Long Non-Coding RNAs in Cancer

Preface
Contents
Contributors
Part I: LncRNA Classification and Utility in Cancer
	Chapter 1: LncRNAs and Available Databases
		1 Introduction
		2 Classification of lncRNAs
			2.1 Classification Based on lncRNA Position Respect Protein Coding Genes
			2.2 Classification Based on lncRNA Genomic Location
			2.3 Classification Based on lncRNA Function
		3 LncRNA Databases
			3.1 Identification of Known and Novel lncRNAs
				3.1.1 RNAcentral (https://rnacentral.org/)
				3.1.2 LncBooK (http://bigd.big.ac.cn/lncbook)
				3.1.3 LNCipedia (https://lncipedia.org)
				3.1.4 FANTOMCAT (https://fantom.gsc.riken.jp/cat/v1/#/)
				3.1.5 NONCODE (http://www.noncode.org)
				3.1.6 LNCat (http://biocc.hrbmu.edu.cn/LNCat/)
			3.2 Prediction of Function Through Subcellular Distribution, Structure Analysis, Interactions, Coexpression
				3.2.1 LncATLAS (https://lncatlas.crg.eu/)
				3.2.2 lncRNome (http://genome.igib.res.in/lncRNome)
				3.2.3 LncTarD (http://biocc.hrbmu.edu.cn/LncTarD/)
				3.2.4 NPInter v4.0 (http://bigdata.ibp.ac.cn/npinter4)
				3.2.5 LncRInter (http://bioinfo.life.hust.edu.cn/lncRInter/)
				3.2.6 DIANA-LncBase v3.0 (www.microrna.gr/LncBase)
				3.2.7 LnCeDB (http://gyanxet-beta.com/lncedb)
			3.3 Prediction of lncRNA-Disease Association
				3.3.1 LncRNADisease (http://www.rnanut.net/lncrnadisease/)
				3.3.2 CCG (http://ccg.xingene.net)
				3.3.3 Lnc2Cancer (http://www.bio-bigdata.net/lnc2cancer)
				3.3.4 EVLncRNAs (http://biophy.dzu.edu.cn/EVLncRNAs)
				3.3.5 CLC (https://www.gold-lab.org/clc)
				3.3.6 LncRNASNP2 (http://bioinfo.life.hust.edu.cn/lncRNASNP2)
				3.3.7 LincSNP 3.0 (http://bioinfo.hrbmu.edu.cn/LincSNP)
			3.4 Databases Focused on Specific Features of lncRNAs
				3.4.1 Lnc2Meth (http://www.bio-bigdata.com/Lnc2Meth)
				3.4.2 RMBase v2.0 (http://mirlab.sysu.edu.cn/rmbase)
				3.4.3 exoRBase (http://www.exoRBase.org)
		4 Conclusions
		References
	Chapter 2: LncRNA as Cancer Biomarkers
		1 Introduction
		2 Guidelines for the Discovery of lncRNAs Behaving as Cancer Biomarkers
			2.1 Cohort Selection
			2.2 Sample Source
			2.3 Sample Composition: Tumor Cell Content and Microenvironment
			2.4 Integrating Patients Material with Cell Lines Samples
			2.5 Choice of the Technological Platform
			2.6 Data Analysis
			2.7 Databases Mining
			2.8 Validation of lncRNAs as Cancer Biomarkers
		3 Conclusions
		References
Part II: LncRNA Profiling and Discovery
	Chapter 3: Bioinformatics Pipeline to Analyze lncRNA Arrays
		1 Introduction
		2 Materials
		3 Methods
			3.1 lncRNA Annotation GeneChip Microarrays
			3.2 Probe Filter Strategy
			3.3 Flat File Creation
			3.4 CDF File Creation
		4 Notes
		References
	Chapter 4: Bioinformatic Pipelines to Analyze lncRNAs RNAseq Data
		1 Introduction
		2 Materials
			2.1 Hardware Specifications
		3 Methods for lncRNA Analysis
			3.1 Target Preparation
			3.2 Data Preprocessing
			3.3 Alignment Pipeline
				3.3.1 Kallisto
				3.3.2 STAR
			3.4 Quantification
			3.5 Downstream Analyses
		4 Notes
		References
	Chapter 5: Single-Cell RNAseq Analysis of lncRNAs
		1 Introduction
		2 Materials
			2.1 Analysis of Single Myofibers
			2.2 miRNA, lncRNA, and mRNA Sequencing Library Based on the SMART Amplification
			2.3 Single-Nucleus RNA Sequencing (snRNA-Seq)
		3 Methods
			3.1 Single-Myofiber RNA Sequencing (Single-Cell RNA-Seq)
