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Cancer and exosomal long non-coding RNAs

Extracellular vesicles, including exosomes and microvesicles, allow early diagnosis, prognosis and potentially targeted treatments of cancer. Circulating exosomes are a source of stable RNAs including mRNAs, microRNAs, and lncRNAs.

Exosomes are cell-derived vesicles present in body fluids such as blood, urine, as well as in cell culture mediums. 
Exosomes are tiny vesicles released from plasma membranes of different cell types. Exosomes are cellular protein complexes that contain enzymes degrading nuclear and cytoplasmic RNA.

In contrast, endosomes are membrane-bound vesicles present in the cytoplasm formed during endocytosis, the process that transports molecules into the cell by engulfing them with its membrane.

Apparently all cell types produce extracellular vesicles (EVs). EVs deliver a variety of biomolecules such as lipids, proteins, DNA, mRNA, microRNA as well as long non-coding RNAs (lncRNAs), into cells, dynamically and bidirectionally.


Classification and definition of extracellular vesicles


(i)    Small nano-sized exosomes are formed within the cytoplasm by inward budding of endosomes pooled into multivesicular bodies (MVBs). The molecular transporting machinery called “the endosomal sorting complex” is involved in sorting and incorporation of the molecular material into multivesicular bodies (MVBs). MVBs fuse with the cell membrane and release exosomes into the extracellular space,

(ii)    Nano- to micro-sized vesicles or microvesicles shed from the plasma membrane, and

(iii)    Micro-sized vesicles are created as byproducts of cell death or apoptotic bodies.

Long non-coding RNAs (lncRNAs)

Long non-coding RNAs (lncRNAs) are a heterogeneous group of non-coding transcripts localized in different cell compartments and are usually longer than 200 nucleotides. lncRNAs are non-protein coding RNAs distinct from housekeeping RNAs such as tRNAs, rRNAs, and snRNAs, and independent from small RNAs with a specific molecular processing machinery, for example, micro- or piwi-RNAs.

lncRNAs are classified into intragenic (intronic or antisense) and intergenic lncRNAs. Some of these are stable, highly expressed and conserved, while others have a high turnover, often are barely detectable and poorly conserved. lncRNAs have diverse functions which they exert by interacting with DNA, RNA and proteins in a sequence-specific and conformational manner, where they can act as a scaffold, a decoy or as enhancer RNA. However, the exact role of exosomal lncRNAs is not at all clear. Dragomir et al. assume that lncRNAs could be a loading vehicle for miRNAs, mRNAs, and other complex molecules into the exosome but this hypothesis will need to be confirmed experimentally. Synthetic lncRNAs may allow more detailed functional studies of these RNA species.

The structural model for the SINEB2 element of the long non-coding RNA activator of translation AS Uchl1 solved by NMR is an excellent example for a long non-coding RNA (Figure 1. Podbevšek et al. 2018). The invSINEB2/183 RNA folds into a structure with mostly helical secondary structure elements. SINE elements have been hypothesized to act as portable domains in lncRNAs contributing to their biological functions.


Figure 1: NMR based model of the structure for the SINEB2 element of the long non-coding RNA activator of translation AS Uchl1 (PDB ID: 5LSN). A: A cartoon model of the structure is shown. B: The surface of the SINEB2 element is shown.

SINEUPs are a new class of natural, synthetic antisense long non-coding RNAs that activate translation. Zuchelli et al. in 2015 reported the discovery of a new functional class of natural and synthetic antisense lncRNAs that stimulate translation of sense mRNAs. The research group named these molecules SINEUPs since for them to function requires the activity of an embedded inverted SINEB2 sequence to UP-regulate translation. The existence of natural SINEUPs suggests that embedded Transposable Elements may represent functional domains in long non-coding RNAs.

Also, the Zuchelli et al. argue that the design of synthetic SINEUPs to target antisense sequences of mRNAs of choice allows for a scalable increase of protein synthesis of potentially any gene of interest.

Examples of the categorization for non-coding RNAs

Category

Description

Example

Intronic

Expressed from the intron of target.

