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Research Techniques Made Simple: Identification and Characterization of Long Noncoding RNA in Dermatological Research

      Long noncoding RNAs (lncRNAs) are a functionally heterogeneous and abundant class of RNAs acting in all cellular compartments that can form complexes with DNA, RNA, and proteins. Recent advances in high-throughput sequencing and techniques leading to the identification of DNA-RNA, RNA-RNA, and RNA-protein complexes have allowed the functional characterization of a small set of lncRNAs. However, characterization of the full repertoire of lncRNAs playing essential roles in a number of normal and dysfunctional cellular processes remains an important goal for future studies. Here we describe the most commonly used techniques to identify lncRNAs, and to characterize their biological functions. In addition, we provide examples of these techniques applied to cutaneous research in healthy skin, that is, epidermal differentiation, and in diseases such as cutaneous squamous cell carcinomas and psoriasis. As with protein-coding RNA transcripts, lncRNAs are differentially regulated in disease, and can serve as novel biomarkers for the diagnosis and prognosis of skin diseases.

      Abbreviations

      CME Activity Dates: February 22, 2017
      Expiration Date: February 22, 2018
      Estimated Time to Complete: 1 hour
      Planning Committee/Speaker Disclosure: All authors, planning committee members, CME committee members and staff involved with this activity as content validation reviewers have no financial relationship(s) with commercial interests to disclose relative to the content of this CME activity.
      Commercial Support Acknowledgment: This CME activity is supported by an educational grant from Lilly USA, LLC.
      Description: This article, designed for dermatologists, residents, fellows, and related healthcare providers, seeks to reduce the growing divide between dermatology clinical practice and the basic science/current research methodologies on which many diagnostic and therapeutic advances are built.
      Objectives: At the conclusion of this activity, learners should be better able to:
      • Recognize the newest techniques in biomedical research.
      • Describe how these techniques can be utilized and their limitations.
      • Describe the potential impact of these techniques.
      CME Accreditation and Credit Designation: This activity has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council for Continuing Medical Education through the joint providership of William Beaumont Hospital and the Society for Investigative Dermatology. William Beaumont Hospital is accredited by the ACCME to provide continuing medical education for physicians.
      William Beaumont Hospital designates this enduring material for a maximum of 1.0 AMA PRA Category 1 Credit(s)™. Physicians should claim only the credit commensurate with the extent of their participation in the activity.
      Method of Physician Participation in Learning Process: The content can be read from the Journal of Investigative Dermatology website: http://www.jidonline.org/current. Tests for CME credits may only be submitted online at https://beaumont.cloud-cme.com/RTMS-Mar17 – click ‘CME on Demand’ and locate the article to complete the test. Fax or other copies will not be accepted. To receive credits, learners must review the CME accreditation information; view the entire article, complete the post-test with a minimum performance level of 60%; and complete the online evaluation form in order to claim CME credit. The CME credit code for this activity is: 21310. For questions about CME credit email [email protected] .

      Introduction

      The sequencing and functional analysis of the human genome has demonstrated that while well-characterized protein-coding genes account for only 2% of the genome, approximately 80% of the genome is transcribed from one or both strands, resulting in a large number of RNAs with little or no protein coding potential. Long noncoding RNAs (lncRNAs) are defined as RNA transcripts equal to or longer than 200 nucleotides that do not encode for proteins (
      • Geisler S.
      • Coller J.
      RNA in unexpected places: long non-coding RNA functions in diverse cellular contexts.
      ). Regulation of their expression occurs by mechanisms similar to those observed for coding genes. Similar to coding genes, lncRNA genes are often spliced, although with fewer exons, capped at their 5′ end, and polyadenylated at their 3′ end, although some lncRNAs do not contain a poly(A) tail.
      LncRNAs have versatile functions due to their ability to pair with other nucleic acids and to form secondary and tertiary structures that can serve as scaffolds for multiple protein complexes. Several studies have revealed the functional relevance of lncRNAs in normal physiology, and their clinical implication in a number of malignancies as well as in other pathologies (
      • Leucci E.
      • Vendramin R.
      • Spinazzi M.
      • Laurette P.
      • Fiers M.
      • Wouters J.
      • et al.
      Melanoma addiction to the long non-coding RNA SAMMSON.
      ,
      • Schmitt A.M.
      • Chang H.Y.
      Long noncoding RNAs in cancer pathways.
      ,
      • Yan X.
      • Hu Z.
      • Feng Y.
      • Hu X.
      • Yuan J.
      • Zhao S.D.
      • et al.
      Comprehensive genomic characterization of long non-coding RNAs across human cancers.
      ).

