Novel potential tumor biomarkers: Circular RNAs and exosomal circular RNAs in gastrointestinal malignancies

Abstract Background Circular RNAs (circRNAs) are structural ubiquitous RNA molecules. Accumulating evidences have elucidated that circRNAs play essential roles in the pathogenesis of diseases including cancers. Exosomal circRNAs are those circRNAs stably existing in exosomes and having high clinical values as novel potential diagnostic biomarkers of many diseases. Gastrointestinal (GI) malignancies, including pancreatic cancer, colorectal cancer, hepatocellular carcinoma (HCC), and gastric cancer, are leading causes of mortality worldwide and a major global health burden. However, no ideal tumor biomarkers of screening early GI cancers are currently available. Methods We collected data through Web of Science. The search terms used were as follows: circular RNA, circRNA, exosomes, exosomal circRNAs, biomarkers, gastrointestinal malignancies, pancreatic cancer, hepatocellular carcinoma, HCC, gastric cancer, colorectal cancer, physiological functions, biogenesis, molecular mechanism. Only articles published in English were included. Results We found that several circRNAs and exosomal circRNAs have been used as potential biomarkers to screen GI cancers including pancreatic cancer (hsa_circ_0001649, circ_0007534, circ_0030235, circRHOT1, circZMYM2, circ‐LDLRAD3, chr14:101402109‐101464448C, chr4:52729603‐52780244C, circ‐IARS, and circ‐PDE8A), HCC (circSETD3, circADAMTS13, hsa_circ_0007874, hsa_circ_104135, circFBLIM1, cSMARCA5, circRNA‐100338, and circPTGR1), colorectal cancer (hsa_circ_0001178, hsa_circ_0000826, hsa_circ_0004771, circDDX17, circITGA7, and circHIPK3), and gastric cancer (hsa_circ_0074362, circNRIP1, circAKT3, circ‐DONSON, circPSMC3, circ‐KIAA1244, circPVRL3, circPVT1, hsa_circ_0000096, ciRS‐133, hsa_circ_0001017, and hsa_circ_0061276). Conclusion CircRNAs and exosomal circRNAs have the potential high clinical diagnostic values for GI malignancies.