DMD lncRNA

H3K4me3

Has a methylated H3K4 promoter.

lincRNA-p21

Antisense

Expressed from the non-coding strand and acts on the complementary target.

BACE1-AS

Enhancer

Expressed to enhance expression at a locus at some distance from target.

p53 eRNAs

Promoter

Acting on and expressed from the promoter of target.

DBE-T

Intergenic

Expressed at some distance from coding genes.

lincRNA00299

Trans-acting

Acting at some distance from target

Evf2

Cis-acting

Acting on an adjacent target

AIRN

Small

Less than 200 bp in size

microRNA 137

Long

Greater than 200 bp in size

Fendrr

5-UTR

Expressed near the 5′UTR of target

5′UTR ELK-1

(Source: Ernst and Morton, 2013). 

Examples of lncRNAs showing diversity of functions

lncRNA

Biological function

Antisense-Uchl1

Enhances translation without interfering with mRNA expression level.

Gomafu/MIAT

Neuron-restricted expression; may hinder spliceosome formation and affect the splicing of mRNAs by sequestering splicing factors.

Hotair

HOX gene regulation via recruitment of PRC2 in order to silence expression.

Kcnq1ot1

Genomic imprinting.

PCAT-1

Promotes cell proliferation; target of PRC2 regulation.

lincRNA-p21

DNA damage response lincRNA; repressor in p53-dependent transcriptional responses.

Malat1

Affects transcriptional and post-transcriptional regulation of cytoskeletal and extracellular matrix genes.

Panda

Transcription factor decoy; sequesters transcription factor NF-YA.

Xist

Dosage compensation, genomic imprinting, inactivation of X chromosome.

 (Source: Kashi et al. 2016).

Many lncRNAs are found protected in human body fluids within circulating tumor cells or EVs. These lncRNAs are quite stable and can be extracted from blood or other body fluids as potential biomarkers for disease diagnosis, prognosis and therapy. The expression of lncRNAs can be blocked with antisense oligonucleotides (ASOs), aptamers, hammerhead ribozymes, siRNAs, or small specific molecules. Since some lncRNAs appear to form scaffolds to bind and recruit protein complexes to specific genomic loci, targeting them may open up new ways to treat certain cancers. ASOs as well as CRISPR based method allow studying unknown functions of lncRNA.

Also, Naderi-Meskin et al. suggested that exosome analysis can be used as a strategy for cancer diagnostics and monitoring dynamic changes during cancer development and therapy. Exosomal lncRNAs as potential diagnosis biomarkers and prognosis indicators in cancer.

Exosomal lncRNAs as potential diagnosis biomarkers and prognosis indicators in cancer 

Cancer type

Exosomal lncRNA

Sample origin (cohort number)

Reported findings

Non-small cell lung cancer (NSCLC)

MALAT-1

Serum (healthy controls: 30; NSCLC: 77)

(I) Diagnosis: Sens =60.1%; Spec =80.9%; AUC: 0.703;

(II) exosomal MALAT-1 was higher in NSCLC patients and positively associated with advanced TNM stage and lymphatic node metastasis status (P<0.001).

Laryngeal squamous cell carcinoma (LSCC)

HOTAIR

Serum (healthy controls: 30; NSCLC: 77)

(I) Diagnosis: Sens =92.3%; Spec =57.1%; AUC: 0.727; combined with miR-21; Sens =94.2%; Spec =73.5%; AUC=0.876;

(II) exosomal HOTAIR levels were higher in LSCC patients (P=0.0264) and positively associated with advanced TNM stage and lymphatic node metastasis status (P<0.01).

Colorectal cancer (CRC)

BCAR4

Serum (healthy controls: 76; colorectal adenoma: 20; CRC: 76)

(I) Diagnosis: the combination of two mRNA, KRTAP5-4 and MAGEA, with BCAR4 provided a high AUC =0.936 in training cohort and an AUC =0.877 in test cohort; (II) exosomal BCAR4 was down-regulated in CRC patients.

