Integration of machine learning to identify diagnostic genes in leukocytes for acute myocardial infarction patients.

BACKGROUND: Acute myocardial infarction (AMI) has two clinical characteristics: high missed diagnosis and dysfunction of leukocytes. Transcriptional RNA on leukocytes is closely related to the course evolution of AMI patients. We hypothesized that transcriptional RNA in leukocytes might provide potential diagnostic value for AMI. Integration machine learning (IML) was first used to explore AMI discrimination genes. The following clinical study was performed to validate the results. METHODS: A total of four AMI microarrays (derived from the Gene Expression Omnibus) were included in bioanalysis (220 sample size). Then, the clinical validation was finished with 20 AMI and 20 stable coronary artery disease patients (SCAD). At a ratio of 5:2, GSE59867 was included in the training set, while GSE60993, GSE62646, and GSE48060 were included in the testing set. IML was explicitly proposed in this research, which is composed of six machine learning algorithms, including support vector machine (SVM), neural network (NN), random forest (RF), gradient boosting machine (GBM), decision trees (DT), and least absolute shrinkage and selection operator (LASSO). IML had two functions in this research: filtered optimized variables and predicted the categorized value. Finally, The RNA of the recruited patients was analyzed to verify the results of IML. RESULTS: Thirty-nine differentially expressed genes (DEGs) were identified between controls and AMI individuals from the training sets. Among the thirty-nine DEGs, IML was used to process the predicted classification model and identify potential candidate genes with overall normalized weights > 1. Finally, two genes (AQP9 and SOCS3) show their diagnosis value with the area under the curve (AUC) > 0.9 in both the training and testing sets. The clinical study verified the significance of AQP9 and SOCS3. Notably, more stenotic coronary arteries or severe Killip classification indicated higher levels of these two genes, especially SOCS3. These two genes correlated with two immune cell types, monocytes and neutrophils. CONCLUSION: AQP9 and SOCS3 in leukocytes may be conducive to identifying AMI patients with SCAD patients. AQP9 and SOCS3 are closely associated with monocytes and neutrophils, which might contribute to advancing AMI diagnosis and shed light on novel genetic markers. Multiple clinical characteristics, multicenter, and large-sample relevant trials are still needed to confirm its clinical value.

Structure-Guided Strategies of Targeted Therapies for Patients with EGFR-Mutant Non-Small Cell Lung Cancer.

Oncogenic mutations within the EGFR kinase domain are well-established driver mutations in non-small cell lung cancer (NSCLC). Small-molecule tyrosine kinase inhibitors (TKIs) specifically targeting these mutations have improved treatment outcomes for patients with this subtype of NSCLC. The selectivity of these targeted agents is based on the location of the mutations within the exons of the EGFR gene, and grouping mutations based on structural similarities has proved a useful tool for conceptualizing the heterogeneity of TKI response. Structure-based analysis of EGFR mutations has influenced TKI development, and improved structural understanding will inform continued therapeutic development and further improve patient outcomes. In this review, we summarize recent progress on targeted therapy strategies for patients with EGFR-mutant NSCLC based on structure and function analysis.

Verbena Attenuates Adriamycin-Induced Renal Tubular Injury via Inhibition of ROS-ERK1/2-NLRP3 Signal Pathway.

Chronic kidney disease (CKD) has become a global public health problem. Tubular epithelial cell injury plays a vital role in the progression and prognosis of CKD. Therapies to protect tubular cells is the key to delaying CKD progression. Our study found that verbena, a natural traditional Chinese herb, has a potential reno-protective role in kidney diseases. However, the detailed mechanism remains unknown. In the current study, we employed adriamycin (ADR)-induced renal tubular cell injury to mimic the conditions of tubular injury in vitro. Results showed that total aqueous exact of verbena (TAEV) ameliorated ADR-induced cell disruption, loss of cellular viability, and apoptosis via inhibition of ROS-ERK1/2-mediated activation of NLRP3 signal pathway, suggesting that TAEV serves as a promising renoprotective agent in delaying the progression of CKD, while ROS-ERK1/2-mediated NLRP3 signal pathway might be a novel target in treating kidney diseases.