      Identification and Validation of lncRNAs

      To generate a comprehensive and global expression profile of lncRNAs in a given cell type or tissue, the most accurate and high-resolution method is RNA sequencing (RNA-seq) that takes advantage of high-throughput next-generation sequencing technologies (Figure 1a). The experimental design is crucial to obtain quantitative data and the appropriate comparison of different samples, especially when considering healthy and patient tissues. The first step is to create a cDNA library from RNA samples by depleting the highly abundant ribosomal RNA (for details see
      • Whitley S.K.
      • Horne W.T.
      • Kolls J.K.
      Research techniques made simple: methodology and clinical applications of RNA sequencing.
      ). After sequencing, basic bioinformatics analysis, and data extraction, the sequenced reads can be mapped to the corresponding genome or transcriptome reference, using several databanks including reference sequence (RefSeq; https://www.ncbi.nlm.nih.gov/refseq/), GENCODE (https://www.gencodegenes.org), lncrbadb (http://lncrnadb.com), and NONCODEv4 (http://noncode.org). An advantage of RNA-seq as opposed to microarrays is the potential of discovering novel transcribed regions and alternatively spliced forms of known genes by transcriptome reconstruction.
      Figure 1
      Figure 1Schematic experimental procedure for the (a) identification, (b) validation, and (c) functional characterization of lncRNAs. RNA interactome analysis with high-throughput sequencing (RIA-Seq); protein microarray analysis (PMA); RNA binding protein (RBP) pull-down. FISH, fluorescence in situ hybridization; lncRNA, long noncoding RNA; qRT-PCR, quantitative reverse transcription polymerase chain reaction.
      Using RNA-seq to compare undifferentiated and differentiated human keratinocytes,
      • Kretz M.
      • Siprashvili Z.
      • Chu C.
      • Webster D.E.
      • Zehnder A.
      • Qu K.
      • et al.
      Control of somatic tissue differentiation by the long non-coding RNA TINCR.
      identified the first lncRNA (terminal differentiation-induced ncRNA [TINCR]) that controls human epidermal differentiation by a post-transcriptional mechanism. PolyA-selected RNA was used to generate cDNA, and high-throughput transcriptome sequencing was undertaken using the Illumina HiSeq platform. Differential expression analysis was performed using human RefSeq transcripts as a reference transcriptome.
      The largest expression dataset of lncRNAs in skin generated to date is from polyA+ RNA-derived cDNA from 216 samples of lesional, nonlesional psoriatic skin, and normal skin (
      • Tsoi L.C.
      • Iyer M.K.
      • Stuart P.E.
      • Swindell W.R.
      • Gudjonsson J.E.
      • Tejasvi T.
      • et al.
      Analysis of long non-coding RNAs highlights tissue-specific expression patterns and epigenetic profiles in normal and psoriatic skin.
      ). More recently,
      • Gupta R.
      • Ahn R.
      • Lai K.
      • Mullins E.
      • Debbaneh M.
      • Dimon M.
      • et al.
      Landscape of long noncoding RNAs in psoriatic and healthy skin.
      performed RNA-seq to compare the expression of lncRNAs in normal skin from healthy individuals and in lesional skin from patients with psoriasis before and after treatment with adalimumab, a humanized monoclonal antibody against tumor necrosis factor-alpha. cDNA was generated from ribosomal-depleted RNA from more than 15 individuals for each group, and sequences were obtained using the Illumina HiSeq. Interestingly, in this case, lncRNAs were mapped using a combined dataset derived from RefSeq, GENCODE, and a previously generated lncRNA catalog (
      • Hangauer M.J.
      • Vaughn I.W.
      • McManus M.T.
      Pervasive transcription of the human genome produces thousands of previously unidentified long intergenic noncoding RNAs.
      ). Data were validated by reanalyzing previously published RNA-seq data obtained from an independent set of psoriatic skin (
      • Li B.
      • Tsoi L.C.
      • Swindell W.R.
      • Gudjonsson J.E.
