Expression profiles and initial confirmation of long non-coding RNAs in Chinese patients with pulmonary adenocarcinoma
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ABSTRACT: 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.
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:BACKGROUND:The purpose of this study was to investigate differentially expressed long noncoding RNAs (lncRNAs) in pulmonary adenocarcinoma tissue and adjacent noncancerous tissue from Chinese patients using lncRNA expression microarray and preliminary analysis. METHODS:RNA extracted from three paired pulmonary adenocarcinoma tissue and adjacent noncancerous tissue specimens was used to synthesize double-stranded complementary DNA after labeling and hybridization. The complementary DNA 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. The expression level of one lncRNA, AK124939, in the paired pulmonary adenocarcinoma/adjacent noncancerous tissue of another 30 patients was measured using real-time quantitative polymerase chain reaction. The experimental data were further analyzed and compared with clinical features. RESULTS:Of 39,000 lncRNAs investigated, 704 were differentially expressed in pulmonary adenocarcinoma tissue; 385 were upregulated and 319 were downregulated compared with those in the adjacent noncancerous tissue (fold change ?2 and ?-2, P<0.05). AK124939 expression levels in poorly differentiated adenocarcinoma tissue were lower than those found in well to moderately differentiated adenocarcinoma tissue (P=0.05). CONCLUSION:There are significant differences in the lncRNA expression profiles in Chinese patients with pulmonary adenocarcinoma. LncRNAs such as AK124939 may be anticancer factors related to the progression of pulmonary adenocarcinoma.
Project description:Study designed to obtain microRNA profile of tumorigenic Sca-1+CD34+ cells during the formation of pulmonary adenocarcinoma. After intranasal infection with adenovirus CRE (Ad-Cre), lung adenocarcinoma was identified pathologically in Lox-stop-lox Kras (LSL-Kras) G12D mice. Sca-1+CD34+ cells were sorted by flow cytometry and tested for the tumor-initiating ability to undergo self-renewal and differentiation. MiRNA profile was obtained from the microarray and further confirmed by qRT-PCR.The function of miRNAs was predicted bioinformatically, and miR-294 was verified to explore its relationship with tumor migration and invasion.
Project description:Study designed to obtain microRNA profile of tumorigenic Sca-1+CD34+ cells during the formation of pulmonary adenocarcinoma. After intranasal infection with adenovirus CRE (Ad-Cre), lung adenocarcinoma was identified pathologically in Lox-stop-lox Kras (LSL-Kras) G12D mice. Sca-1+CD34+ cells were sorted by flow cytometry and tested for the tumor-initiating ability to undergo self-renewal and differentiation. MiRNA profile was obtained from the microarray and further confirmed by qRT-PCR.The function of miRNAs was predicted bioinformatically, and miR-294 was verified to explore its relationship with tumor migration and invasion. We taken lung tissues from the LSL-Kras G12D mouse on the 0, 15th, 30th days, and defined as Group A, B, C respectively. These 3 samples were analyzed. Each kind of probe has 4 repeat spots on the microarray. Total RNA are harvested using TRIzol (Invitrogen) and RNeasy mini kit (QIAGEN). All processing conducted at the Kang Cheng Biological chip Company Limited. Microarray was synthesized using μParafloTM microfluidic chip technology, and the information of miRNA probe sequence of each region was obtained from the database of Sanger miRBase version 11.0.
Project description:Idiopathic pulmonary fibrosis (IPF) and lung cancer share common risk factors, epigenetic and genetic alterations, cellular and molecular aberrations, the activation of similar signaling pathways and poor survival. The aim of this study was to examine the gene expression profiles of stromal cells from patients with IPF and lung adenocarcinoma (ADC) as well as from normal lung.
Project description:Pulmonary enteric adenocarcinoma (PEAD) is a rare non-small cell lung cancer subtype. It is poorly characterized and cannot be distinguished from metastatic colorectal or upper gastrointestinal adenocarcinomas (ADCs) by means of routine pathological methods. As DNA methylation patterns are known to be highly tissue specific, we aimed to develop a methylation-based algorithm to differentiate these entities. To this end, genome wide methylation profiles of 600 primary pulmonary, colorectal and upper gastrointestinal ADCs obtained from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) database were used as a reference cohort to train a machine learning algorithm. The resulting classifier correctly classified all samples from a validation cohort consisting of 680 primary pulmonary, colorectal and upper gastrointestinal ADCs from TCGA and the GEO database, demonstrating the ability of the algorithm to reliably distinguish these three entities. We then analyzed DNA methylation data of 15 PEADs as well as four pulmonary metastases and four primary colorectal ADCs with the algorithm. All 15 PEADs were reliably classified as primary pulmonary tumors and all four metastases as well as all four primary colorectal ADC samples were identified as primary colorectal ADCs. In a t-distributed stochastic neighbor embedding analysis, the PEAD samples did not form a separate methylation subclass but rather diffusely intermixed with other pulmonary ADCs. Additional characterization of the PEAD series using fluorescence in-situ hybridization, next generation sequencing and copy number analysis revealed KRAS mutations in nine of 15 samples (60%) and a high number of structural chromosomal changes. Except for an unusually high rate of chromosome 20 gain (66.7%) the molecular data was mostly reminiscent of standard pulmonary ADCs. In conclusion, we provide sound evidence of the pulmonary origin of PEAD and in addition provide a publicly available machine learning based algorithm to reliably distinguish PEAD from metastatic colorectal cancer and upper gastrointestinal adenocarcinomas.
Project description:Small non-coding RNAs (sRNAs) play key roles in plant development, growth and responses to biotic and abiotic stresses. At least four classes of sRNAs have been well characterized in plants, including repeat-associated siRNAs (rasiRNAs), microRNAs (miRNAs), trans-acting siRNAs (tasiRNAs) and natural antisense transcript-derived siRNAs. Chinese fir (Cunninghamia lanceolata) is one of the most important coniferous evergreen tree species in China. No sRNA from Chinese fir has been described to date. To obtain sRNAs in Chinese fir, we sequenced a sRNA library generated from seeds, seedlings, leaves, stems and calli, using Illumina high throughput sequencing technology. A comprehensive set of sRNAs were acquired, including conserved and novel miRNAs, rasiRNAs and tasiRNAs. With BLASTN and MIREAP we identified a total of 115 conserved miRNAs comprising 40 miRNA families and one novel miRNA with precursor sequence. The expressions of 16 conserved and one novel miRNAs and one tasiRNA were detected by RT-PCR. Utilizing real time RT-PCR, we revealed that four conserved and one novel miRNAs displayed developmental stage-specific expression patterns in Chinese fir. In addition, 209 unigenes were predicted to be targets of 30 Chinese fir miRNA families, of which five target genes were experimentally verified by 5' RACE, including a squamosa promoter-binding protein gene, a pentatricopeptide (PPR) repeat-containing protein gene, a BolA-like family protein gene, AGO1 and a gene of unknown function. We also demonstrated that the DCL3-dependent rasiRNA biogenesis pathway, which had been considered absent in conifers, existed in Chinese fir. Furthermore, the miR390-TAS3-ARF regulatory pathway was elucidated. We unveiled a complex population of sRNAs in Chinese fir through high throughput sequencing. This provides an insight into the composition and function of sRNAs in Chinese fir and sheds new light on land plant sRNA evolution.