Allele-specific activation, enzyme kinetics, and inhibitor sensitivities of EGFR exon 19 deletion mutations in lung cancer.

Oncogenic mutations within the epidermal growth factor receptor (EGFR) are found in 15 to 30% of all non-small-cell lung carcinomas. The term exon 19 deletion (ex19del) is collectively used to refer to more than 20 distinct genomic alterations within exon 19 that comprise the most common EGFR mutation subtype in lung cancer. Despite this heterogeneity, clinical treatment decisions are made irrespective of which EGFR ex19del variant is present within the tumor, and there is a paucity of information regarding how individual ex19del variants influence protein structure and function. Herein, we identified allele-specific functional differences among ex19del variants attributable to recurring sequence and structure motifs. We built all-atom structural models of 60 ex19del variants identified in patients and combined molecular dynamics simulations with biochemical and biophysical experiments to analyze three ex19del mutations (E746_A750, E746_S752 > V, and L747_A750 > P). We demonstrate that sequence variation in ex19del alters oncogenic cell growth, dimerization propensity, enzyme kinetics, and tyrosine kinase inhibitor (TKI) sensitivity. We show that in contrast to E746_A750 and E746_S752 > V, the L747_A750 > P variant forms highly active ligand-independent dimers. Enzyme kinetic analysis and TKI inhibition experiments suggest that E746_S752 > V and L747_A750 > P display reduced TKI sensitivity due to decreased adenosine 5'-triphosphate Km. Through these analyses, we propose an expanded framework for interpreting ex19del variants and considerations for therapeutic intervention.

Erratum: Epigenomic characterization of a p53-regulated 3p22.2 tumor suppressor that inhibits STAT3 phosphorylation via protein docking and is frequently methylated in esophageal and other carcinomas: Erratum.

[This corrects the article DOI: 10.7150/thno.20893.].

Structure-function analysis of oncogenic EGFR Kinase Domain Duplication reveals insights into activation and a potential approach for therapeutic targeting.

Mechanistic understanding of oncogenic variants facilitates the development and optimization of treatment strategies. We recently identified in-frame, tandem duplication of EGFR exons 18 - 25, which causes EGFR Kinase Domain Duplication (EGFR-KDD). Here, we characterize the prevalence of ERBB family KDDs across multiple human cancers and evaluate the functional biochemistry of EGFR-KDD as it relates to pathogenesis and potential therapeutic intervention. We provide computational and experimental evidence that EGFR-KDD functions by forming asymmetric EGF-independent intra-molecular and EGF-dependent inter-molecular dimers. Time-resolved fluorescence microscopy and co-immunoprecipitation reveals EGFR-KDD can form ligand-dependent inter-molecular homo- and hetero-dimers/multimers. Furthermore, we show that inhibition of EGFR-KDD activity is maximally achieved by blocking both intra- and inter-molecular dimerization. Collectively, our findings define a previously unrecognized model of EGFR dimerization, providing important insights for the understanding of EGFR activation mechanisms and informing personalized treatment of patients with tumors harboring EGFR-KDD. Finally, we establish ERBB KDDs as recurrent oncogenic events in multiple cancers.

Tumor suppressive BTB/POZ zinc-finger protein ZBTB28 inhibits oncogenic BCL6/ZBTB27 signaling to maintain p53 transcription in multiple carcinogenesis.