				3.1.1 Myofiber Isolation
				3.1.2 Long RNA and Short RNA Separation Using RNeasy Kits (Qiagen)
				3.1.3 Long RNA Purification Retained in the Column
				3.1.4 miRNA Purification Using miRNeasy  Kit
				3.1.5 miRNA, lncRNA, and mRNA Sequencing Library Based on the SMART Amplification
					miRNA Polyadenylation
					Retrotranscription
					Amplification of Retrotranscribed  RNA
			3.2 Improvement with Single-Nucleus RNA Sequencing (snRNA-seq)
				3.2.1 Single Nucleus Preparation
				3.2.2 Single-Nucleus Library Preparation for RNAseq (Bio-Rad snRNAseq)
			3.3 Bioinformatic Analyses of Single-Cell or -Nucleus RNAseq
		4 Notes
		References
Part III: Validation of Identified LncRNAs
	Chapter 6: Measuring lncRNA Expression by Real-Time PCR
		1 Introduction
		2 Materials
			2.1 General Equipment and Facilities
			2.2 Primer Design
			2.3 RNA Isolation
			2.4 Reverse Transcription
			2.5 Real-Time PCR Setup, Cycling, and Data Analysis
		3 Methods
			3.1 Primer Design
			3.2 RNA Isolation from Human Cell Lines Using TRI Reagent
				3.2.1 Cell Lysis
				3.2.2 RNA Precipitation and Extraction from 1 ml of TRI-Reagent Lysate
				3.2.3 DNase Treatment, Reprecipitation, and Extraction (Optional, See Note 13)
				3.2.4 RNA Integrity Analysis
			3.3 Reverse Transcription
			3.4 Real-Time PCR Setup, Cycling, and Data Analysis
		4 Notes
		References
	Chapter 7: Quantitation of Long Noncoding RNA Using Digital PCR
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 cDNA Synthesis
			2.3 ddPCR Reaction Setup
			2.4 Droplet Generation and PCR Amplification
		3 Methods
			3.1 Sample Preparation
			3.2 ddPCR Reaction Setup for TaqMan
			3.3 ddPCR Reaction Setup for EvaGreen
			3.4 Droplet Generation
			3.5 PCR Amplification
			3.6 Droplet Reading
			3.7 Data Analysis
		4 Notes
		References
	Chapter 8: Detection of lncRNA by LNA-Based In Situ Hybridization in Paraffin-Embedded Cancer Cell Spheroids
		1 Introduction
		2 Materials
			2.1 Reagents and Buffers
			2.2 Equipment
		3 Methods
			3.1 HT29 Cell Spheroid Preparation and Embedding of Cells
			3.2 Tissue Sections
			3.3 In Situ Hybridization
		4 Notes
		References
Part IV: Functional Studies To Decipher the Role of Identified LncRNAs
	Chapter 9: In Vitro Silencing of lncRNA Expression Using siRNAs
		1 Introduction
		2 Materials
			2.1 Manual Annotation of the lncRNA of Interest
			2.2 Analysis of Cellular Localization in the Relevant Cell  Line
			2.3 Tissue Culture and Transfection of siRNAs
			2.4 Validation of lncRNA Silencing by Quantitative PCR and Single-Molecule In Situ Hybridization
		3 Methods
			3.1 Manual Annotation of the lncRNA of Interest
			3.2 Analysis of Cellular Localization in the Relevant Cell Line (Adapted from)
			3.3 Tissue Culture and Transfection of siRNAs
			3.4 Confirmation of lncRNA Knockdown by Quantitative RT-PCR and Single-Molecule In Situ Hybridization
		4 Notes
		References
	Chapter 10: In Vitro Silencing of lncRNAs Using LNA GapmeRs
		1 Introduction
		2 Materials
		3 Methods
			3.1 LNA GapmeR Electroporation for Suspension Cells Using the Neon Transfection System
			3.2 LNA GapmeR Gymnotic Delivery for Suspension Cells
		4 Notes
		References
	Chapter 11: Methods Used to Make Lipid Nanoparticles to Deliver LNA Gapmers Against lncRNAs into Acute Myeloid Leukemia (AML) ...
