The emerging roles of exosomal long non‐coding RNAs in bladder cancer

Abstract Extracellular vesicles (EVs), especially exosomes, have been reported to play essential roles as extracellular messengers by transporting goods in various diseases, while their potential roles in bladder cancer (BC) still remain to be further studied. BC exhibits a high degree of chemoresistance and metastatic ability, which may be affected by cancer‐derived exosomes that carry proteins, lipids and RNA. To date, the most studied exosomal molecular cargo is long non‐coding RNA (lncRNA). Although there is increasing interest in its role and function, there is relatively little knowledge about it compared with other RNA transcripts. Nevertheless, in the past ten years, we have witnessed increasing interest in the role and function of lncRNA. For example, lncRNAs have been studied as potential biomarkers for the diagnosis of BC. They may play a role as a therapeutic target in precision medicine, but they may also be directly involved in the characteristics of tumour progression, such as metastasis, epithelial‐mesenchymal transition and drug resistance. Cancer cells are on chemotherapy acting. The function of lncRNA in various cancer exosomes has not yet been determined. In this review, we summarize the current studies about the prominent roles of exosomal lncRNAs in genome integrity, BC progression and carcinogenic features.


| INTRODUC TI ON
Bladder cancer (BC) is one of the most common malignant tumours of the genitourinary system, accounting for the 9th most common malignant tumour in the world. 1,2 According to pathological classification, 90% of patients with BC have urothelial cancer. About one-third of these patients are first diagnosed with muscle invasive bladder cancer (MIBC). 3,4 In some patients, even if the first diagnosis is non-muscle invasive bladder cancer (NMIBC), 10%-30% of patients progress to MIBC. 4,5 BC has become a disease that seriously affects human health. 6,7 At present, its early diagnosis and treatment have made great progress, 8,9 but its specific mechanism of occurrence and development is still unclear.
In recent years, non-coding RNAs (ncRNAs) have become a research hotspot. NcRNAs can be divided into housekeeping ncRNAs and regulatory ncRNAs. Among them, regulatory ncRNAs can be mainly divided into microRNA (miRNA), long noncoding RNA (ln-cRNA) and circular RNA (circRNA). [10][11][12][13] LncRNA is a general term for single-stranded nucleotide sequences exceeding 200 bp. 14 Although it does not have the function of encoding proteins, it can participate in gene regulation at the epigenetic level, transcription level and post-transcriptional level, [15][16][17] affect tumour occurrence, development, metastasis and malignant progression of drug resistance. [18][19][20][21][22][23] Based on the current research on the mechanism of lncRNA, the competitive endogenous 'ceRNA' mechanism is the most common type and a widely recognized regulatory mechanism, that is, some | 967 LIU ncRNAs have binding sites with microRNAs. 24,25 The cell acts as a miRNA sponge, thereby releasing the inhibition of miRNA on the target gene, thereby increasing the expression level of the target gene. [26][27][28][29] For instances, elevated LINC00909 can promote tumour progression of ovarian cancer via regulating the miR-23b-3p/MRC2 axis. 30 Long noncoding RNA IL6-AS1 is upregulated in chronic obstructive pulmonary disease and is interrelated to interleukin 6 via sponging miR-149-5p and regulating early B-cell factor 1 expression 31 ; Long non-coding RNA (lncRNA) DLEU2 can drive epithelialmesenchymal transition (EMT) genes and glycolysis in endometrial cancer through modulating the miR-455/HK2 and EZH2/miR-181a pathway. 32 Exosomes are small extracellular vesicles (EVs) (30-100 nm in diameter). 33,34 They are secretory vesicles containing cytoplasmic protein and RNA in a bilayer of phospholipids and exist in all eukaryotic and prokaryotic cells. [35][36][37] Through their role as transporters, exosomes form complex networks that connect tumour cells in the tumour microenvironment and play a crucial role in these networks. [38][39][40] Carrying substances through autocrine, paracrine, endocrine and other signalling pathways, exosomes transport specific proteins, DNA and RNA to recipient cells, thereby regulating the biological characteristics of the recipient cells. [41][42][43][44] For example, exosomes can promote tumour development, inhibit tumour cell apoptosis and immune escape, stimulate tumour angiogenesis and transfer genetic material. [45][46][47][48][49][50] Recently, it has been shown that the release of exosomes may act as a vital role in the chemotherapy resistance of cancer cells by mediating the transfer of drugs, nucleic acids and proteins. [51][52][53] In recent years, studies have shown that exosomal-lncRNAs also play an indispensable role in the occurrence and progression of many cellular processes. Besides, exosomal lncRNAs can regulate the tumour microenvironment by modulating the expression of various key signalling pathways and molecular and play important regulatory roles in cancer metastasis. Moreover, due to their specificity and sensitivity, exosomal lncRNAs can also be released into tumour microenvironments and act as potential tumour markers. Ni et al. show that breast cancer-derived exosomal lnc-SNHG16 can enhance the activation of the TGF-β1/SMAD5 pathway through the miR-16-5p/SMAD5 regulatory axis, thereby inducing the expression of CD73 in Vδ1 T cells and leading to malignant tumour progression. 54 Li et al. identify that the expression of Lnc-FMR1-AS1 is increased in the tissues of patients with oesophageal cancer and is related to the poor prognosis of patients. 55 Lnc-FMR1-AS1 can be packaged into exosomes and released into the tumour microenvironment, and maintain the dynamic interconversion state of tumour stem cells by activating TLR7-NFκB signalling and up-regulating c-Myc levels in recipient cells. Guo et al. found that we can detect the occurrence of gastric cancer (GC) and predict the later progression of GC by detecting the expression of circulating exosomes lncRNA-GC1. 56 Combining the detection of circulating exosomes lncRNA-GC1 with endoscopy can improve the early diagnosis rate of GC. Lin et al. reveal that the AUC values of lncUEGC1 in distinguishing EGC patients from healthy individuals and patients with precancerous chronic atrophic gastritis were 0.8760 and 0.8406, respectively, which were higher than the diagnostic accuracy of carcinoembryonic antigen and were a good marker for early diagnosis of GC. 57 In this review, we focused on the latest evidence of major exosomal lncRNAs related to BC, and discussed the latent biological role of exosomal lncRNAs in the development, treatment and clinical applications of BC.