Zinc-finger and BTB/POZ domain-containing family proteins (ZBTB) are important transcription factors functioning as tumor suppressors or oncogenes, such as BCL6/ZBTB27 as a key oncoprotein for anti-cancer therapy. Through epigenome study, we identified ZBTB28/BCL6B/BAZF, a BTB/POZ domain protein highly homologous to BCL6, as a methylated target in multiple tumors. However, the functions and mechanism of ZBTB28 in carcinogenesis remain unclear. Methods: ZBTB28 expression and methylation were examined by reverse-transcription PCR and methylation-specific PCR. The effects and mechanisms of ectopic ZBTB28 expression on tumor cells were assessed with molecular biological and cellular approaches in vitro and in vivo. Results: Albeit broadly expressed in multiple normal tissues, ZBTB28 is frequently downregulated in aero- and digestive carcinoma cell lines and primary tumors, and correlated with its promoter CpG methylation status. Further gain-of-function study showed that ZBTB28 functions as a tumor suppressor inhibiting carcinoma cell growth in vitro and in vivo, through inducing cell cycle arrest and apoptosis of tumor cells. ZBTB28 suppresses cell migration and invasion by reversing EMT and cell stemness. ZBTB28 transactivates TP53 expression, through binding to the p53 promoter in competition with BCL6, while BCL6 itself was also found to be a direct target repressed by ZBTB28. Conclusion: Our results demonstrate that ZBTB28 functions as a tumor suppressor through competing with BCL6 for targeting p53 regulation. This newly identified ZBTB28/BCL6/p53 regulatory axis provides further molecular insight into carcinogenesis mechanisms and has implications in further improving BCL6-based anticancer therapy.

On-target Resistance to the Mutant-Selective EGFR Inhibitor Osimertinib Can Develop in an Allele-Specific Manner Dependent on the Original EGFR-Activating Mutation.

PURPOSE: The third-generation EGFR inhibitor, osimertinib, is the first mutant-selective inhibitor that has received regulatory approval for the treatment of patients with EGFR-mutant lung cancer. Despite the development of highly selective third-generation inhibitors, acquired resistance remains a significant clinical challenge. Recently, we and others have identified a novel osimertinib resistance mutation, G724S, which was not predicted in in vitro screens. Here, we investigate how G724S confers resistance to osimertinib.Experimental Design: We combine structure-based predictive modeling of G724S in combination with the 2 most common EGFR-activating mutations, exon 19 deletion (Ex19Del) and L858R, with in vitro drug-response models and patient genomic profiling. RESULTS: Our simulations suggest that the G724S mutation selectively reduces osimertinib-binding affinity in the context of Ex19Del. Consistent with our simulations, cell lines transduced with Ex19Del/G724S demonstrate resistance to osimertinib, whereas cells transduced with L858R/G724S are sensitive to osimertinib. Subsequent clinical genomic profiling data further suggest G724S occurs with Ex19Del but not L858R. Furthermore, we demonstrate that Ex19Del/G724S retains sensitivity to afatinib, but not to erlotinib, suggesting a possible therapy for patients at the time of disease relapse. CONCLUSIONS: Altogether, these data suggest that G724S is an allele-specific resistance mutation emerging in the context of Ex19Del but not L858R. Our results fundamentally reframe the problem of targeted therapy resistance from one focused on the "drug-resistance mutation" pair to one focused on the "activating mutation-drug-resistance mutation" trio. This has broad implications across clinical oncology.

MiR-148b-3p inhibits renal carcinoma cell growth and pro-angiogenic phenotype of endothelial cell potentially by modulating FGF2.

MicroRNAs (miRNAs) have been implicated in a large number of biological processes such as tumor angiogenesis. MiR-148b-3p has been identified as a tumor suppressor in multiple cancer types and the function of miR-148b-3p in renal carcinoma remains unidentified. In this study, we found that the expression of miR-148b-3p was decreased in renal carcinoma based on GEO analysis and the gain-of-function experiments revealed that miR-148b-3p promoted renal carcinoma cell apoptosis and suppressed cell proliferation, migration in vitro and tumor growth in vivo. Functionally, the tube formation, invasion and migration capabilities of human umbilical vein endothelial cells (HUVECs) were suppressed by conditioned media derived from renal carcinoma 786-O cells that were transfected with miR-148b-3p mimics. Meanwhile, these conditioned media inhibited the proliferation and promoted apoptosis of HUVECs. The key angiogenesis inducer hypoxia inducible factor-1α (HIF-1α) and the pro-angiogenic mediators were decreased in 786-O cells that were transfected with miR-148b-3p mimics. Mechanistically, miR-148b-3p could target fibroblast growth factor-2 (FGF2) and further impaired the activation of fibroblast growth factor receptor 2 (FGFR2). Taken together, our findings demonstrate that miR-148b-3p attenuates renal carcinoma cell growth, the invasion and tube formation of endothelial cell potentially via regulating FGF2-FGFR2 signaling pathway.

