Differences In Expression Of lncRNAs Research Articles

Abstract 1396: A novel approach of target enriched next generation sequencing reveals differences in expression of lncRNAs in stage II colorectal cancer formalin fixed paraffin embedded (FFPE) tumors

Abstract The aim of this study was to identify and quantify lncRNAs in formalin-fixed paraffin-embedded (FFPE) samples from stage II colorectal cancer (CRC) patients. Colorectal cancer (CRC) is the third most common cancer worldwide, and has high metastasis and recurrence rates. Use of adjuvant chemotherapy in stage II CRC patients is challenging, and new biomarkers are required to identify patients with high probability of relapse. Long noncoding RNAs (lncRNA) play an important role in cancer, awakening especial interest as potential novel cancer biomarkers and therapeutic targets. Resected tissues and biopsy preserved as formalin-fixed, paraffin-embedded (FFPE) samples are the most abundant supply for translational research. Unfortunately, long-chain RNA molecules, such as lncRNAs, may suffer degradation in those conditions. This fact coupled with their low expression levels challenges their detection with current techniques. Thus, in this study we developed a new approach which couples target enrichment and RNAseq to enhance detection of lncRNAs. Our three main goals are: I) evaluate the efficiency of this methodology on FFPE samples, II) investigate the lncRNAs involved in stage II CRC, and III) check for the presence of lncRNA differentially expressed in tumors of individuals who relapsed after surgery and not. We designed a custom-made target enrichment based on Nimblegen (Roche) technology to sequence a total of 8,048 lncRNAs (258 of interest for CRC). We tested 36 paired stage II CRC tumor FFPE tissues and their adjacent normal tissue, 12 from patients who relapsed after surgery. We quantified the expression of the targeted lncRNA and performed a differential expression analysis. Our approach detected expression of numerous lncRNA from FFPE samples. Importantly, we validated 5 target lncRNAs expression values by real time qPCR using two independent housekeeping genes, and found significant correlation between RNAseq (log2 (TPM)) to the qPCR (deltaCT) values. In addition, we identified a total of 83 lncRNAs which are differentially expressed between normal and tumoral samples. This list is enriched in genes previously reported as being involved in CRC, but includes many novel candidates of unknown function. Finally, the expression profile of tumors from patients with and without relapse seems quite similar, although we identified 5 lncRNAs that are significantly differentially expressed. Further validation in a larger cohort is needed to determine whether they could have prognostic value. In summary, our study shows that SeqCap Enrichment System is a good strategy to determine lncRNAs expression in FFPE tissue. Of note, we identified a list of new lncRNAs that are deregulated in stage II CRC tumor. Further research on these lncRNAs may lead to novel clinical applications in cancer biogenesis and prognosis. Citation Format: Anna Brunet-Vega, Cinta Pegueroles, Susana Iraola-Guzmán, Laia Vilà, Alex Casalots, Paula Ribera, Ismael Macias, Hrant Hovhannisyan, Ester Saus, Carles Pericay, Toni Gabaldón. A novel approach of target enriched next generation sequencing reveals differences in expression of lncRNAs in stage II colorectal cancer formalin fixed paraffin embedded (FFPE) tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1396.

Genome-wide identification and integrated analysis of lncRNAs in rice backcross introgression lines (BC2F12)

BackgroundDistant hybridization is an important way to create interspecific genetic variation and breed new varieties in rice. A lot of backcross introgression lines (BILs) had been constructed for the scientific issues in rice. However, studies on the critical regulatory factor lncRNA in cultivated rice, wild rice and their BIL progenies were poorly reported.ResultsHere, high-throughput RNA sequencing technology was used to explore the functional characteristics and differences of lncRNAs in O. sativa, O. longistaminata and their three BC2F12 progenies. A total of 1254 lncRNAs were screened out, and the number of differentially expressed lncRNAs between progenies and O. sativa were significantly less than that between progenies and O. longistaminata. Some lncRNAs regulated more than one mRNA, and 89.5% of lncRNAs regulated the expression of target genes through cis-acting. A total of 78 lncRNAs and 271 mRNAs were targeted by 280 miRNAs, and 22 lncRNAs were predicted to be the precursor of 20 microRNAs. Some miRNAs were found to target their own potential precursor lncRNAs. Over 50% of lncRNAs showed parental expression level dominance (ELD) in all three progenies, and most lncRNAs showed ELD-O. sativa rather than ELD-O. longistaminata. Further analysis showed that lncRNAs might regulate the expression of plant hormone-related genes and the adaptability of O. sativa, O. longistaminata and their progenies.ConclusionsTaken together, the above results provided valuable clues for elucidating the functional features and expression differences of lncRNAs between O. sativa, O. longistaminata and their BIL progenies, and expanded our understanding about the biological functions of lncRNAs in rice.