| Biochemical characteristics of exosomes
There are two main secretion mechanisms of exosomes: con- Transmembrane proteins (such as CD9, CD63, CDS1), apoptosislinked gene 2 interacting protein X (ALIX), heat shock proteins (such as HSP70, HSP90). 45,58-60 Exosomes carry many nucleic acid molecules, such as miRNA, ncRNA and mRNA. 47,50,58 In addition, it also carries cytokines and growth factor proteins similar to the source cells. 60,61 The biological process of exosome biogenesis and release was showed in Figure 1.
The separation methods of exosomes have not yet been unified, including sucrose gradient centrifugation, differential ultracentrifugation, filtration centrifugation, immunoaffinity capture technology, chromatography technology, microfluidic chip technology and PEG polymer precipitation. [62][63][64] The appropriate combination of these technologies may be effective. Even better, there are more commercial kits based on the above principles. The gold standard method is differential ultracentrifugation. 65,66 The exosomes obtained by the sucrose gradient centrifugation method have high purity, but the preliminary preparation is time-consuming, the extraction process is very time-consuming and the yield is low. Exosomes can be stored at 4°C for a short term (within 1-2 days) and stored at 80°C for a long term.
The identification of exosomes relies on morphological observation and protein composition analysis. 53,67,68 Observe the morphology of exosomes under an electron microscope. It can be seen that they are cup-shaped or flat balloons. Nanoparticle tracking analysis (NTA) can also be used to measure their diameters; protein composition analysis usually uses Western blot to detect exosomes. The protein expression level of body enrichment, as usual, chooses to detect CD63 and CD81.

| Exosomes and tumours
Exosomes were first discovered to participate in antigen presentation and immune activation and suppression. 69 Table 1).

| Exosomal lncRNAs and epithelialmesenchymal transition (EMT) in BC
Epithelial-mesenchymal transition is intimately interrelated to the development of tissues or organs during embryogenesis. Additionally, this phenomenon is significantly associated with tumour development 87 and is a trigger for invasion, migration and acquisition of stem cell-like phenotype in cells of diverse cancers, including BC. 88