| INTRODUC TI ON
In the recent few decades, with the accomplishment of Human Genome Project, approximately 20 000 genes encoding proteins have been identified. 1 However, only lower than 2% of genome actually codes proteins. The vast majority of genome, which accounts for almost 98%, is transcribed to non-coding RNAs (ncRNAs). 2 Compared with microRNAs (miRNAs) and traditional linear RNAs, circular RNAs (circRNAs) resist to ribonuclease R. 3 Because of their covalently closed structure, circRNAs are stable and present in high abundance in blood. 4,5 CircRNAs are now considered as potential biomarkers in gastrointestinal (GI) malignancies. [6][7][8] Thanks to the advancement of high-throughput functional genomic screening biotechnology, an increasing number of researches have confirmed tumor-associated circRNAs. 6,9 Exosomes are vesicles generated and released by cells. Exosomes, carrying a cargo of lipids, genetic materials, proteins, and their derivatives, modulate cells' behaviors, influence the extracellular system, and may be a source of human disease biomarkers. 10,11 Exosomal circRNAs (exo-circRNAs) have unique profiles reflecting the characteristics of tumors. 10 They would be new GI cancers biomarkers.
Gastrointestinal malignancies, which mainly include gastric cancer (GC), colorectal cancer (CRC), hepatocellular carcinoma (HCC), and pancreatic cancer, are main causes of mortality worldwide and a global burden. For example, pancreatic cancer with only approximately 5% patients surviving over 5 years is the deadliest cancer; CRC is the third most frequent tumor and the second major origin of deaths related to cancers in the USA; HCC is the fourth major origin of cancer-associated death worldwide; GC is the fifth most frequent tumor and the third major burden associated with cancers in modern society. 12 Nevertheless, most of patients with GI malignancies miss the therapeutic window. 12 When found early, GI malignancies are highly curable. However, the early diagnosis of GI malignancies continues to be a major challenge.
Recent years, researchers have gradually realized that the developments of new technology for non-invasive early detection are likely to be the most effective method for cutting down mortality of GI malignancies.
Commonly used biomarkers such as carbohydrate antigen 125 (CA125), CA 19-9, squamous cell carcinoma antigen (SCCA), α fetoprotein (AFP), tissue inhibitor of metalloproteinases 1 (TIMP-1), cytokeratin 19 fragments (CyFra21-1), and carcinoembryonic antigen (CEA) could be found not only in serum of patients with GI malignancies but also in serum of healthy individuals. 13 For example, the specificity and sensitivity of CEA to detect GC were 0.686 and 0.593, respectively, and those for CA19-9 were 0.605 and 0.559, respectively. 14 Obviously, these biomarkers interfere the diagnostic accuracy and sensitivity.
CircRNAs and exo-circRNAs extracted from plasma are high-efficient blood-based biomarkers with great clinical significance. In recent years, with dramatic successes of considerable technological advances and the progression of techniques, such as real-time and high-sensitivity liquid biopsy assays, sensitive sequencing, high-depth targeted next-generation sequencing, sequencing somatic mutations, polymerase chain reaction (PCR), next-generation sequencing, and droplet digital PCR (ddPCR) analysis, circRNAs and exo-circRNAs have appeared potential high clinical values and could be used to detect various GI malignancies. 15,16 Compared with lower specificity and sensitivity of CEA and CA19-9 to detect GC, we explored that the specificity and sensitivity of hsa_circ_0001017 in blood reached 0.794 and 0.811, respectively. 4 Therefore, there is a great hope and promising broad clinical applications in screening GI malignancies through circRNA-based approaches.
This review summarizes GI cancer-associated circRNAs and exo-circRNAs and their possible application in the diagnosis of GI cancers.

| The history of circRNA research
CircRNAs were first discovered in viruses 41 years ago. 17 For decades, researchers considered circRNAs as products of splicing errors. Later, in 1996, circRNAs were described as self-replicating molecules in rodent and human cancer cells. 18 Nonetheless, circR-NAs have been re-evaluated mainly owing to the great advances in deep sequencing and computational approaches. 19

| The biogenesis and physiological functions of circRNAs
Nowadays, a major group of circRNAs have been reported to be generated by different mechanisms. 9,19 Most circRNAs are arisen from precursor mRNAs (pre-mRNAs) through "out-of-order" splicing (a process called back-splicing). 9,20 Multiple known circRNAs are from exons of protein-encoding genes. 19 circRNAs are crucial players involved in numerous biological processes, such as sponging of miRNAs, regulating parental gene transcription and tumorigenicity, protein kinase activation, angiogenic sprouting, generation of short proteins, and post-transcriptional regulation in the pathogenesis of malignancies. [20][21][22][23][24] Additionally, recent functional studies have reported many tissue-specific and cell-specific circRNAs, which were functionally characterized as efficient biomarkers in human diseases. 25 For example, ciRS-7 (also termed CDR1as) has been demonstrated to be a risk factor through sponging corresponding miRNAs. 26,27 CircSETD3 (hsa_circ_0000567) and circADAMTS13 also act as sponges of miRNAs and participate in HCC tumorigenesis. 28,29 Strikingly, hundreds of circRNAs including ciRS-7 are highly abundant in the mammalian brains. 30 Some researchers believe that ciRS-7 could be possibly used as therapeutic target agents against Alzheimer's neuronal injury. 31 Moreover, several studies showed that the expression of circRNAs elucidated various cardiovascular and cerebrovascular diseases. 32,33 Furthermore, some circRNAs may be potential therapeutic targets of gestational diabetes mellitus. 34 Similarly, circHIPK3 was identified as an applicable therapeutic intervention for diabetic proliferative retinopathy. 35

