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author:("TANG, hengli")
1.  Oral Epithelium as a Surrogate Tissue for Assessing Smoking-Induced Molecular Alterations in Lungs 
Both the lungs and oral cavity are exposed to tobacco carcinogens in smokers. We hypothesized that the oral epithelium undergoes molecular alterations similar to those in lungs and therefore may be used as a surrogate tissue to assess tobacco-induced molecular alterations.
Promoter methylation of p16 and FHIT genes was analyzed with methylation-specific PCR in 1,774 oral and bronchial brush specimens (baseline and 3 months after intervention) from 127 smokers enrolled in a prospective randomized placebo-controlled chemoprevention trial. The association between methylation patterns in oral tissues and bronchial methylation indices (methylated sites/total sites per subject) was analyzed blindly.
At baseline, promoter methylation was observed in 23%, 17%, and 35% of the bronchial tissues for p16, FHIT, and either of the two genes, respectively, which were comparable to the 19%, 15%, and 31% observed in the oral tissues. Among the 125 individuals with available data from both oral and bronchial tissues, strong correlations were observed between tissues from the two sites (P<0.0001 for both p16 and FHIT). Among the 39 individuals with oral tissue methylation in either of the two genes, the mean bronchial methylation index was 0.53 (± 0.29) compared with only 0.27 (± 0.26) for the 86 subjects without oral tissue methylation (P<0.0001). Similar correlations were also observed in samples obtained at 3 months after chemopreventive intervention.
The oral epithelium may be used as a surrogate tissue to assess tobacco-induced molecular damage in lungs, which has an important implication in conducting biomarker-based lung cancer prevention trials.
PMCID: PMC4183362  PMID: 19138934
2.  Impact of smoking cessation on global gene expression in the bronchial epithelium of chronic smokers 
Cigarette smoke is the major cause of lung cancer and can interact in complex ways with drugs for lung cancer prevention or therapy. Molecular genetic research promises to elucidate the biologic mechanisms underlying divergent drug effects in smokers versus non-smokers and to help in developing new approaches for controlling lung cancer. The present study compared global gene expression profiles (determined via Affymetrix microarray measurements in bronchial epithelial cells) between chronic smokers, former smokers, and never smokers. Smoking effects on global gene expression were determined from a combined analysis of three independent datasets. Differential expression between current and never smokers occurred in 591 of the 13,902 genes measured on the microarrays (P < 0.01 and >2 fold change; pooled data)—a profound effect. In contrast, differential expression between current and former smokers occurred in only 145 of the measured genes (P < 0.01 and >2 fold change; pooled data). Nine of these 145 genes showed consistent and significant changes in each of the three datasets (P < 0.01 and >2 fold change), with 8 being down-regulated in former smokers. Seven of the 8 down-regulated genes, including CYP1B1 and 3 AKR genes, influence the metabolism of carcinogens and/or therapeutic/chemopreventive agents. Our data comparing former and current smokers allowed us to pinpoint the genes involved in smoking–drug interactions in lung cancer prevention and therapy. These findings have important implications for developing new targeted and dosing approaches for prevention and therapy in the lung and other sites, highlighting the importance of monitoring smoking status in patients receiving oncologic drug interventions.
PMCID: PMC4181408  PMID: 19138944
3.  Next Generation Sequencing Analysis of Circulating RNAs in Human Plasma 
Management of metastases, a major challenge in cancer therapy, requires constant monitoring of tumor burden in response to treatment. The importance of circulating tumor DNA (ctDNA), as biomarker for metastatic cancer, is increasingly evident as new studies have shown greater correlation of changes in tumor burden with ctDNA levels than with traditional markers like cancer antigen 15-3. The presence of tumor-associated RNA in plasma of cancer patients has also brought attention to the use of circulating RNA as markers. Recently, expression profiling of circulating RNAs have identified differentially expressed transcripts in cancer patients. These tumor-associated transcripts can potentially provide a non-invasive approach to identify new targets for cancer detection. To characterize the performance of RNA-seq methods with circulating RNAs, we evaluated the efficacy of three commercial library preparation protocols. Using two different plasma RNA samples, we constructed and sequenced five libraries and compared them to libraries from frozen tissue and FFPE samples. Our data show that over 40% genes in plasma samples, with 30X depth of mean coverage, were detected compared to 70% genes from frozen tissue sample with 44X depth of mean coverage. Although 64% genes were detected in FFPE sample, the mean coverage was only 10X. More than 50% of lncRNA detected in frozen tissue were also detected in plasma samples. We theorize that the lower percentage of detected genes in plasma sample is probably due to incomplete RNA spectrum in circulating RNAs. Among the library preparation kits we used for plasma RNAs, TruSeq Stranded Total RNA kit performed the best with up to 60% of reads uniquely mapping to mRNA exonic region. Our preliminary analysis suggests that current plasma RNA-seq methods are robust and the data generated from plasma RNA is comparable to frozen tissue.
