| Background:Lung cancer is a molecularly-heterogeneous disease and the leading cause of cancer mortality. The molecular basis for this clinical heterogeneity remains incompletely understood. Over the past decade, research has primarily focused on the deregulation of protein-coding genes to identify oncogenes and tumor suppressors that could serve as diagnostic and therapeutic targets for lung cancer. Identification of oncogenic driver mutations have helped improve the outcomes in specific subtypes of patients with non-small cell lung cancer(NSCLC), however the majority of the patients with lung cancer do not have an actionable molecular aberration. Hence, there is an urgent need for reliable biomarkers and identification of alternative treatment options. Increasing appreciation of the role of long non-coding RNAs(lnc RNAs) in cancer progression has fostered efforts to characterize their role in disease biology and to evaluate them as novel biomarkers, as well as potential therapeutic targets.Genome-wide analyses indicate that Inc RNAs frequently demonstrate restricted tissue-specific and cancerspecific expression patterns, distinguishing them from most mi RNAs and protein-coding m RNAs. Moreover, lnc RNA expression may confer clinical information about patient outcomes and have utility in diagnostic tests. High-throughput RNA sequencing(RNA-Seq) in human cancer shows remarkable potential to identify both novel markers of disease and uncharacterized aspects of tumor biology, particularly lnc RNA species. We hypothesize that lnc RNAs are both drivers of lung cancer biology and biomarkers for diseasedetectable in patient tissues. The innovation of this study includes(1) Using next-generation sequencing(RNA-Seq) to identify lnc RNAs from a large cohort of lung cancer tissues;(2) Developing lnc RNAbased biomarkers in patient tissues for detection and prognosis of early lung cancer;(3) Defining the biological function/pathway and underlying mechanisms for important lnc RNA in lung cancer carcinogenesis.Methords:1. Identify lnc RNAs in lung cancer by RNA-Seq.2. Identify lnc RNAs differentially expressed in lung cancer.3. Identify lung cancer lnc RNAs associated with patient clinical outcomes.4. Validate diagnostic/prognostic lnc RNA signatures in an independent cohort of tumors by q RT-PCR.5. Kaplan-Meier with log-rank test and Cox proportional hazards model will be used for determining correlation to patient survival for linc00857.6. Performed q RT-PCR using RNA isolated from total, nuclear and cytoplasmicfractions to determine the cellular localization of linc00857.7. To determine the biologic function of linc00857 in regulating in tumor growth and invasion in vitro.8. To determine tumor formation potential of linc00857 in vivo by mouse in vivo xenograft tumor growth assay.9. Utilized flow cytometry analysis to examine the effect of linc00857 knockdown on the cell cycle.10.To investigate potential genes specifically regulated by linc00857, we performed whole transcriptome analysis using Affymetrix ST2.1 exon arrays.11.Validated several dysregulated genes using q RT-PCR and Western blot.Results:A. Differentially expressed lung lnc RNAs discovery and cross-validation.1. 21,560 transcripts were found in lung tissues by RNA-Seq including 16,017 protein-coding genes(74%), 1,726 pseudogenes(8%), and 3,136 lnc RNAs(15%). 2. A total of 281 lnc RNAs were discovered differently-expressed in UM, TCGA, Seo RNA-Seq datas. 3. A total of 127 survival-related lnc RNAswere discovered from RNA-seq data based on 67 ADs using both uni- and multi-variable Cox model with p < 0.05. A total of 54 of 127 lnc RNAs were found in Affymetrix U133 plus 2.0 array and 34 of 54 lnc RNAs were verified with p<0.01 by Cox model based on 226 early stage lung ADs(Okayma M. Cancer Res., 2011)4. Among the most overexpresssed lnc RNAs, linc00857 was found to be significantly increased in LUADs and was associated with worse survival (p<0.001) in our patient cohort(UM 67 LUADs).5. Validation linc00857 expression in an expanded UM cohort(101 lung ADs and 19 normal lung tissues) by an independent q RT-PCR assay. And found linc00857 expression levels were significantly higher in LUAD and higher expression of linc00857 was significantly related to worse patient survival.B. Functional assays to determine cancer-specific roles for linc00857 1. A significant decrease in cell proliferation in both H1299 and H838 cell lines and reduced cancer cell invasion(as measured by Boyden chamber matrigel invasion assays) was noted when linc00857 was knocked down. Similarly linc00857 abrogation resulted in a significant loss in cell migration and colony formation.2. Subcutaneous injections in nude mice with linc00857 stable knock down H1299 cells showed impaired tumor growth.C. Evaluation of linc00857-mediated cellular pathways and gene expression patterns.1. q RT-PCR of total RNA isolated from total, nuclear and cytoplasmic fraction of H1299 and H838 cell lines revealed linc00857 enrichment in the cytoplasmic fraction.2. Whole transcriptome analysis using Affymetrix ST2.1 exon arrays was performed to investigate potential genes specifically regulated by linc00857. 136 genes were down-regulated in H1299 and H838 cells, and 116 genes were up-regulated in both cell lines after linc00857 si RNA treated.3. Knockdown of linc00857 by si RNA did not affect gene expression levels of neighboring genes ANXA11 and MAT1 A.4. linc00857 positively correlated genes including CCNE1 and CDK2 in primary tumors were significantly involved in cell cycle regulation.5. The expression CCNE1 and CDK2 were decreased after linc00857 knockdown supporting the premise that linc00857 may affect the cell cycle at G1/S through regulation of CCNE1 and CDK2 genes.Conclusion:1. Identified 281 differently expressed lnc RNAs in LUAD and present our results from an in depth characterization of our top candidate linc00857. 2. Validated the differential expression in multiple independent datasets and established the prognostic importance of linc00857 expression. 3. Functional characterization indicated that linc00857 plays an important role in tumor proliferation, migration and invasion. 4. linc00857 regulated the cell cycle by causing G1/S arrest through regulating CCNE1 and CDK2 expression. Taken together, our study provides a comprehensive analysis of newly characterized linc00857 in lung cancer. It establishes the role of linc00857 as potential driver of lung cancer pathogenesis and a diagnostic or prognostic biomarker of disease. |
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