Mechanisms of receptor tyrosine kinase activation in cancer.

Receptor tyrosine kinases (RTKs) play an important role in a variety of cellular processes including growth, motility, differentiation, and metabolism. As such, dysregulation of RTK signaling leads to an assortment of human diseases, most notably, cancers. Recent large-scale genomic studies have revealed the presence of various alterations in the genes encoding RTKs such as EGFR, HER2/ErbB2, and MET, amongst many others. Abnormal RTK activation in human cancers is mediated by four principal mechanisms: gain-of-function mutations, genomic amplification, chromosomal rearrangements, and / or autocrine activation. In this manuscript, we review the processes whereby RTKs are activated under normal physiological conditions and discuss several mechanisms whereby RTKs can be aberrantly activated in human cancers. Understanding of these mechanisms has important implications for selection of anti-cancer therapies.

Epigenomic characterization of a p53-regulated 3p22.2 tumor suppressor that inhibits STAT3 phosphorylation via protein docking and is frequently methylated in esophageal and other carcinomas.

Rationale: Oncogenic STAT3 signaling activation and 3p22-21.3 locus alteration are common in multiple tumors, especially carcinomas of the nasopharynx, esophagus and lung. Whether these two events are linked remains unclear. Our CpG methylome analysis identified a 3p22.2 gene, DLEC1, as a methylated target in esophageal squamous cell (ESCC), nasopharyngeal (NPC) and lung carcinomas. Thus, we further characterized its epigenetic abnormalities and functions. Methods: CpG methylomes were established by methylated DNA immunoprecipitation. Promoter methylation was analyzed by methylation-specific PCR and bisulfite genomic sequencing. DLEC1 expression and clinical significance were analyzed using TCGA database. DLEC1 functions were analyzed by transfections followed by various cell biology assays. Protein-protein interaction was assessed by docking, Western blot and immunoprecipitation analyses. Results: We defined the DLEC1 promoter within a CpG island and p53-regulated. DLEC1 was frequently downregulated in ESCC, lung and NPC cell lines and primary tumors, but was readily expressed in normal tissues and immortalized normal epithelial cells, with mutations rarely detected. DLEC1 methylation was frequently detected in ESCC tumors and correlated with lymph node metastasis, tumor recurrence and progression, with DLEC1 as the most frequently methylated among the established 3p22.2 tumor suppressors (RASSF1A, PLCD1 and ZMYND10/BLU). DLEC1 inhibits carcinoma cell growth through inducing cell cycle arrest and apoptosis, and also suppresses cell metastasis by reversing epithelial-mesenchymal transition (EMT) and cell stemness. Moreover, DLEC1 represses oncogenic signaling including JAK/STAT3, MAPK/ERK, Wnt/ß-catenin and AKT pathways in multiple carcinoma types. Particularly, DLEC1 inhibits IL-6-induced STAT3 phosphorylation in a dose-dependent manner. DLEC1 contains three YXXQ motifs and forms a protein complex with STAT3 via protein docking, which blocks STAT3-JAK2 interaction and STAT3 phosphorylation. IL-6 stimulation enhances the binding of DLEC1 with STAT3, which diminishes their interaction with JAK2 and further leads to decreased STAT3 phosphorylation. The YXXQ motifs of DLEC1 are crucial for its inhibition of STAT3 phosphorylation, and disruption of these motifs restores STAT3 phosphorylation through abolishing DLEC1 binding to STAT3. Conclusions: Our study demonstrates, for the first time, predominant epigenetic silencing of DLEC1 in ESCC, and a novel mechanistic link of epigenetic DLEC1 disruption with oncogenic STAT3 signaling in multiple carcinomas.