PMCID: PMC4162285
4.  Visualizing the Transcriptome: A Comparison of Different RNA Library Preparation Methods 
RNA-seq is a powerful tool used to obtain in-depth information on expression profiling, gene annotation, and transcript discovery. With the growing popularity of RNA sequencing, new library preparation techniques are becoming commercially available. These techniques are improvements on the classic poly-A selection and rRNA reduction methods, and in some cases sensitive enough to analyze the transcriptome of a single cell. However, limited information is available on comparative analysis of these methods and their appropriate application for the transcriptome studies. We utilized Illumina's HiSeq technology to compare the merits of four commercial sample preparation kits: NuGen's Ovation RNA-seq system v2, Illumina's TruSeq RNA Sample Preparation kit v2, Epicentre's ScriptSeq RNA-seq kit v2 and Clontech's SMARTer Ultra Low RNA kit. We found that the quality of input RNA was critical for optimum performance of SMARTer Ultra Low RNA kit. Ovation and ScriptSeq kits, on the other hand, worked well with moderate quality input RNA as well. Based on analysis of the sequencing data, 12% of reads from ScriptSeq mapped to the mitochondrial genes as compared to 24% reads from Ovation. The library complexity and percentage of reads aligning to non-exonic region was similar between both kits. However, 28% reads aligned to the coding region for ScriptSeq versus 18% for Ovation. While TruSeq and SMARTer kits are designed for Poly-A containing RNAs only, ScriptSeq and Ovation kits provide more global analysis of the transcriptome. Analyzing the differences between these methods provides a better understanding of their specific advantage over the other. This information is especially useful for Sequencing Core Facilities, to recommend and apply appropriate methods to different transcriptome studies.
PMCID: PMC3635385
5.  Frequent expression of MAGE1 tumor antigens in bronchial epithelium of smokers without lung cancer 
Melanoma antigens (MAGE) are frequently expressed in lung cancer and are promising targets of anticancer immunotherapy. Our preliminary data suggested that MAGE may be expressed during early lung carcinogenesis, raising the possibility of targeting MAGE as a lung cancer prevention strategy. The purpose of this study was to investigate MAGE activation patterns in the airways of chronic smokers without lung cancer. MAGE-A1, -A3 and -B2 gene expression was determined in bronchial brush cells from chronic former smokers without lung cancer by reverse transcription-PCR (RT-PCR). The results were correlated with clinical parameters. The 123 subjects had a median age of 57 years, a median of 40 pack-years smoking history, and had quit smoking for at least one year prior to enrollment. Among the subjects, 31 (25%), 38 (31%), and 46 (37%) had detectable MAGE-A1, -A3 and -B2 expression, respectively, in their bronchial brush samples. Expression of MAGE-A1 and -B2 positively correlated with pack-years smoking history (P=0.03 and 0.03, respectively). The frequency of expression did not decrease despite a prolonged smoking cessation period. In conclusion, MAGE-A1, -A3 and -B2 genes are frequently expressed in the bronchial epithelial cells of chronic smokers without lung cancer, suggesting that chronic exposure to cigarette smoke activates these genes even before the malignant transformation of bronchial cells in susceptible individuals. Once activated, the expression persists despite long-term smoking cessation. These data support the targeting of MAGE as a novel lung cancer prevention strategy.
PMCID: PMC3440643  PMID: 22977481
melanoma antigens; airway; smokers; lung cancer; prevention

Results 1-5 (5)