		1 Introduction
		2 Materials (See Note 1)
			2.1 For the Synthesis of Transferrin-PEG2000-DSPE
			2.2 For Preparing Cationic Liposomes
			2.3 For Preparing Transferrin-Conjugated LNA Lipid Nanoparticles (Tf-LNPs)
			2.4 Nanoparticle Characterization
				2.4.1 Gel Retardation by Agarose Gel Electrophoresis
				2.4.2 Dynamic Light Scattering (DLS) and Electrophoretic Light Scattering (ELS)
		3 Methods
			3.1 Preparation of Transferrin-PEG2000-DSPE
				3.1.1 Sulfhydryl Transferrin (Tf-SH) Formation
				3.1.2 Sulfhydryl Transferrin Purification
				3.1.3 Transferrin-PEG2000-DSPE (Tf-PEG2000-DSPE) Conjugation
			3.2 Preparation of Cationic Liposomes
			3.3 Transferrin-Conjugated LNA Lipid Nanoparticle Formation
			3.4 Transferrin-Conjugated LNA Lipid Nanoparticle Characterization
				3.4.1 LNA Oligonucleotide Encapsulation Assessment by Gel Retardation (See Notes 3 and 4)
				3.4.2 Particle Size and Zeta Potential Measurements by DLS and ELS
			3.5 Application Considerations
		4 Notes
		References
	Chapter 12: CRISPR/Cas9 to Silence Long Non-Coding RNAs
		1 Introduction
		2 Materials
			2.1 Electroporation Agent
			2.2 sgRNA
			2.3 Cas9
			2.4 Electroporation Apparatus
			2.5 Cell Culturing Reagent
			2.6 Flow Sorting to Establish Single Cell Colonies
			2.7 PCR Screening
		3 Methods
			3.1 Establish a Knockout Strategy
			3.2 Identification of Target Region for CRISPR/Cas9
			3.3 Design of PCR Primers for Validation of Clones
			3.4 Cultivation of Cells and Collection of Conditioned Media
			3.5 Transfection of sgRNAs and Cas9
			3.6 Seeding of Single-Cell Colonies
			3.7 Expansion of Single-Cell Colonies and Genotype Screening
		4 Notes
		References
	Chapter 13: CRISPR Interference (CRISPRi) and CRISPR Activation (CRISPRa) to Explore the Oncogenic lncRNA Network
		1 Introduction
		2 Materials
			2.1 dCAS9 and gRNA Vectors
			2.2 Virus Generation
			2.3 Testing CRISPRi/a in Multiple Myeloma (MM) Cells
			2.4 Equipment
		3 Methods
			3.1 Stable Expression of dCAS9 Fusion Proteins in MM Cell Lines
				3.1.1 Production of Lentiviral Particles
				3.1.2 Transduction of MM Cells
			3.2 gRNA Design and Cloning
				3.2.1 Design
				3.2.2 Cloning
			3.3 Modulation of lncRNA Expression in MM Cells
		4 Notes
		References
	Chapter 14: In Vivo Silencing/Overexpression of lncRNAs by CRISPR/Cas System
		1 Introduction
			1.1 LncRNAs
			1.2 CRISPR/Cas System
			1.3 Mouse Models
				1.3.1 Zygotic Microinjection
				1.3.2 mESC In Vitro Manipulation and Blastocyst Transplantation
				1.3.3 Embryos Electroporation
		2 In Vivo Studies of lncRNAs by CRISPR/Cas System
			2.1 In Vivo Loss-of-Function Studies of lncRNAs Using CRISPR/Cas-Mediated Genome Editing
				2.1.1 Impairment of lncRNA Transcription
				2.1.2 Elimination of lncRNA Sequence from the Genome (Knockout)
			2.2 In Vivo Knockin of lncRNAs by CRISPR/Cas System
			2.3 In Vivo Transcriptional Regulation of lncRNAs by CRISPRi and CRISPRa
		3 Conclusions and Future Perspectives
		References