      • Tejasvi T.
      • Johnston A.
      • et al.
      Transcriptome analysis of psoriasis in a large case-control sample: RNA-seq provides insights into disease mechanisms.
      ) with the combined database. This approach validated the top-scoring lncRNA identified by Gupta et al., underlying the crucial importance of publicly available RNA-seq raw data that can be reanalyzed in subsequent experiments by other groups.
      Once specific lncRNAs of interest have been identified, a validation step is required (Figure 1b). Quantitative reverse transcription polymerase chain reaction approaches are useful to confirm lncRNA expression levels under different conditions. Although less quantitative and sensitive, Northern blot can provide reliable visual evidence for the abundance and length of the transcripts (
      • Kretz M.
      • Siprashvili Z.
      • Chu C.
      • Webster D.E.
      • Zehnder A.
      • Qu K.
      • et al.
      Control of somatic tissue differentiation by the long non-coding RNA TINCR.
      ). A useful technique to determine the subcellular localization of lncRNAs is single-molecule RNA fluorescence in situ hybridization (RNA FISH) analysis (
      • Kretz M.
      • Siprashvili Z.
      • Chu C.
      • Webster D.E.
      • Zehnder A.
      • Qu K.
      • et al.
      Control of somatic tissue differentiation by the long non-coding RNA TINCR.
      ,
      • Piipponen M.
      • Nissinen L.
      • Farshchian M.
      • Riihilä P.
      • Kivisaari A.
      • Kallajoki M.
      • et al.
      Long noncoding RNA PICSAR promotes growth of cutaneous squamous cell carcinoma by regulating ERK1/2 activity.
      ) (Figure 2c). Visualization of the subcellular localization of a given lncRNA by FISH technology can shed light on its putative functions. This fluorescent method led to the demonstration that the lncRNA TINCR, a crucial regulator of keratinocyte differentiation, is present at low levels both in the nucleus and in the cytoplasm of undifferentiated human keratinocytes, whereas its expression becomes abundant in the cytoplasm of differentiated human keratinocytes, indicating that it plays a cytoplasmic function as confirmed by its ability to bind to and stabilize differentiation mRNAs (
      • Kretz M.
      • Siprashvili Z.
      • Chu C.
      • Webster D.E.
      • Zehnder A.
      • Qu K.
      • et al.
      Control of somatic tissue differentiation by the long non-coding RNA TINCR.
      ).
      Figure 2
      Figure 2Expression of the lncRNA PICSAR is specifically upregulated in cSCC cells and its depletion suppresses growth of cSCC xenografts. (a) The heatmap of whole transcriptome analysis showing significantly (P < 0.05) regulated lncRNAs in primary (Prim; n = 5) and metastatic (Met; n = 3) cSCC cell lines and in NHEKs (n = 4). (b) Expression of PICSAR in cSCC (n = 6) and in normal skin (n = 7) was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). (c) Expression of PICSAR in cSCC and NHEK was determined by RNA FISH (in red). Scale bar = 10 μm. (d) PICSAR siRNA or negative control transfected cSCC cells were injected subcutaneously into the back of immunodeficient mice. Xenografts were harvested 18 days after inoculation and weighed. Mean ± SEM is shown; *P < 0.05. (e) Histology of the tumors was analyzed by hematoxylin and eosin (H&E) staining. The proliferation marker Ki-67 was detected in xenografts by immunohistochemistry. Hematoxylin was used as a counterstain. Scale bar = 100 μm. cSCC, cutaneous squamous cell carcinoma; FISH, fluorescence in situ hybridization; lncRNA, long noncoding RNA; NHEK, normal human keratinocyte; PICSAR, p38 inhibited cutaneous squamous cell carcinoma associated lincRNA; SEM, standard error of the mean; siRNA, small interfering RNA.
      Figure reproduced with permission from
      • Piipponen M.
      • Nissinen L.
      • Farshchian M.
      • Riihilä P.
      • Kivisaari A.
      • Kallajoki M.
      • et al.
      Long noncoding RNA PICSAR promotes growth of cutaneous squamous cell carcinoma by regulating ERK1/2 activity.
      .