| The functions of exosomes
Exosomes (diameter, 30-160 nm) act on many biological processes and participate in substance delivery and signal transduction. 36 Exosome formation occurs spontaneously within the endosomal network via re-routing of multivesicles and are released through exocytosis. 37 43,44 For instance, an exosomebased drug delivery system with high application potential in cancer targeted therapy was reported. 43 Additionally, cancer-derived exosomes show a great potential for acting as biomarkers. 45 These studies have provided new ideas about how exosomes may be used to treat cancers efficiently and effectively. However, exosomes may present as two-edged sword. For instance, researchers found that exosomes play critical roles in neurological disorders and could spread pathological proteins in these neuropsychiatric deficits, such as ischemic stroke, Parkinson neurodegeneration, brain infarction, and Alzheimer's neuronal injury. 46 Unfortunately, some tumorderived exosomes may promote cancer progression and accelerate metastasis by modulating the primary site of tumors and assisting metastatic cancer cells escape from immunologic surveillance. 47

| circRNA s A S B I OMARKER S IN G I MALIG NAN CIE S
Most advanced GI malignancies and their metastases are difficult to be cured. In contrast, patients with early GI cancer can be treated effectively. However, the main clinical imaging equipments such as digital X-ray radiography, magnetic resonance imaging (MRI), and positron emission tomography-computed tomography (PET-CT) are not only expensive but also often quite subjective. Therefore, circRNAs and exo-circRNAs as potential effective biomarkers may

| circRNAs as biomarkers of gastric cancer
Gastric cancer is a main origin of death in the modern society. 48 Recent years, researchers have investigated circRNAs as biomarkers of GC. Based on RNA sequencing biotechnology, circNRIP1 and circAKT3 (hsa_circ_0000199) were explored to be increasingly expressed in human GC tissues; the increments of circNRIP1 and F I G U R E 1 Gastrointestinal cancer-associated circular RNAs circAKT3 in human GC tissues were related to the metastasis potential and recurrence risk. 49,50 After purifying exosomes, Zhang et al found upregulated expression of circNRIP1 in GC patients. 49 Their findings suggested that high levels of circNRIP1 indicated reduced survival. 49 Huang et al found that the upregulation of cir-cAKT3 in GC patients elucidated reduced five-year disease-free survival (DFS). 50 The area under the curve (AUC) is 0.91, which elucidated that circAKT3 could be biomarker to predict survival. 50 In addition, circ-DONSON (hsa_circ_0004339) was markedly increased in GC tissues. 51 The expression of circ-DONSON was related to DFS, confirming circ-DONSON as a novel GC biomarker. 51 In contrast, the expression of circPSMC3 and circ-KIAA1244 was significantly reduced in GC patients' plasmas. 52,53 Mechanism study elucidated that circPSMC3 might regulate the tumor progression through sponging miR-296-5p and might be applied as a potential therapeutic target of GC. 52 Sun et al found that the attenuated expression of circPVRL3 could boost GC cells growth. 54 Our group measured the expression profiles of circRNAs in cancer plasma from GC patients. 4,6 Aberrant expression of 308 circRNAs was demonstrated in GC tissues. 6 Total 343 circRNAs in plasma from GC patients were further found. 4 The above studies elucidated that circRNAs have advantages on the potential novel biomarkers of GC.

| circRNAs as biomarkers of hepatocellular carcinoma
Currently, the incidence of HCC is supreme in Central America. 55 Thousands of patients with HCC were diagnosed at late stages and lost the best opportunity of a surgical cure. Reduced circMTO1 expression in HCC was related to irreversible miss of therapeutic window for HCC patients. 56 Bai et al discovered that silencing of circFBLIM1 could markedly inhibit HCC cells by directly sponging miR-346. 57 Additionally, underlying mechanism experiments showed that circSETD3 suppresses tumor growth by activating the cascade pathways. 29 Furthermore, cSMARCA5 is known to act an essential role in the tumorigenesis of HCC. 58 Our group found that hsa_circ_0068669 expression elucidated stages of HCC patients. 8 These studies helped us uncover the great clinical value of circRNAs as biomarkers of HCC.

