The rat lncRNA microarray analysis of the 8 samples from control and NNK treatment group
ABSTRACT: In order to assess the alteration in lncRNA expression in rat lung carcinogenesis induced by NNK, we determined the lncRNA expression profiles in 3 rat lung tumor samples and matched normal lung tissues and 2 blood samples from the control and NNK treatment group in the 95th week using Arraystar Rat lncRNA Microarray. We induced lung cancer using 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in a rat model and determined the lncRNA expression profiles in lung cancer tissues and rat blood samples.
Project description:Purpose: Chronic pulmonary inflammation in the form of chronic obstructive pulmonary disease (COPD) has been consistently shown to increase the risk of lung cancer. Therefore, identification of chemopreventive agents with anti-inflammatory effects, in addition to antiproliferative and apoptotic activities, is indispensable. Recently, we found that combinations of silibinin (Sil) and indole-3-carbinol (I3C) significantly inhibited lung tumorigenesis induced by 4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone (NNK) and enhanced by chronic treatment with the inflammatory agent lipopolysaccharide (LPS). In this study, we described gene expression profiling of lung tissues using RNA-seq to determine the gene expression signature in inflammation-driven lung tumors and modulation of this signature by the chemopreventive agents Sil and I3C. Methods: Total RNA extracted from lung tissues of control and treated mice were processed for mRNA sequencing, in triplicate, using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed for transcript abundance at the gene level using CLC Bio Genomics Workbench and differential gene expression analysis was performed using the built-in Empirical Analysis of Differential Gene Expression (DGE) based on 'Exact Test' method. qRT–PCR validation was performed using SYBR Green assays. Results and conclusions: We found that 330, 2,957, and 1,143 genes were differentially regulated in mice treated with NNK, LPS, and NNK + LPS, respectively. The expression of inflammation-and immunity-related genes was significantly more deregulated in lung tissues of mice treated with LPS alone compared to mice treated with NNK + LPS. Among 1,143 genes differentially regulated in the NNK + LPS group, the expression of 162 genes and associated signaling pathways were significantly modulated by I3C and/or Sil + I3C. These genes include cytokines, chemokines, and genes with a well-established role in inflammation and/or tumorigenesis such as c-ros oncogene 1 (Ros1), the EGFR ligands amphiregulin and epiregulin, Cyp1a1, and the circadian rhythm genes Arntl, and Npas2. To our knowledge, this is the first report that provides insight into genes that are differentially expressed during inflammation-driven lung tumorigenesis and the modulation of these genes by chemopreventive agents. Lung tissue mRNA profiles of mice treated with control vehicle, NNK, LPS, NNK+LPS, or NNK+LPS supplemented with chemopreventive agent(s) were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000.
Project description:This study evaluated transcriptional effects of the lung carcinogen NNK ( 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) injection and selenocystine consumption on the murine lung. Female A/J mice at 5 weeks of age were obtained from The Jackson Laboratory. Animals were stabilized on an unsupplemented AIN-76 diet for one week prior to being given the selenium supplemented diet. The basal level of selenium in the diet is 0.35 ppm Se, the selenocytine supplemention was at 15 ppm. NNK was administered i.p. as a single 10 uM injection in 0.2 mL saline. With this protocol, 100% of animals reproducibly develop lung tumors after 3 months. Three days after NNK administration, animals were provided AIN-76A diets supplemented with selenocystine at 15 ppm selenium ad libitum for 10 days. Animals were sacrificed thirteen days after NNK administration. Lung tissue was harvested, immediately homogenized in Trizol and frozen. The organic extracted RNAs were run over Qiagen RNeasy columes before quantifying and qualifying them. All samples had RNA quality index's greater than 9. Four groups of A/J mice were utilized with 4 biological replicates per group. 1) Untreated - controls on the AIN-76 diet (0.35 ppm Se). 2) NNK treated - single injection of NNK, maintained on norma AIN-76 diet, sacraficed after 13 days. 3) SECY - selenocystine supplemented (15 ppm) for 10 days on a AIN-76 diet then sacraficed. 4) NNK plus SECY - single injection of NNK, after 3 days, selenocystine supplemented (15 ppm) diet for 10 days then sacraficed. RNAs from the the four untreated mice were combined to phenotypically anchor the dual color expression profile. mouse lung responses to NNK injection and/or selenocystine dietary supplementation
Project description:The hepatocyte growth factor (HGF)/c-Met signaling pathway is known to mediate vascularization. We have previously demonstrated that expression of a human HGF transgene in the small airways produced mice (HGF TG) that were more susceptible to the tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). We also have observed that HGF TG mice display significantly enhanced vascularization in the lungs that increases over time compared to wild-type (WT) littermates. To analyze which genes might contribute to increased vascularization from HGF overexpression in the airways, RNA and protein were isolated from whole lungs of individual HGF TG and WT adult mice. We profiled the mRNA expression of several hundred genes representative of six biological pathways involved in transformation, angiogenesis, and tumorigenesis using two commercial microarrays. Significant changes in expression over a 1.5-fold boundary were also observed in lung tumors derived from NNK-treated HGF TG mice. Lung tumors were induced by exposing mice to four weekly i.p. injections of 3mg NNK (15μg/μl) over 2 weeks. Whole lungs from control untreated animals were dissected after sacrifice at 10, 20, or 40 wks of age, and NNK induced lung tumors were dissected from the animals at 20 or 40 weeks of age. Total RNA was extracted from whole lung or isolated tumors from HGF TG or WT mice using TRIzol reagent and the Array Grade Total RNA Isolation Kit. The cDNA was generated and labeled using the TrueLabeling-AMP Linear RNA Amplication Kit. RNA was analyzed from 28 mice in total. The angiogenesis array was used to analyze 10 samples taken from 40 week old mice (HGF TG untreated [n=4], WT untreated [n=4], HGF TG NNK treated [n=1], WT NNK treated [n=1]) and 6 samples from 20 week old mice (HGF TG untreated [n=2], WT untreated [n=2], HGF TG NNK treated [n=1], WT NNK treated [n=1]). The cancer gene array was used to analyze 8 samples taken from 40 week old mice (HGF TG untreated [n=2], WT untreated [n=2], HGF TG NNK treated [n=2], WT NNK treated [n=2]) and 4 samples from 20 week old mice (HGF TG untreated [n=2], WT untreated [n=2]).