      Functional Studies of lncRNAs

      LncRNAs have been shown to participate in many biological processes including the control of gene transcription, DNA replication, RNA splicing and stability, protein synthesis, and protein modification. LncRNAs’ function is not easily predictable by their primary structure, given their ability to fold into complicated secondary and tertiary structures that can depend on their interacting molecules, which may be part of different complexes. In the nucleus, lncRNAs can recruit proteins to chromatin sites through RNA-DNA base pairing, can function as scaffolds to create discrete protein complexes, or act as decoys to remove proteins from target DNA. These multifaceted functions are due to the biochemical versatility of RNA, which can directly pair by the base-base interaction with other nucleic acids, and fold into three-dimensional structures providing complex and dynamic recognition surfaces. Although so far molecular and functional studies have been performed only for a limited number of lncRNAs, several approaches can be undertaken, including isolation of other nucleic acids or proteins with which the lncRNA interacts (Figure 1c).
      If the lncRNA is chromatin bound and interacts with genomic DNA, RNA-DNA FISH can be used to simultaneously reveal the localization of a specific genomic region with the RNA of interest. In addition, various genomic methods are being developed to map the functional association of lncRNAs to distinct regions of the genome. Chromatin isolation by RNA purification followed by deep sequencing is based on pull-down assays, a method for selectively isolating macromolecules based on affinity purification. In this specific case, biotinylated oligonucleotides complementary to the lncRNA of interest are used as a “handle” to bring down associated chromatin to catalog the binding sites of novel RNA molecules in a genome (
      • Chu C.
      • Spitale R.C.
      • Chang H.Y.
      Technologies to probe functions and mechanisms of long noncoding RNAs.
      ).
      To discover RNA interacting with the lncRNA of interest, RNA interactome analysis followed by deep sequencing is also based on a pull-down assay. RNA interactome analysis followed by deep sequencing has been used to identify RNA binding to the lncRNA TINCR (
      • Kretz M.
      • Siprashvili Z.
      • Chu C.
      • Webster D.E.
      • Zehnder A.
      • Qu K.
      • et al.
      Control of somatic tissue differentiation by the long non-coding RNA TINCR.
      ), revealing that TINCR interacts with and stabilizes a number of differentiation-associated mRNAs through a 25-nucleotide “TINCR box” motif.
      Another important strategy that can shed light on the biological function of lncRNAs is the analysis of interacting protein partners. LncRNA pull-down assays allow purification of RNA-protein complexes after which proteins can be detected by mass spectrometry (
      • Feng Y.
      • Zhang L.
      Long non-coding RNAs: methods and protocols, vol. 1402.
      ). As an alternative approach,
      • Kretz M.
      • Siprashvili Z.
      • Chu C.
      • Webster D.E.
      • Zehnder A.
      • Qu K.
      • et al.
      Control of somatic tissue differentiation by the long non-coding RNA TINCR.
      have used TINCR RNA-labeled probes to hybridize commercially available human protein microarrays containing 9,400 spotted proteins. Data obtained from protein microarray analysis revealed that Staufen1, a RNA-binding protein, displayed the strongest TINCR RNA binding signal (
      • Kretz M.
      • Siprashvili Z.
      • Chu C.
      • Webster D.E.
      • Zehnder A.
      • Qu K.
      • et al.
      Control of somatic tissue differentiation by the long non-coding RNA TINCR.
      ). Impaired differentiation of epidermal tissue was observed in the absence of both TINCR and STAU1, suggesting that the TINCR-STAU1 complex mediates stabilization of differentiation mRNAs and is required for epidermal differentiation.
      In another recent study,
      • Piipponen M.
      • Nissinen L.
      • Farshchian M.
      • Riihilä P.
      • Kivisaari A.
      • Kallajoki M.
      • et al.
      Long noncoding RNA PICSAR promotes growth of cutaneous squamous cell carcinoma by regulating ERK1/2 activity.
      identified PICSAR (p38 inhibited cutaneous squamous cell carcinoma associated lincRNA, or LINC00162) as the highest expressed lncRNA in cutaneous squamous cell carcinoma (cSCC) cells compared with normal human keratinocytes (Figure 2a). Increased expression levels of PICSAR were also demonstrated in tissue derived from cSCCs, compared with normal skin (Figure 2b).
      To assess the biological function of aberrant expression of PICSAR, Piipponen et al. designed siRNA specifically to knock down PICSAR in cSCC cells. They determined that PICSAR knockdown caused a significant inhibition in tumor growth associated with reduced cell proliferation in xenograft models (Figure 2d and e) (
      • Piipponen M.
      • Nissinen L.
      • Farshchian M.
      • Riihilä P.
      • Kivisaari A.
      • Kallajoki M.
      • et al.
      Long noncoding RNA PICSAR promotes growth of cutaneous squamous cell carcinoma by regulating ERK1/2 activity.
      ). Accordingly, PICSAR-depleted cSCC cells exhibited impaired proliferation and migration, and decreased extracellular signal-regulated kinase 1/2 activity. To determine the molecular effects of PICSAR on cSCC cells, Piipponen et al. performed RNA-seq analysis in PICSAR-depleted cSCC cells compared with controls. Among the most upregulated genes after PICSAR depletion were the dual-specificity phosphatases DUSP1 and DUSP6 whose product dephosphorylate and inactivate mitogen-activated protein kinases (
      • Piipponen M.
      • Nissinen L.
      • Farshchian M.
      • Riihilä P.
      • Kivisaari A.
      • Kallajoki M.
      • et al.
      Long noncoding RNA PICSAR promotes growth of cutaneous squamous cell carcinoma by regulating ERK1/2 activity.
      ). Interestingly, in the presence of a DUSP6 inhibitor, PICSAR knockdown had no effect on extracellular signal-regulated kinase 1/2 activation, indicating that DUSP6 may be the link between PICSAR and the extracellular signal-regulated kinase 1/2 signaling pathway (
      • Piipponen M.
      • Nissinen L.
      • Farshchian M.
      • Riihilä P.
      • Kivisaari A.
      • Kallajoki M.
      • et al.
      Long noncoding RNA PICSAR promotes growth of cutaneous squamous cell carcinoma by regulating ERK1/2 activity.
      ). The exact molecular mechanism by which PICSAR regulates mRNA expression of several genes remains to be explored, and further studies will benefit from the identification of PICSAR-interacting molecules.
      In conclusion, in this overview, we discuss how to determine the relevance of lncRNAs in normal physiology and diseases of the skin, using multidisciplinary approaches including RNA-seq, RNA FISH, and RNA and protein interactome analysis. Future studies aimed at determining lncRNAs altered in skin diseases will help identifying novel potential biomarkers of these diseases as well as targets of therapeutic treatments.