Genetic diagnosis of a Chinese multiple endocrine neoplasia type 2A family through whole genome sequencing.

Approximately 98% of patients with multiple endocrine neoplasia type 2A (MEN 2A) have an identifiable RET mutation. Prophylactic or early total thyroidectomy or pheochromocytoma/parathyroid removal in patients can be preventative or curative and has become standard management. The general strategy for RET screening on family members at risk is to sequence the most commonly affected exons and, if negative, to extend sequencing to additional exons. However, different families with MEN 2A due to the same RET mutation often have significant variability in the clinical exhibition of disease and aggressiveness of the MTC, which implies additional genetic loci exsit beyond RET coding region. Whole genome sequencing (WGS) greatly expands the breadth of screening from genes associated with a particular disease to the whole genome and, potentially, all the information that the genome contains about diseases or traits. This is presumably due to additive effect of disease modifying factors. In this study, we performed WGS on a typical Chinese MEN 2A proband and identified the pathogenic RET p.C634R mutation. We also identified several neutral variants within RET and pheochromocytoma-related genes. Moreover, we found several interesting structural variants including genetic deletions (RSPO1, OVCH2 and AP3S1, etc.) and fusion transcripts (FSIP1-BAZ2A, etc.).

Dickkopf-related protein 2 induces G0/G1 arrest and apoptosis through suppressing Wnt/ß-catenin signaling and is frequently methylated in breast cancer.

Dickkopf-related protein 2 (DKK2) is one of the antagonists of Wnt/ß-catenin signaling, with its downregulation reported in multiple cancers. However, how DKK2 contributes to breast tumorigenesis remains unclear. We examined its expression and promoter methylation in 10 breast tumor cell lines, 98 primary tumors, and 21 normal breast tissues. Compared with normal tissues, DKK2 was frequently silenced in breast cell lines (7/8). DKK2 promoter methylation was detected in 77.8% of cell lines and 86.7% of breast tumors; while rarely detected in normal breast tissues (19%), indicating common DKK2 methylation in breast cancer. Ectopic expression of DKK2 changed breast tumor cell morphology, inhibited cell proliferation and colony formation by inducing G0/G1 cell cycle arrest and apoptosis, and suppressed tumor cell migration by reversing epithelial-mesenchymal transition (EMT) and downregulating stem cell markers. Moreover, restored expression of DKK2 in MCF7 cells disrupted the microtube formation of human umbilical vein endothelial cells on Matrigel®. In vivo, the growth of MDA-MB-231 cells in nude mice was markedly decreased after stable expression of DKK2. DKK2 suppressed canonical Wnt/ß-catenin signaling by inhibiting ß-catenin activity with decreased active ß-catenin protein. Thus, our findings demonstrate that DKK2 functions as a tumor suppressor through inhibiting cell proliferation and inducing apoptosis via regulating Wnt signaling during breast tumorigenesis.

Epigenetic inactivation of the CpG demethylase TET1 as a DNA methylation feedback loop in human cancers.

Promoter CpG methylation is a fundamental regulatory process of gene expression. TET proteins are active CpG demethylases converting 5-methylcytosine to 5-hydroxymethylcytosine, with loss of 5 hmC as an epigenetic hallmark of cancers, indicating critical roles of TET proteins in epigenetic tumorigenesis. Through analysis of tumor methylomes, we discovered TET1 as a methylated target, and further confirmed its frequent downregulation/methylation in cell lines and primary tumors of multiple carcinomas and lymphomas, including nasopharyngeal, esophageal, gastric, colorectal, renal, breast and cervical carcinomas, as well as non-Hodgkin, Hodgkin and nasal natural killer/T-cell lymphomas, although all three TET family genes are ubiquitously expressed in normal tissues. Ectopic expression of TET1 catalytic domain suppressed colony formation and induced apoptosis of tumor cells of multiple tissue types, supporting its role as a broad bona fide tumor suppressor. Furthermore, TET1 catalytic domain possessed demethylase activity in cancer cells, being able to inhibit the CpG methylation of tumor suppressor gene (TSG) promoters and reactivate their expression, such as SLIT2, ZNF382 and HOXA9. As only infrequent mutations of TET1 have been reported, compared to TET2, epigenetic silencing therefore appears to be the dominant mechanism for TET1 inactivation in cancers, which also forms a feedback loop of CpG methylation during tumorigenesis.

