. Results showed that NEUROG2 and NID2 were hypermethylated in stage I NSCLC tissues (31.07 and 46.60%, respectively) and unmethylated in normal lung tissues (0/15) and non-cancerous tissues (0/26). Following recombination, an optimized 5-gene panel (NEUROG2, NID2, RASSF1A, APC and HOXC9) achieved a sensitivity of 91.26% with a specificity of 84.62% in the detection of stage I NSCLC. The optimized 5-gene panel greatly improved the diagnostic power for stage I NSCLC.
doi:10.3892/ol.2012.587
PMCID: PMC3362429  PMID: 22741015
NEUROG2; NID2; DNA methylation; non-small cell lung cancer; stage I
7.  Global Analysis of DNA Methylation by Methyl-Capture Sequencing Reveals Epigenetic Control of Cisplatin Resistance in Ovarian Cancer Cell 
PLoS ONE  2011;6(12):e29450.
Cisplatin resistance is one of the major reasons leading to the high death rate of ovarian cancer. Methyl-Capture sequencing (MethylCap-seq), which combines precipitation of methylated DNA by recombinant methyl-CpG binding domain of MBD2 protein with NGS, global and unbiased analysis of global DNA methylation patterns. We applied MethylCap-seq to analyze genome-wide DNA methylation profile of cisplatin sensitive ovarian cancer cell line A2780 and its isogenic derivative resistant line A2780CP. We obtained 21,763,035 raw reads for the drug resistant cell line A2780CP and 18,821,061reads for the sensitive cell line A2780. We identified 1224 hyper-methylated and 1216 hypomethylated DMRs (differentially methylated region) in A2780CP compared to A2780. Our MethylCap-seq data on this ovarian cancer cisplatin resistant model provided a good resource for the research community. We also found that A2780CP, compared to A2780, has lower observed to expected methylated CpG ratios, suggesting a lower global CpG methylation in A2780CP cells. Methylation specific PCR and bisulfite sequencing confirmed hypermethylation of PTK6, PRKCE and BCL2L1 in A2780 compared with A2780CP. Furthermore, treatment with the demethylation reagent 5-aza-dC in A2780 cells demethylated the promoters and restored the expression of PTK6, PRKCE and BCL2L1.
doi:10.1371/journal.pone.0029450
PMCID: PMC3245283  PMID: 22216282
8.  Hypomethylation of the hsa-miR-191 Locus Causes High Expression of hsa-miR-191 and Promotes the Epithelial-to-Mesenchymal Transition in Hepatocellular Carcinoma12 
Neoplasia (New York, N.Y.)  2011;13(9):841-853.
hsa-miR-191 is highly expressed in hepatocellular carcinoma (HCC), but the factors regulating this elevated expression are unknown. This study aimed to investigate the epigenetic mechanisms of increased hsa-miR-191 expression by analyzing the relationship between the DNA methylation status of hsa-miR-191 and miR-191 expression. Methylation-specific polymerase chain reaction (PCR), bisulfite sequencing PCR, Northern blot, and quantitative real-time PCR were performed to examine hsa-miR-191 methylation and expression levels. Western blot, transwell, and scratch assays were performed to examine the function and molecular mechanisms of hsa-miR-191. Approximately 58.9% of hsa-miR-191 expression was higher in HCC tissues than in adjacent noncancerous tissues; this high expression was associated with poor prognosis. The hypomethylation observed in some HCC cell lines and HCC tissues was correlated with the hsa-miR-191 expression level. This correlation was validated by treatment with the 5-aza-DAC demethylation agent. The level of hypomethylation was 63.0% in 73 clinical HCC tissue samples and was associated with increased (2.1-fold) hsa-miR-191 expression. The elevated expression of hsa-miR-191 in the SMMC-771 HCC cell line induced the cells to transition into mesenchymal-like cells; they exhibited characteristics such as loss of adhesion, down-regulation of epithelial cell markers, up-regulation of mesenchymal cell markers, and increased cell migration and invasion. Inhibiting hsa-miR-191 expression in the SMMC-7721 cell line reversed this process (as assessed by cell morphology and cell markers). Furthermore, hsa-miR-191 probably exerted its function by directly targeting TIMP metallopeptidase inhibitor 3 and inhibiting TIMP3 protein expression. Our results suggest that hsa-miR-191 locus hypomethylation causes an increase in hsa-miR-191 expression in HCC clinical tissues and that this expression induces HCC cells to transition into mesenchymal-like cells.