Screening and verification of key long chain noncoding RNA of gastric cancer with genetic risk

Objective To screen and verify the expression profiles of long non-coding RNAs (lncRNAs) in gastric cancer (GC) with genetic risk. Methods Microarray analysis was performed in 5 pairs GC tissues with Agilent Array platform to measure the expression levels of lncRNAs and mRNAs and to investigate the expression differences of lncRNAs in GC, and hierarchical clustering used to screen out the differently expressed lncRNAs. In order to verify the reliability of the chip, 4 abnormal expression of lncRNAs were randomly selected, and real time polymerase chain reaction (RT-PCR) was detected in 72 pairs of gastric cancer tissues and their matched normal tissues. Results Compared with the normal tissues, the expression of 2 times higher than those in the gastric cancer tissues was determined as the differential expression. It were found that 1 300 of them were up-regulated in lncRNAs and down-regulated in 1 807 lncRNAs. If this standard was 10 times, it was found that 55 of them were up-regulated and down-regulated in 372 lncRNAs. The results of 4 lncRNAs (H19, HOTAIRM1, HCG18 and LNC00261) showed that there was no significant difference between RT-PCR detection and chip detection (H19: t=1.332, P=0.314; HOTAIRM1: t=0.913, P=0.458; HCG18: t=1.517, P=0.269; LINC00261: t=0.176, P=0.876). Conclusion Some of the lncRNAs are abnormally expressed in tissues with a genetic risk of gastric cancer, which are likely to be the key risk factors for gastric cancer. Key words: Gastric Carcinoma; Heredity; Risk; Long non-coding RNAs; Microchip

Impact of high fat diet on long non-coding RNAs and messenger RNAs expression in the aortas of ApoE(-/-) mice.

Atherosclerosis is a chronic multifactorial inflammatory disease with high prevalence worldwide, and has become the leading cause of death. The present study was designed to investigate the impact of high-fat diet on ApoE(−/−) mice exhibiting atherosclerosis by detecting the genome-wide expression profile of lncRNAs and mRNAs. A total of 354 differentially expressed lncRNAs were identified (≥2.0 folds). Simultaneously, 357 differentially expressed mRNAs from the same chip were found. The expression differences of lncRNAs and mRNAs were consistent in both qPCR and microarray detection. Annotation results of the mRNAs which correlated with lncRNAs showed that the commonly related pathways were metabolism and inflammation. Hypergeometric distribution analysis indicated that the differentially expressed lncRNAs had been mostly regulated by transcription factors (TFs) such as Myod1, Rxra, Pparg, Tcf3, etc. Additional lncRNA-target-TFs network analysis was conducted for the top 20 differentially expressed lncRNAs. The results indicated Hnf4a, Ppara, Vdr, and Runx3 as the TFs most likely to regulate the production of these lncRNAs, and might play roles in inflammatory and metabolic processes in atherosclerosis. In a nutshell, the present study identified a panel of dysregulated lncRNAs and mRNAs that may be potential biomarkers or drug targets relevant to the high-fat diet related atherogenesis.

Identification of Long Noncoding RNA Associated with Osteoarthritis in Humans

Long noncoding RNAs (lncRNAs) are an important class of genes involved in various biological functions; however, knowledge about lncRNAs in osteoarthritis (OA) is limited. Therefore, the present study aimed to identify which lncRNAs are expressed in OA versus normal cartilage. To identify lncRNAs specifically expressed in OA cartilage, expression of lncRNAs in OA cartilage was compared with that in normal cartilage using microarray analysis. The identified differences in expression of lncRNAs were validated by real time polymerase chain reaction (RT-PCR). Furthermore, expression of several key mRNAs associated with OA, including those for matrix metalloproteinase (MMP)-9, MMP-13, bone morphogenetic protein (BMP)-2, COL2A1 and ADAMTS5, was investigated by RT-PCR in OA and normal cartilage. Microarray analysis identified 121 lncRNAs that were up- or down-regulated in OA compared with normal tissue, 73 being upregulated and 48 downregulated compared with normal cartilage. Twenty-one of the above differently expressed lncRNAs were up-regulated twofold. Expression of six lncRNAs, including HOTAIR, GAS5, PMS2L2, RP11-445H22.4, H19 and CTD-2574D22.4, was up-regulated in OA compared with normal tissue as validated by RT-PCR after microarray analysis. Expression of mRNA for MMP-9, MMP-13, BMP-2, and ADAMTS5 in OA was significantly greater than in normal cartilage. However, expression of mRNA for COL2A1 was lower in OA than in normal cartilage. The differently expressed lncRNAs may be associated with the pathogenesis of OA. Further functional studies are critical to confirming the function of lncRNAs in OA and to exploring new potential targets for therapy.