The epigenetic modifier CHD5 functions as a novel tumor suppressor for renal cell carcinoma and is predominantly inactivated by promoter CpG methylation.

Renal cell carcinoma (RCC) is the most common urological cancer with steadily increasing incidence. A series of tumor suppressor genes (TSGs) have been identified methylated in RCC as potential epigenetic biomarkers. We identified a 1p36.3 TSG candidate CHD5 as a methylated target in RCC through epigenome study. As the role of CHD5 in RCC pathogenesis remains elusive, we further studied its expression and molecular functions in RCC cells. We found that CHD5 was broadly expressed in most normal genitourinary tissues including kidney, but frequently silenced or downregulated by promoter CpG methylation in 78% of RCC cell lines and 44% (24/55) of primary tumors. In addition, CHD5 mutations appear to be rare in RCC tumors through genome database mining. In methylated/silenced RCC cell lines, CHD5 expression could be restored with azacytidine demethylation treatment. Ectopic expression of CHD5 in RCC cells significantly inhibited their clonogenicity, migration and invasion. Moreover, we found that CHD5, as a chromatin remodeling factor, suppressed the expression of multiple targets including oncogenes (MYC, MDM2, STAT3, CCND1, YAP1), epigenetic master genes (Bmi-1, EZH2, JMJD2C), as well as epithelial-mesenchymal transition and stem cell markers (SNAI1, FN1, OCT4). Further chromatin immunoprecipitation (ChIP) assays confirmed the binding of CHD5 to target gene promoters. Thus, we demonstrate that CHD5 functions as a novel TSG for RCC, but is predominantly inactivated by promoter methylation in primary tumors.

A Small Indel Mutant Mouse Model of Epidermolytic Palmoplantar Keratoderma and Its Application to Mutant-specific shRNA Therapy.

Epidermolytic palmoplantar keratoderma (EPPK) is a relatively common autosomal-dominant skin disorder caused by mutations in the keratin 9 gene (KRT9), with few therapeutic options for the affected so far. Here, we report a knock-in transgenic mouse model that carried a small insertion-deletion (indel) mutant of Krt9, c.434delAinsGGCT (p.Tyr144delinsTrpLeu), corresponding to the human mutation KRT9/c.500delAinsGGCT (p.Tyr167delinsTrpLeu), which resulted in a human EPPK-like phenotype in the weight-stress areas of the fore- and hind-paws of both Krt9(+/mut) and Krt9(mut/mut) mice. The phenotype confirmed that EPPK is a dominant-negative condition, such that mice heterozygotic for the K9-mutant allele (Krt9(+/mut)) showed a clear EPPK-like phenotype. Then, we developed a mutant-specific short hairpin RNA (shRNA) therapy for EPPK mice. Mutant-specific shRNAs were systematically identified in vitro using a luciferase reporter gene assay and delivered into Krt9(+/mut) mice. shRNA-mediated knockdown of mutant protein resulted in almost normal morphology and functions of the skin, whereas the same shRNA had a negligible effect in wild-type K9 mice. Our results suggest that EPPK can be treated by gene therapy, and this has significant implications for future clinical application.

A novel CRX mutation by whole-exome sequencing in an autosomal dominant cone-rod dystrophy pedigree.