Project description:There are significant differences in the lncRNA expression profiles in Chinese patients with pulmonary adenocarcinoma. LncRNAs such as AK124939 may be anti-cancer factors related to the progress of pulmonary adenocarcinoma. RNA extracted from three paired pulmonary adenocarcinoma tissue and adjacent normal lung tissue specimens was used to synthesize double-stranded complementary DNA (cDNA) after labeling and hybridization. The cDNA was labeled and hybridized to the lncRNA expression microarray, and array data were analyzed for hierarchical clustering. Gene coexpression networks were constructed to identify interactions among genes. To validate the microarray findings, we measured the relative expression levels of four random differentially expressed lncRNAs in the same tissue used for microarray using real-time quantitative polymerase chain reaction (qRT-PCR). The expression level of one lncRNA AK124939, in the paired pulmonary adenocarcinoma/adjacent normal lung tissue of another 30 patients was measured using qRT-PCR. The experimental data were further analyzed and compared with clinical features.
Project description:Long noncoding RNAs (lncRNAs) are known to regulate the development and progression of various cancers, however, few lncRNAs have been well characterized in lung adenocarcinoma (LUAD). Understanding the expression profile of lncRNAs and protein-coding genes is critical to develop new diagnosis and treatment strategies for LUAD and improving the prognosis of diagnosed patients. Five female LUAD patients with no smoking history were selected to profile lncRNA and protein-coding gene expression with microarrays. Paired tumor tissues and adjacent nontumor tissues were collected and confirmed by pathologists.
Project description:We describe the lncRNA expression profiles of two HCC cell lines, one with high potential for metastasis to the lung (HCCLM3) and the other to lymph nodes (HCCLYM-H2). The HCCLM3(LM3) and HCCLM6 cell lines were derived from the same parental cell line MHCC-97H. LM3 metastasizes to the lung, while HCCLM6 can metastasize to multiple organs in a mouse model. By subcloning HCCLM6, we established the HCCLYM-H2(H2) cell line, which showed stable and high metastatic potential specific to the lymph nodes. In the present study, we compared the expression profiles of HCC cell lines with a similar genetic background but different potential for lung or lymph node metastasis. We found that expression signatures comprising lncRNAs and protein-coding mRNAs were significantly associated with organ-specific HCC metastasis. To further validate microarray data, we selected six lncRNAs (CR613944, BC058547, RP5-1014O16.1, NCRNA00173, lincRNA-CALCA, lincRNA-TSPAN8) and two mRNAs (TSPAN8, CALCB) in the two HCC cell lines using qRT-PCR. Data obtained from qRT-PCR and the microarray was consistent. Differentially expressed lncRNAs between high lymphatic metastatic potential HCC cell H2 and high lung metastatic potiential HCC cell LM3 were identified by microarray and validated using quantitative real-time polymerase chain reaction.
Project description:Identification of genes associated with exposure to the carcinogen Nitrosamine (NNK) in mouse lungs of susceptible and resistant strains. Microarrays were used to capture all of the up and down regulated genes in two strains of mice. Lungs were excised and analyzed between 3-12 weeks after exposure to NNK before mature tumors had formed
Project description:The lncRNA expression profiles in three pairs of hTERT-positive gastric cancer tissue sand hTERT-negative para-cancerous tissues. The para-cancerous tissue is at least 5cm away from the cancer tissue. The expression of hTERT of identified by immunohistochemistry before RNA extraction for lncRNA assay. LncRNAs/mRNAs in 3 gastric cancer tissue and 3 paired para-cancerous tissue (Control) by microarray using Arraystar Human LncRNA Microarray v2.0
Project description:LncRNA and mRNA microarrays were performed to identify differentially expressed lncRNAs and mRNAs in fibroblast-like synoviocytes from rheumatoid arthritis patients compared with fibroblast-like synoviocytes from trauma patients. Fibroblast-like synoviocytes were isolated from synovial tissues. LncRNA and mRNA microarrays were performed using fibroblast-like synoviocytes at passage 3.