PMCID: PMC3182276  PMID: 21969817
9.  Is Shared Housing a Way to Reduce Homelessness? The Effect of Household Arrangements on Formerly Homeless People 
Journal of housing economics  2010;19(1):1-12.
Most single adults share housing with other adults, and living alone is considerably more expensive than living with someone else. Yet policies that discourage shared housing for formerly homeless people or people at risk of becoming homeless are common, and those that encourage it are rare. This would be understandable if such housing adversely affected its users in some way. We ask whether shared housing produces adverse effects. Our provisional answer is no. For the most part, whether a person lives alone or shares housing seems to make no difference to the outcomes we studied although shared housing is associated with reduced psychotic symptomology. We use data from ACCESS, a 5-year, 18-site demonstration project with over 6,000 formerly homeless individuals as participants.
doi:10.1016/j.jhe.2009.10.001
PMCID: PMC2862009  PMID: 20440383
homelessness; shared housing; mental health; psychotic symptomology
10.  The DNA Methylome of Human Peripheral Blood Mononuclear Cells 
PLoS Biology  2010;8(11):e1000533.
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.
DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome) analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold per strand), we report a comprehensive (92.62%) methylome and analysis of the unique sequences in human peripheral blood mononuclear cells (PBMC) from the same Asian individual whose genome was deciphered in the YH project. PBMC constitute an important source for clinical blood tests world-wide. We found that 68.4% of CpG sites and <0.2% of non-CpG sites were methylated, demonstrating that non-CpG cytosine methylation is minor in human PBMC. Analysis of the PBMC methylome revealed a rich epigenomic landscape for 20 distinct genomic features, including regulatory, protein-coding, non-coding, RNA-coding, and repeat sequences. Integration of our methylome data with the YH genome sequence enabled a first comprehensive assessment of allele-specific methylation (ASM) between the two haploid methylomes of any individual and allowed the identification of 599 haploid differentially methylated regions (hDMRs) covering 287 genes. Of these, 76 genes had hDMRs within 2 kb of their transcriptional start sites of which >80% displayed allele-specific expression (ASE). These data demonstrate that ASM is a recurrent phenomenon and is highly correlated with ASE in human PBMCs. Together with recently reported similar studies, our study provides a comprehensive resource for future epigenomic research and confirms new sequencing technology as a paradigm for large-scale epigenomics studies.
Author Summary
Epigenetic modifications such as addition of methyl groups to cytosine in DNA play a role in regulating gene expression. To better understand these processes, knowledge of the methylation status of all cytosine bases in the genome (the methylome) is required. DNA methylation can differ between the two gene copies (alleles) in each cell. Such allele-specific methylation (ASM) can be due to parental origin of the alleles (imprinting), X chromosome inactivation in females, and other as yet unknown mechanisms. This may significantly alter the expression profile arising from different allele combinations in different individuals. Using advanced sequencing technology, we have determined the methylome of human peripheral blood mononuclear cells (PBMC). Importantly, the PBMC were obtained from the same male Han Chinese individual whose complete genome had previously been determined. This allowed us, for the first time, to study genome-wide differences in ASM. Our analysis shows that ASM in PBMC is higher than can be accounted for by regions known to undergo parent-of-origin imprinting and frequently (>80%) correlates with allele-specific expression (ASE) of the corresponding gene. In addition, our data reveal a rich landscape of epigenomic variation for 20 genomic features, including regulatory, coding, and non-coding sequences, and provide a valuable resource for future studies. Our work further establishes whole-genome sequencing as an efficient method for methylome analysis.
doi:10.1371/journal.pbio.1000533
PMCID: PMC2976721  PMID: 21085693

Results 1-10 (10)