| Exosomal lncRNAs and cell proliferation in BC
Cell proliferation is a precision control process, which is vital for embryonic and postnatal development. 94 Under pathological conditions, abnormal cell proliferation is a central mechanism attributing to disease progressions. Abnormal cell proliferation includes both F I G U R E 2 Role and functions of deregulated exosomal lncRNAs in BC progression. Exosomal lncRNAs can participate in the progression of BC by regulating the EMT (A), cell proliferation (B), lymphangiogenesis (C) and chemoresistance (D). Long noncoding RNA, lncRNA; bladder cancer, BC; HOX transcript antisense RNA, HOTAIR; epithelial-to-mesenchyme transition, EMT; urothelial cancer-associated 1, UCA1; phosphatase and tensin homologue deleted on chromosome ten, PTEN; micro RNA, miRNA; lymph node metastasis-associated transcript 2, LNMAT2; heterogeneous nuclear ribonucleoprotein A2B1, hnRNPA2B1; prospero homeobox 1, PROX1; ATP binding cassette subfamily B member 1, ABCB1; brain cytoplasmic RNA 1, BCYRN1; integration site family member 5A, Wnt5a; Vascular endothelial growth factor receptor 3, VEGF3; cancer-associated fibroblasts, CAFs; human lymphatic endothelial cells, HLECs. Long noncoding RNA, lncRNA; bladder cancer, BC; orthodenticle homeobox 2 antisense 1, OTX2-AS1; metastasis-associated lung adenocarcinoma transcript 1, MALAT1; prostate cancer-associated transcript 1, PCAT1; SPRY4 intronic transcript 1, SPRY4-IT1; phosphatase and tensin homolog pseudogene 1, PTENP1; small nucleolar RNA host gene 16, SNHG16; H19 imprinted maternally expressed transcript, H19 abnormal cell division and abnormal cell differentiation. 95 Besides, cell proliferation is also a main characteristic of cancer cells and the base of metastasis. 96 Studies have shown that exosomal lncRNAs can regulate the cell proliferation of BC ( Figure 2B). Zheng and colleagues showed that exosomal PTENP1 was a novel biomarker that could be applied for clinical detection of BC. 97 Exosomes secreted by normal cells could transfer PTENP1 to BC cells and suppress cell growth and metastasis. The results indicated that exosomal PTENP1 might participate in the communication between normal cells and BC cells in the process of BC carcinogenesis. In addition, exosome-mediated could transfer LINC01133 inhibits the progression of BC by regulating the Wnt signalling pathway. 98

| Exosomal lncRNAs and lymphangiogenesis in BC
Tumour-induced lymphangiogenesis plays an essential role in promoting tumour growth and metastasis. 99 Tumour-associated lymphatic vessel density is closely correlated with sentinel lymph node metastasis, distant metastasis and patient survival. 100 In addition, lymph endothelial cells can interact with various immune cells to modulate immune cell activity. 101

| Exosomal lncRNAs and chemoresistance in BC
Chemoresistance can be divided into primary drug resistance and multiple drug resistance (MDR). The former refers to cancer cells that are resistant to induced drugs, whereas the latter refers to cancer cells that develop resistance to induced drugs or other chemotherapeutic agents. 104 The establishment of chemoresistance in cancer cells involves various mechanisms, including downregulation of apoptosis, increased DNA repair, altered drug targets and overexpression of MDR proteins. 105 Studies have shown that exosomal lncRNAs can regulate the chemoresistance of BC ( Figure 2D). Luo et al. proved that the CAFderived exosome LINC00355 could promote the resistance of BC cells to cisplatin by regulating the miR-34b-5p/ABCB1 axis. 107

| Exosomal lncRNAs could act as diagnostic and prognostic Biomarkers in BC
Previous studies have shown that exosomal lncRNAs play a crucial role in the early diagnosis and prognostic evaluation of tumours. [108][109][110][111] We summarized the diagnostic and prognostic value of exosomal-lncRNAs in BC (Table 2).

| FUTURE E XPEC TATI ON S OF E XOSOME IN BC
No matter how hard the current multidisciplinary treatment is, the high recurrence rate of BC is still the biggest obstacle for treatment. [116][117][118] The

| Future prospects of exosomal lncRNAs in BC
At present, studies have found that exosomal lncRNA has an important biological role in BC, but more research is still needed to explore the clinical translational value of exosomal lncRNAs in BC. Many studies have confirmed that exosomal lncRNAs can promote the malignant progression of tumours by promoting angiogenesis. [119][120][121] In addition, exosomal lncRNA can also mediate immunosuppressive microenvironments, 122,123 regulate cell radioresistance 124 and mediate metabolic reprogramming, 125

| CON CLUS ION
Due to its very aggressive nature, BC has the lower survival rate of urology cancers. [126][127][128] This extremely high mortality rate is primarily the result of its early asymptomatic development, so it is diagnosed as late. 129,130 Therefore, there is an urgent need for new diagnostic tools and new treatment strategies. In recent years, the potential role of lncRNAs as biomarkers, therapeutic targets and therapeutics in cancer research have attracted increasing interest. However, the pathophysiological function of lncRNAs still remains unknown, and whether they are the cause or consequence of cancer remains to be determined. Additionally, the same lncRNA can play completely distinct roles in various cancer environments, which makes the characterization of lncRNA particularly difficult. Although most studies are still in the preclinical stage, the diagnostic and prognostic applications of lncRNA related to exosomes are very promising for BC treatment. New advances in lncRNAs-related studies in specific fields, such as bioinformatics, pharmacokinetics, and improved nanotechnology to deliver lncRNAs-containing exosomes to the tumour microenvironment, will lay the foundation for future clinical applications.
Understanding the function and role of lncRNAs is essential for their effective use as biomarkers, precision medicine or therapeutic targets.

ACK N OWLED EG M ENTS
We thank the generous support by Liaoning Cancer Hospital & Institute (Shenyang).

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
Qiang Liu: Original draft preparation, allocation, revision, supplement and edition.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data in the current study are available from the corresponding authors on reasonable request.