	Chapter 15: Experimental Validation of the Noncoding Potential for lncRNAs
		1 Introduction
		2 Experimental Methods to Determine the Translation Status of lncRNAs
			2.1 Polysome Fractionation and Ribosome Profiling
			2.2 CRISPR/Cas9-Based Approaches
			2.3 Additional Methods to Access lncRNA Coding Potential
			2.4 Proteomics-Based Techniques to Study Translation
		3 Summary
		References
	Chapter 16: Identification of lncRNA-Protein Interactions by CLIP and RNA Pull-Down Assays
		1 Introduction
		2 Materials
			2.1 Cross-Linking and Immunoprecipitation (IP)
				2.1.1 Lysis Buffers
				2.1.2 Wash Buffers and Elution Buffer
			2.2 RNA Pull-Down
			2.3 Additional Reagents and Equipment
		3 Methods
			3.1 Cross-Linking and Immunoprecipitation
				3.1.1 Protein-RNA Cross-Linking
				3.1.2 Lysates Preparation
				3.1.3 Beads Preparation
				3.1.4 RNA Immunoprecipitation
				3.1.5 Immune Complex Washes
				3.1.6 Complex Elution and Decrosslink
				3.1.7 RNA Detection
			3.2 RNA Pull-Down Assay
				3.2.1 In Vitro Biotin-Labeled RNA Synthesis
				3.2.2 RNA and Protein Binding Reaction
				3.2.3 RNA Pull-Down
				3.2.4 Protein Detection
		4 Notes
		References
	Chapter 17: Empirical Validation of Overlapping Virus lncRNAs and Coding Transcripts by Northern Blot
		1 Introduction
		2 Materials
			2.1 RNA Extraction
			2.2 Formaldehyde Agarose Gel Electrophoresis
			2.3 Transfer, Blotting, Radiolabeling and Hybridization
		3 Methods
			3.1 RNA Extraction
			3.2 Formaldehyde Gel Electrophoresis
			3.3 Transfer to Membrane
			3.4 Preparation of the Radiolabeled RNA Probe and Hybridization
		4 Notes
		References
Part V: LncRNA Analysis in Liquid Biopsy
	Chapter 18: Phospho-RNAseq Profiling of Extracellular mRNAs and lncRNAs
		1 Introduction
		2 Materials
			2.1 Instruments
			2.2 Reagents for RNA 5′ Phosphorylation and Removal of 3′ Phosphoryl Groups
			2.3 Reagents for RNA Purification
			2.4 Reagents for Library Preparation
			2.5 Reagent for Gel Purification
			2.6 Reagents for Library Validation
		3 Methods
			3.1 Blood Specimen Processing for Plasma and RNA Isolation
			3.2 RNA 5′ Phosphorylation and Removal of 3′ Phosphoryl Groups
			3.3 Purification of PNK-Treated Samples
				3.3.1 Prepare RNA Binding Buffer
				3.3.2 Prepare Vacuum Manifold (See Note 4)
				3.3.3 Column Purification Using Vacuum Manifold
			3.4 Library Preparation (See Note 12)
				3.4.1 3′ Adapter Ligation
				3.4.2 5′ Adapter Ligation
				3.4.3 Reverse Transcription
				3.4.4 PCR Amplification
			3.5 Gel Purification (See Note 22)
		4 Notes
		References
	Chapter 19: Detection of Circulating RNA Using Nanopore Sequencing
		1 Introduction
		2 Materials
			2.1 RNA Extraction
			2.2 RNA Poly-A Tailing
			2.3 RNA Clean and Concentrator
			2.4 cDNA-PCR Sequencing
		3 Methods
			3.1 RNA Extraction from PAXgene Blood RNA Tubes
			3.2 RNA Poly-A Tailing
			3.3 RNA Clean and Concentrator
			3.4 cDNA-PCR Sequencing
				3.4.1 Library Preparation
				3.4.2 Selecting for Full-Length Transcripts
				3.4.3 Adapter Addition