Expression of long non-coding RNA associated with radiotherapy-resistance in colorectal cancer cell lines with different radiosensitivity

To screen long non-coding RNA (lncRNA) associated with radiosensitivity in colorectal carcinoma cell lines. Colony formation assay was performed in colorectal cancer cell lines HT29, SW480, RKO, Lovo and HCT116 after irradiation with different radiation doses. Radiation sensitivity of these 5 cell lines was detected through survival fraction at 2 Gy (SF2 value). High-throughput lncRNA chip was used to screen lncRNA genes with expression differences more than 2 folds among SW480, RKO and Lovo. Further experiment on the expression differences of lncRNAs selected was conducted by realtime PCR. The radiosensitivity order of these 5 cell lines from low to high (SF2 value from high to low) was HT29 (0.83 ± 0.03), SW480 (0.69 ± 0.02), RKO(0.53 ± 0.02), Lovo (0.47 ± 0.05), HCT116 (0.32 ± 0.03) (P < 0.01). Five lncRNAs associated with radiation sensitivity were screened. Among them, expression levels of R05532, NR_015441, and NR_033374 were positively correlated with radiation resistance(all P < 0.01), and expression levels of the other 2 lncRNAs, NR_073156 and AA745020, were not correlated with radiation resistance of colorectal cancer cells (both P>0.05). lncRNA R05532, NR_015441 and NR_033374 may be used as the predictive marker of radiosensitivity of colorectal cancer cells. Higher expression of these genes shows radiation resistance.

Abstract 3268: Differences in expression of lncRNAs in breast tumor, adjacent normal-appearing breast tissue, and normal breast tissue from healthy donors

Abstract Long noncoding RNAs (lncRNAs) are an emerging class of key regulatory RNAs that are greater than 200nt and do not code for protein. They appear to be critical for various biological processes including gene regulation. Increasing evidence has suggested that aberrantly expressed lncRNAs play a key role in the initiation and progression of breast cancer. In most studies of breast cancer, breast tumor tissue was compared to adjacent normal-appearing tissue to identify differentially expressed lncRNAs. However, expression of lncRNAs in normal breast tissue from healthy donors has rarely been studied, and the differences in expression of lncRNAs in breast tumor and normal breast tissue from healthy donors remain largely unknown. Using RNA-sequencing data generated from freshly-frozen breast tissue samples, we examined the expression of lncRNAs in 119 breast tumor, 47 adjacent normal-appearing breast tissue samples, and 23 normal breast tissue samples from healthy donors. Bowtie and RESM algorithms were used to align raw reads and quantify transcript abundance. Differential expression analyses were performed using edgeR software. We assessed the expression of a total of 7,844 mapped lncRNAs using normalized transcript counts. Unsupervised Principal Components Analysis (PCA) demonstrated that the lncRNA expression profile of adjacent normal-appearing tissue was different compared to normal breast tissue from healthy donors, and partially overlapped with the tumor. At various fold change thresholds, more differentially expressed lncRNAs can be detected when the tumor tissue is compared to normal tissue than to adjacent non-tumorous tissue. With a false discovery rate (FDR) &amp;lt; 0.05 and a fold change (FC) of more than ±2, we identified 107 differentially expressed lncRNAs for the tumor vs. normal breast tissue, 53 lncRNAs for tumor vs. adjacent normal-appearing tissue, and 20 lncRNAs for adjacent normal-appearing vs. normal breast tissue. In our analyses of tumor vs. adjacent normal-appearing tissue and tumor vs. normal tissue from healthy donors, we identified differentially expressed lncRNAs including HOTAIR, GAS5 and ANRIL/CDKN2B-AS1 that were previously implicated in breast cancer, with considerably higher fold changes in latter comparison. When using normal breast tissue from healthy donors as the baseline, we identified novel putative breast cancer-associated lncRNAs including EMX2OS, TERC, HOTAIRM1, and WT1-AS that are aberrantly expressed in breast tumor. These lncRNAs were not identifiable in a direct comparison of tumor and adjacent normal-appearing tissue. Our results suggest that adjacent normal-appearing tissue undergoes changes in lncRNAs expression similar to those observed in tumor tissue to some extent, and a number of new and potentially important lncRNAs can only be detected using normal tissue from healthy donors as an optimal baseline tissue. Citation Format: Erin Wagner, Yunlong Liu, Bryan Schneider, Anna Maria Storniolo, Jiali Han, Chunyan He. Differences in expression of lncRNAs in breast tumor, adjacent normal-appearing breast tissue, and normal breast tissue from healthy donors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3268. doi:10.1158/1538-7445.AM2014-3268