Colorectal cancer (CRC)

CRNDE-h

Serum (healthy controls: 80; hyperplastic polyp: 80; inflammatory bowel disease: 80; colorectal adenoma: 80; CRC: 148)

(I) Diagnosis: Sens =70.3%; Spec =94.4%; AUC: 0.892;

(II) exosomal CRNDE-h levels were higher in CRC patients (P<0.01) and positively correlated with regional lymph node metastasis (P=0.019) and distant metastasis (P=0.003);

(III) high exosomal CRNDE-h levels predict shorter overall survival.

Colorectal cancer (CRC)

ZFAS1

Serum (healthy controls: 37; CRC: 40)

(I) Diagnosis: Sens =80.0%; Spec =75.7%; AUC: 0.837;

(II) exosomal ZFAS1 levels were higher in GC patients (P<0.001) and positively associated with lymphatic metastasis (P=0.002) and TNM stage (P=0.025).

Cervical cancer

HOTAIR, MALAT1, MEG-3

Cervicovaginal lavage (HPV negative healthy controls: 30; HPV positive healthy controls: 30; cervical cancer: 30)

Exosomal HOTAIR and MALAT1 levels were higher, while MEG3 levels were lower in cervical cancer patients compared to HPV negative or positive controls.

Prostate cancer

PCA3

Urine (pairs before and after digital rectal examination: 30)

Diagnosis: AUC of exosomal PCA3 after DRE: 0.52; AUC after exosomal PCA3 values were normalized to PSA: 0.64.

Prostate cancer

lncRNA-p21

Urine (benign prostatic hyperplasia: 49; prostate cancer: 30)

(I) Diagnosis: LincRNA-21, Sens =67%; Spec =63%, AUC: 0.663;

(II) exosomal lincRNA-p21 levels were higher in patients (P=0.016).

Urothelial bladder cancer (UBC)

HOTAIR, HOX-AS-2, MALAT-1, lincRoR HYMA1, LINC00477, LOC100506688, OTX2-AS1

Urine (Cohort 1 — healthy controls: 5; UBC: 8; Cohort 2— healthy controls: 7; UBC: 10)

All of these exosomal lncRNAs were higher in UBC patients (P<0.01).

 (Source: Dragomir et al. 2018).

Reference

Dragomir, M., Chen, B., & Calin, G. A. (2018). Exosomal lncRNAs as new players in cell-to-cell communication. Translational Cancer Research, 7(Suppl 2), S243–S252.

Ernst, C., & Morton, C. C. (2013). Identification and function of long non-coding RNA. Frontiers in Cellular Neuroscience, 7, 168.

Hewson C, Morris KV; Form and Function of Exosome-Associated Long Non-coding RNAs in Cancer. Curr Top Microbiol Immunol. 2016;394:41-56. doi: 10.1007/82_2015_486.

Hojjat Naderi-Meshkin, Xin Lai, Raheleh Amirkhah, Julio Vera, John E J Rasko, Ulf Schmitz; Exosomal lncRNAs and cancer: connecting the missing links, Bioinformatics, , bty527.

Kaori Kashi, Lindsey Henderson, AlessandroBonetti, PieroCarninci;  Discovery and functional analysis of lncRNAs: Methodologies to investigate an uncharacterized transcriptome. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, Volume 1859, Issue 1, January 2016, Pages 3-15. 

Podbevšek, P., Fasolo, F., Bon, C., Cimatti, L., Reißer, S., Carninci, P., … Gustincich, S. (2018). Structural determinants of the SINE B2 element embedded in the long non-coding RNA activator of translation AS Uchl1. Scientific Reports, 8, 3189. http://doi.org/10.1038/s41598-017-14908-6.

Zhang, J., Li, S., Li, L., Li, M., Guo, C., Yao, J., & Mi, S. (2015). Exosome and Exosomal MicroRNA: Trafficking, Sorting, and Function. Genomics, Proteomics & Bioinformatics, 13(1), 17–24. http://doi.org/10.1016/j.gpb.2015.02.001.

Zucchelli, S., Cotella, D., Takahashi, H., Carrieri, C., Cimatti, L., Fasolo, F., … Gustincich, S. (2015). SINEUPs: A new class of natural and synthetic antisense long non-coding RNAs that activate translation. RNA Biology, 12(8), 771–779. 

 
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