AIM: To identify the disease-causing gene mutation in a Chinese pedigree with autosomal dominant cone-rod dystrophy (adCORD). METHODS: A southern Chinese adCORD pedigree including 9 affected individuals was studied. Whole-exome sequencing (WES), coupling the Agilent whole-exome capture system to the Illumina HiSeq 2000 DNA sequencing platform was used to search the specific gene mutation in 3 affected family members and 1 unaffected member. After a suggested variant was found through the data analysis, the putative mutation was validated by Sanger DNA sequencing of samples from all available family members. RESULTS: The results of both WES and Sanger sequencing revealed a novel nonsense mutation c.C766T (p.Q256X) within exon 5 of CRX gene which was pathogenic for adCORD in this family. The mutation could affect photoreceptor-specific gene expression with a dominant-negative effect and resulted in loss of the OTX tail, thus the mutant protein occupies the CRX-binding site in target promoters without establishing an interaction and, consequently, may block transactivation. CONCLUSION: All modes of Mendelian inheritance in CORD have been observed, and genetic heterogeneity is a hallmark of CORD. Therefore, conventional genetic diagnosis of CORD would be time-consuming and labor-intensive. Our study indicated the robustness and cost-effectiveness of WES in the genetic diagnosis of CORD.

Characterization of the nasopharyngeal carcinoma methylome identifies aberrant disruption of key signaling pathways and methylated tumor suppressor genes.

AIMS: Nasopharyngeal carcinoma (NPC) is a common tumor consistently associated with Epstein-Barr virus infection and prevalent in South China, including Hong Kong, and southeast Asia. Current genomic sequencing studies found only rare mutations in NPC, indicating its critical epigenetic etiology, while no epigenome exists for NPC as yet. MATERIALS & METHODS: We profiled the methylomes of NPC cell lines and primary tumors, together with normal nasopharyngeal epithelial cells, using methylated DNA immunoprecipitation (MeDIP). RESULTS: We observed extensive, genome-wide methylation of cellular genes. Epigenetic disruption of Wnt, MAPK, TGF-ß and Hedgehog signaling pathways was detected. Methylation of Wnt signaling regulators (SFRP1, 2, 4 and 5, DACT2, DKK2 and DKK3) was frequently detected in tumor and nasal swab samples from NPC patients. Functional studies showed that these genes are bona fide tumor-suppressor genes for NPC. CONCLUSION: The NPC methylome shows a special high-degree CpG methylation epigenotype, similar to the Epstein-Barr virus-infected gastric cancer, indicating a critical epigenetic etiology for NPC pathogenesis.

Adenovirus-mediated NDRG2 inhibits the proliferation of human renal cell carcinoma cell line OS-RC-2 in vitro.

OBJECTIVE: To investigate the inhibitory effects of adenovirus-mediated NDRG2 on the proliferation of human renal cell carcinoma cell line OS-RC-2 in vitro. METHOD: NDRG2 was harvested by RT-PCR, confirmed by DNA sequencing, and then cloned into the eukaryotic expression vector pIRES2-EGFP, which encodes green fluorescent protein (GFP), to construct pIRES2-EGFP-NDRG2 plasmid. OS-RC-2 cells with NDRG2 negative expression were transfected with pIRES2-EGFP-NDRG2 plasmid. The growth of transfected OS-RC-2 cells was observed under light and fluorescence microscopes. After colony-forming cell assays, cell proliferation detection and MTT assays, the growth curves of cells in each group were plotted to investigate the inhibitory effects of adenovirus-mediated NDRG2 on the proliferation of OS-RC-2 cells. Cell cycle was determined by flow cytometry. Confocal laser scanning microscopy showed that NDRG2 protein was specifically located on subcellular organelle. RESULTS: A eukaryotic expression vector pIRES2-EGFP-NDRG2 was successfully constructed. After NDRG2 transfection, the growth of OS-RC-2 cells was inhibited. Flow cytometry showed that cells were arrested in S phase but the peak of cell apoptosis was not present, and confocal laser scanning microscopy showed that NDRG2 protein was located in mitochondrion. CONCLUSIONS: NDRG2 can significantly inhibit the proliferation of OS-RC-2 cells in vitro and its protein is specifically expressed in the mitochondrion.