				3.4.4 Priming and Loading SpotON Flow Cell
				3.4.5 Sequencing and Data Analysis
		4 Notes
		References
	Chapter 20: LncRNA Quantification from Extracellular Vesicles Isolated from Blood Plasma or Conditioned Media
		1 Introduction
		2 Materials
			2.1 Blood Plasma Collection
			2.2 Cell Lines, Cell Culture, and Conditioned Medium Collection
			2.3 Preprocessing and Ultracentrifugation
			2.4 RNA Isolation
			2.5 LncRNA Quantification Using Hydrolysis Probes
		3 Methods
			3.1 Collection of Blood Plasma and Preprocessing
			3.2 Collection of Conditioned Media and Preprocessing
			3.3 Ultracentrifugation
			3.4 RNA Isolation
			3.5 Expected Recovery Yield of EVs and EVs-Derived RNA
			3.6 EVs-Derived lncRNA Quantification Using Hydrolysis Probes
				3.6.1 Reverse Transcription
				3.6.2 Hydrolysis Probes Design
				3.6.3 Real-Time PCR Assay
				3.6.4 Housekeeping Genes for EV lncRNA Expression Assessment
			3.7 Comparing lncRNA Expression Between EVs and Secreting Cells
			3.8 Performing an RTqPCR Standard Curve for Efficiency Calculation
		4 Notes
		References
Part VI: Circular RNAs
	Chapter 21: The Use of circRNAs as Biomarkers of Cancer
		1 Introduction
		2 CircRNA Classification and Biogenesis
		3 Biological Function/Mechanisms of Action
			3.1 miRNA sponges
			3.2 Effect on alternative splicing
			3.3 Effect on Protein Translation/mRNA Tramps
			3.4 circRNAs as regulators of transcription
			3.5 Interaction with proteins
			3.6 Protein Production
		4 circRNAs as cancer biomarkers
			4.1 Commonly Dysregulated Cancer-Associated circRNAs
		5 Discovering and analyzing circRNAs
			5.1 RNA-Seq
			5.2 Microarrays
			5.3 qRT-PCR
			5.4 Northern blot
			5.5 FISH
			5.6 Assessing the value of circRNAs as cancer biomarkers
		6 Summary and Future Perspectives
		References
	Chapter 22: Bioinformatic Analysis of Circular RNA Expression
		1 Introduction
		2 Materials
			2.1 Hardware Requirements
			2.2 CircRNA Detection
				2.2.1 Software Requirements and Installation
				2.2.2 Input  Data
			2.3 CircRNA Expression Analysis
				2.3.1 Software Requirements and Installation
				2.3.2 Input  Data
			2.4 CircRNA Characterization
				2.4.1 Software Requirements and Installation
				2.4.2 Input  Data
		3 Methods
			3.1 Input Preprocessing and circRNA Detection
			3.2 CircRNA Expression Analysis
			3.3 Circular to Linear Transcript Differential Proportion Analysis
			3.4 CircRNA Characterization
			3.5 CircRNA Function Databases
			3.6 CircRNA Custom Functional Predictions
				3.6.1 CircRNA Sequence Reconstruction
				3.6.2 Functional Predictions
		4 Notes
		References
	Chapter 23: Study of Circular RNA Expression by Nonradioactive Northern Blot Procedure
		1 Introduction
		2 Materials
			2.1 Reagents, Buffers, and Solutions
			2.2 Equipment
		3 Methods
			3.1 Agarose Gel Electrophoresis
			3.2 Transfer of RNA from Gel to Membrane Using Capillary Blotting
			3.3 Probe Preparation and Purification
			3.4 Hybridization and Signal Detection
		4 Notes
		References
Index