| circRNAs as biomarkers of colorectal cancer
Colorectal cancer is common malignancies and the second cancer killer in the USA. 12 Increasing the screening rate of CRC among people and finding effective treatment targets of CRC is imperative.
Recently, scholars have identified circRNAs as effective biomarkers of CRC. Known to be associated with unfavorable clinicopathological factors, hsa_circ_0001178 and hsa_circ_0000826 clearly upregulated in CRC mucosae compared with adjacent normal mucosae. 59 The AUCs were 0.816 and 0.945 for hsa_circ_0000826 and hsa_circ_0001178, respectively. 59 Moreover, circDDX17 (hsa_circ_0002211) expression was lower in the CRC mucosae; and knockdown of circDDX17 could accelerate CRC cell growth and differentiation as well as suppress apoptosis. 60 These results suggest that circDDX17 could serve as a tumor suppressor. Li et al further found that reduced circDDX17 expression was related to distant stage of CRC. 60 Additionally, circITGA7 markedly inhibited CRC tumorigenesis. 61 Interestingly, the level of circHIPK3 was notably higher. 62 Moreover, increased expression of circHIPK3 was related to CRC distant metastasis, T status of tumor, and advanced clinical stage. 62 These findings highlight circRNAs as biomarkers for CRC.

| circRNAs as biomarkers of pancreatic cancer
Pancreatic cancer is largely incurable cancer with the lowest survival in the worldwide. 12,63 This challenge has attracted the interest F I G U R E 2 CircRNAs and exosomal circRNAs used as biomarkers. In the nuclei of cancer cells, circRNAs are produced by pre-mRNA after transcription. The formation of circRNAs may involve one exon, several exons, several introns, or only one intron. CircRNAs may be released into the blood directly or with exosomes were demonstrated to be novel potential biomarkers and drug targets of pancreatic ductal adenocarcinoma (PDAC). 64 Circ_0007534 and circ_0030235 were markedly upregulated in PDAC; the expression of these circRNAs elucidated the poor prognosis of PDAC patients. 65,66 Furthermore, the expression of hsa_circ_0007534 indicated lymph node invasion. 65 Xu et al found that positive lymph node invasion and higher tumor stage were associated with high expression of hsa_circ_0030235. 66 CircRHOT1 (hsa_circ_0005397) and circ-LDLRAD3 were also overexpressed in pancreatic cancer cells. 67,68 The circ-LDLRAD3

TA B L E 1 Overview of identified gastrointestinal malignancy-associated circRNAs
in plasmas of PDAC patients was associated with stages. 68 In addition, hsa_circ_0001649 was significantly downregulated in cells and tissue specimens of PDAC. 69 Jiang et al confirmed that the low expression of hsa_circ_0001649 elucidated irreversible differentiation grade and stage through Fisher's exact tests. 69 Jiang et al found that high hsa_circ_0001649 expression elucidated high overall survival (OS) rate. 69 These studies verified that hsa_circ_0001649 could be applied as a biomarker of PDAC.

| E XOSOMAL circRNA s A S B I OMARK ER S OF G I MALIG NAN CIE S
Exosomes have been the increasingly interesting particles in medicine and biotechnology, especially for using as biomarkers in clinical diagnosis. 37,70 Their molecular constituents such as pro- Tissues and cells [99] TA B L E 1 (Continued)

| CON CLUS I ON S AND FUTURE DIREC TIONS
There is great hope that circRNAs and exo-circRNAs may be used in medical oncology (Table 1). Our understanding of exosomes has rapidly advanced over the last few years. We look forward to the day of application of circRNAs and exo-circRNAs in the diagnosis and treatment of GI malignancies.
Here, we speculate on two potential future directions for cir-cRNAs and exo-circRNAs. First, circRNAs and exo-circRNAs as novel clinical biomarkers of GI malignancies will become the cornerstones