Wenling Zheng

CHD5, a tumor suppressor that is epigenetically silenced in lung cancer

Chromodomain helicase DNA binding protein 5 (CHD5) is a potent tumor suppressor that serves as a master regulator of a tumor-suppressive network. Examination of the role played by CHD5 in a wide range of human cancers is warranted. In this study, we focused on the epigenetic modification and tumor-suppressive role of CHD5 in lung cancer. We measured CHD5 mRNA and protein expression in lung cancer cells, lung cancer tissues, and their corresponding noncancerous lung tissues using real-time PCR and Western blot analysis. We then determined the methylation status of the CHD5 promoter in these samples using methylation-specific sequencing and analyzed CHD5 re-expression in lung cancer cells treated with or without 5-aza-2-deoxycytidine, an inhibitor of DNA methylation. Next, the lung cancer cell clones stably expressing EGFP-CHD5 protein or EGFP protein, respectively, were obtained and the effects of restored CHD5 expression on cell proliferation, colony formation, and tumorigenicity were assessed. CHD5 expression ranged from low to absent in the lung cancer cell lines and tissues examined; the CHD5 promoter was hyperethylated in these samples. Treatment with 5-aza-dC resulted in a localized decrease in methylation density and an increase in CHD5 expression. Clonogenicity and tumor growth were abrogated in A549 and H1299 cells upon restoration of CHD5 expression. A significant reduction in clonogenicity was observed; an average of 47.83 ± 4.6% reduction for A549-EGFP-CHD5 was observed compared to A549-EGFP, and an average of 56.39 ± 5.3% reduction for H1299-EGFP-CHD5 was observed compared to H1299-EGFP. A549-EGFP exhibited an average tumor size of 452.3 ± 36.5 mm(3), whereas A549-EGFP-CHD5 exhibited an average tumor size of only 57.7 ± 18.5 mm(3). Thus, our findings indicate that CHD5 is a potential tumor suppressor gene that is inactivated via an epigenetic mechanism in lung cancer.

MicroRNA-32 promotes cell proliferation, migration and suppresses apoptosis in breast cancer cells by targeting FBXW7

BackgroundMicroRNAs are a class of small non-coding RNAs that are involved in many important physiological and pathological processes by regulating gene expression negatively. The purpose of this study was to investigate the effect of miR-32 on cell proliferation, migration and apoptosis and to determine the functional connection between miR-32 and FBXW7 in breast cancer.MethodsIn this study, quantitative RT-PCR was used to evaluate the expression levels of miR-32 in 27 breast cancer tissues, adjacent normal breast tissues and human breast cancer cell lines. The biological functions of miR-32 in MCF-7 breast cancer cells were determined by cell proliferation, apoptosis assays and wound-healing assays. In addition, the regulation of FBXW7 by miR-32 was assessed by qRT-PCR, Western blot and luciferase reporter assays.ResultsMiR-32 was frequently overexpressed in breast cancer tissue samples and cell lines as was demonstrated by qRT-PCR. Moreover, the up-regulation of miR-32 suppressed apoptosis and promoted proliferation and migration, whereas down-regulation of miR-32 showed an opposite effect. Dual-luciferase reporter assays showed that miR-32 binds to the 3′-untranslated region of FBXW7, suggesting that FBXW7 is a direct target of miR-32. Western blot analysis showed that over-expression of miR-32 reduced FBXW7 protein level. Furthermore, an inverse correlation was found between the expressions of miR-32 and FBXW7 mRNA levels in breast cancer tissues. Knockdown of FBXW7 promoted proliferation and motility and suppressed apoptosis in MCF-7 cells.ConclusionsTaken together, the present study suggests that miR-32 promotes proliferation and motility and suppresses apoptosis of breast cancer cells through targeting FBXW7.

Identification of genes related to carcinogenesis of oral leukoplakia by oligo cancer microarray analysis

The aim of this study was to identify genes that are predictive of carcinogenic change in patients of oral leukoplakia (OLK) using a cancer-related microarray. Candidate biomarkers were discovered using the Oligo GEArray OHS-802 and validated on independent samples by semi-quantitative reverse transcription-PCR (RT-PCR) and real-time RT-PCR. Both the discovery and the validation cohorts of samples included normal oral tissues, OLK tissues and oral squamous cell carcinoma (OSCC) tissues. Based on the microarray results, we found that there were nine genes successively up-regulated or down-regulated more than 2-fold between the normal group and OLK group and then again between the OLK group and OSCC group. The expression levels of the nine signature genes had statistically significant differences (p<0.05) between the OLK and normal groups and between the OSCC and OLK groups. In summary, the expression of the 9 signature genes might be representative of OLK carcinogenesis. A cancer-related microarray was used to identify a panel of candidate biomarkers for determining carcinogenesis of OLK lesions, in combination with semi-quantitative and real-time RT-PCR to validate the results. Our data indicated that alterations in gene expression that result in carcinogenesis can be identified in precancerous oral tissues.