      Summary

      Advantages:
      • RNA-seq coupled with advanced bioinformatics tools allow detection of even low abundance of known or previously unidentified lncRNAs, determining their primary structure and expression pattern.
      • Well-established techniques such as quantitative reverse transcription polymerase chain reaction, Northern blots, and RNA FISH can be applied to validate expression, length, and to identify the localization of lncRNAs.
      • Recent advances in genomics and proteomics can be applied to the study of lncRNAs by identifying in a high-throughput fashion lncRNA-associating DNA, RNA, and proteins.
      • Because lncRNAs can serve as crucial components of large complexes, their functional characterization can be instrumental for therapeutic intervention.
      Limitations:
      • lncRNAs have highly heterogeneous functions requiring ad hoc studies for each single lncRNA
      • lncRNAs form complicated secondary and tertiary structures that can depend on their interacting molecules; therefore the function is not easily predictable by their primary structure.
      • lncRNAs may be part of different complexes; therefore identification of protein, RNA, and DNA partners may not be entirely predictive of their functions.

      Multiple Choice Questions

      • 1.
        Identification of a novel lncRNA may be performed by:
        • A.
          mQuantitative reverse transcription polymerase chain reaction (qRT-PCR)
        • B.
          RNA fluorescence in situ hybridization (RNA FISH)
        • C.
          Chromatin isolation by RNA purification followed by deep sequencing (ChIRP-seq)
        • D.
          RNA sequencing (RNA-seq)
      • 2.
        Validation of novel identified lncRNAs may be determined by:
        • A.
          Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
        • B.
          RNA interactome analysis followed by deep sequencing (RIA-seq)
        • C.
          Chromatin isolation by RNA purification followed by deep sequencing (ChIRP-seq)
        • D.
          lncRNA interacting protein analysis
      • 3.
        RNA fluorescence in situ hybridization (RNA FISH) is capable of:
        • A.
          Identifying RNA-interacting proteins
        • B.
          Identifying the RNA interactome
        • C.
          Determining the subcellular localization of RNA
        • D.
          Identifying the functional role of RNA
      • 4.
        Identification of lncRNA-binding proteins can be achieved by:
        • A.
          RNA interactome analysis followed by deep sequencing (RIA-seq)
        • B.
          Chromatin isolation by RNA purification followed by deep sequencing (ChIRP-seq)
        • C.
          RNA pulldown followed by Mass Spectrometry
        • D.
          Specific knockdown of lncRNA
      • 5.
        RNA interactome analysis followed by deep sequencing (RIA-seq) is capable of:
        • A.
          Localizing the cellular compartment in which the lncRNA is expressed
        • B.
          Identifying the RNAs that interact with lncRNA
        • C.
          Identifying novel transcribed regions and alternative spliced forms of annotated genes
        • D.
          Identifying the RNA interacting proteins

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      This work was supported by grants from the Italian Association for Cancer Research (AIRC IG2015-17079) and from Fondazione Telethon (GEP15096).

      Supplementary Material

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