Analysis of Physiological Characteristics of Abscisic Acid Sensitivity and Salt Resistance in Arabidopsis ANAC Mutants (ANAC019, ANAC072 and ANAC055)

ABSTRACT This study was carried out to investigate the sensitivity to abscisic acid (ABA) and the resistance to salt of homologous ANAC (Arabidopsis thaliana NAM/ATAF/CUC protein) mutants of Arabidopsis, namely anac019, anac072 and anac055. Three Arabidopsis mutants were identified by a genome and gene expression approach before carrying out any physiological experiments. Multiple sequence alignment of the amino acids of NAC genes showed that the ANAC proteins share the highest degree of homology with other proteins of the ATAF subfamily. The three physical signs of seed germination rate, cotyledon greening and root growth of anac019 were lower compared to the WT (wild type) under different concentrations of ABA. However, for anac055 these signs were higher, indicating insensitivity to ABA. In tests of root length and cotyledon greening, anac072 was sensitive to ABA, however the germination rates were reversed. In the survival rate and the chlorophyll content experiments, ANAC mutants showed lower resistance to salt stress than WT under high salt treatment. In conclusion, the three homologous genes of Arabidopsis (ANAC019, ANAC072 and ANAC055) may play synergistic or antagonistic roles in ABA signaling and ionic osmotic stress.

Identification and functional analysis of a core gene module associated with hepatitis C virus-induced human hepatocellular carcinoma progression

Hepatitis C virus (HCV)-induced human hepatocellular carcinoma (HCC) progression may be due to a complex multi-step processes. The developmental mechanism of these processes is worth investigating for the prevention, diagnosis and therapy of HCC. The aim of the present study was to investigate the molecular mechanism underlying the progression of HCV-induced hepatocarcinogenesis. First, the dynamic gene module, consisting of key genes associated with progression between the normal stage and HCC, was identified using the Weighted Gene Co-expression Network Analysis tool from R language. By defining those genes in the module as seeds, the change of co-expression in differentially expressed gene sets in two consecutive stages of pathological progression was examined. Finally, interaction pairs of HCV viral proteins and their directly targeted proteins in the identified module were extracted from the literature and a comprehensive interaction dataset from yeast two-hybrid experiments. By combining the interactions between HCV and their targets, and protein-protein interactions in the Search Tool for the Retrieval of Interacting Genes database (STRING), the HCV-key genes interaction network was constructed and visualized using Cytoscape software 3.2. As a result, a module containing 44 key genes was identified to be associated with HCC progression, due to the dynamic features and functions of those genes in the module. Several important differentially co-expressed gene pairs were identified between non-HCC and HCC stages. In the key genes, cyclin dependent kinase 1 (CDK1), NDC80, cyclin A2 (CCNA2) and rac GTPase activating protein 1 (RACGAP1) were shown to be targeted by the HCV nonstructural proteins NS5A, NS3 and NS5B, respectively. The four genes perform an intermediary role between the HCV viral proteins and the dysfunctional module in the HCV key genes interaction network. These findings provided valuable information for understanding the mechanism of HCV-induced HCC progression and for seeking drug targets for the therapy and prevention of HCC.

Differential expressed genes in ECV304 Endothelial-like Cells infected with Human Cytomegalovirus

BACKGROUND Human cytomegalovirus (HCMV) is a virus which has the potential to alter cellular gene expression through multiple mechanisms. OBJECTIVE With the application of DNA microarrays, we could monitor the effects of pathogens on host-cell gene expression programmes in great depth and on a broad scale. METHODS Changes in mRNA expression levels of human endothelial-like ECV304 cells following infection with human cytomegalovirus AD169 strain was analyzed by a microarray system comprising 21073 60-mer oligonucleotide probes which represent 18716 human genes or transcripts. RESULTS The results from cDNA microarray showed that there were 559 differential expressed genes consisted of 471 upregulated genes and 88 down-regulated genes. Real-time qPCR was performed to validate the expression of 6 selected genes (RPS24, MGC8721, SLC27A3, MST4, TRAF2 and LRRC28), and the results of which were consistent with those from the microarray. Among 237 biology processes, 39 biology processes were found to be related significantly to HCMV-infection. The signal transduction is the most significant biological process with the lowest p value (p=0.005) among all biological process which involved in response to HCMV infection. CONCLUSION Several of these gene products might play key roles in virus-induced pathogenesis. These findings may help to elucidate the pathogenic mechanisms of HCMV caused diseases. [This corrects the article on p. 243 in vol. 13, PMID: 24235919.].