The role of Transfer RNA-Derived Small RNAs (tsRNAs) in Digestive System Tumors

Transfer RNA-derived small RNA(tsRNA) is a type of non-coding tRNA undergoing cleavage by specific nucleases such as Dicer. TsRNAs comprise of tRNA-derived fragments (tRFs) and tRNA halves (tiRNAs). Based on the splicing site within the tRNA, tRFs can be classified into tRF-1, tRF-2, tRF-3, tRF-5, and i-tRF. TiRNAs can be classified into 5′-tiRNA and 3′-tiRNA. Both tRFs and tiRNAs have important roles in carcinogenesis, especially cancer of digestive system. TRFs and tiRNAs can promote cell proliferation and cell cycle progression by regulating the expression of oncogenes, combining with RNA binding proteins such as Y-box binding protein 1 (YBX1) to prevent transcription. Despite many reviews on the basic biological function of tRFs and tiRNAs, few have described their correlation with tumors especially gastrointestinal tumor. This review focused on the relationship of tRFs and tiRNAs with the biological behavior, clinicopathological characteristics, diagnosis, treatment and prognosis of digestive system tumors, and would provide novel insights for the early detection and treatment of digestive system tumors.


Introduction
Transfer RNAs (tRNAs) have important roles in protein biosynthesis, which has a secondary cloverleaf structure containing three hairpin loops: a D loop, an anti-codon loop, and a TψC loop. TRNA may recognize codons of messenger RNA (mRNA) via its anti-codon loop and then the corresponding amino acid is transferred to the polypeptide synthesized in the ribosome (1). Recently, in-depth analyses of new-generation data have revealed a large number of non-coding RNAs with unique function, including microRNA (miRNA), piwi-interacting RNA (piRNA), long non-coding RNA (lncRNA) and circle RNA (circRNA). Among them, transfer RNA-derived small RNAs (tsRNAs) have been paid increasing attention.
TsRNAs can be found in a variety of organisms (2,3). Numerous researches have demonstrated that tsRNAs could regulate cell proliferation, invasion, metastasis and gene expression, and thus play a crucial role in human cancer (4,5). Aberrant regulation of tsRNAs has been reported in multiple cancers. Five tsRNAs were down-regulated in chronic lymphocytic leukemia and lung cancer, including ts-46, ts-47, ts-49, ts-53 and ts-101, while ts-4 was up-regulated in them (6). Ts-86 was down-regulated in breast cancer whereas ts-66 was up-regulated (6). Differentially expressed tsRNAs have the potential to be diagnostic markers and therapeutic targets of cancer (7,8).

Ivyspring
International Publisher Figure 1. The main classification of tRNA-derived fragments (tRFs) and tRNA halves(tiRNAs). In this figure, the tRFs or tiRNAs were shown the source from tRNAs, according to the color which is the same with tRNAs'.

Differences and correlation between tRFs and tiRNAs
Under specific conditions, tRFs and tiRNAs are similar but not identical in terms of their structure and function. First, both of them are derived from tRNA. However, due to different enzymes and cleavage sites involved, the products differ in terms of their length and structure (17). Second, tRFs and tiRNAs are mostly located within cells except for a small part in peripheral circulation; however, their intracellular original sites and distribution are different. TRF-5s are produced in cytoplasm and transported to nucleus via a similar mechanism for tRNA transportation (10,17,30). TRF-1 might be generated in cytoplasm or nucleus. Some scholars reported that tRF-1s were generated in cytoplasm(4). However, others believed that tRF-1s was likely to be produced in nucleus and transported to cytoplasm via specific mechanism (17). TRF-3s are mainly distributed in cytoplasm (10,17). TiRNAs are mainly located in cytoplasm with a small part in nucleus and mitochondria, while tiny levels are found in circulatory system (10). However, whether the location of tRFs and tiRNAs is associated with their diverse function remains unknown. Third, both tRFs and tiRNAs have important roles in cellular transcription, gene expression and viral infection (11,31), with various mechanisms. For instance, regarding transcriptional regulation, tRF-5s were suggested to mainly inhibit transcriptional elongation, whereas 5'-tiRNAs mainly inhibits transcriptional initiation (6,32,33). TRFs and tiRNA have many features in common, though further investigations are required to explore the correlation between their characteristics and disease development, especially tumor development and progression.

Function of tRFs and tiRNAs
Although the function of most tRFs and tiRNAs have not been elucidated, accumulating evidence has suggested that they are involved in regulating cell proliferation and apoptosis, DNA damage response, gene expression and post-transcriptional modifications. TRFs and tiRNAs have the following function (Table 2).
TsRNAs were also indicated to induce or inhibit apoptosis. During apoptosis induction, inhibition of a specific tsRNA, 3'-tsRNA-LeuCAG, has been shown to induce apoptosis in rapidly dividing cells in vitro and a patient-derived orthotopic hepatocellular carcinoma model of mice. The tsRNA binds to at least two ribosomal protein mRNAs (RPS28 and RPS15) to enhance their translation. A decrease in the translation of RPS28 mRNA blocks pre-18S ribosomal RNA processing, resulting in a reduction in the number of 40S ribosomal subunits to induce apoptosis (34). During apoptosis inhibition, tsRNAs and tRNAs have similar roles. Early studies of tRNA showed that mature tRNA bind to cytochrome C to inhibit the formation of apoptotic bodies and cysteine-aspartic protease (caspase-9) activity to stimulate cellular survival (35). Recent studies showed that tsRNAs derived from tRNA also bind to cytochrome C(36), which then bind to apoptotic protease activating factor 1 (APAF1) and form apoptotic bodies (36). In response to hyperosmotic stress, ANG mediates the competitive binding of 5'-tiRNA and 3'-tiRNA to cytochrome C generating the ribonucleoprotein complex, inhibits the formation and activity of apoptotic bodies, and stimulates cellular survival (10,(36)(37)(38) (Figure 3). However, the complete biological mechanisms remain unclear and require further investigation.

Function similar to miRNA and piRNA
Certain tRFs may act as miRNAs or piRNAs to bind to AGO protein, which is an important RISC (34) with biological function. TRF-1s tend to interact with AGO3 and AGO4 (6,39). TRF-5s and tRF-3s interact with AGO1, AGO3 and AGO4 (17,31,39). Like miRNAs, they have seed sequences complementary to target mRNAs and can be recruited to AGO complexes to regulate target RNA expression and function (17). TRF-5 and tRF-3 both bind to miRNA via the seed region (16,40), which has been confirmed in many other tRFs. For instance, ts-53 and ts-101 not only bind to AGO proteins but also PIWIL2 (a molecule similar to piRNA), regulating target gene expression and post-transcriptional modification (6,30). However, interactions between tiRNA and AGO remain unknown and need further investigation.  Figure 2. The naming rules of tRFs and tiRNAs. The name described the tRFs or tiRNAs should contain the type (tRF-5, tRF-3, tRF-1, 5'-tiRNA, 3'-tiRNA), Chromosome number, tRNA number, the amino acid carried and the anticodon carried. The anticodon is the codon which tRNA carried, then this anticodon will be translated into the codon which is matched amino acid. And every tRFs has a name in tRFdb (tRFdatabase), for example, tRF-1001 is Chr10. Trna2-SerTGA and it belongs to tRF-1 type, and the number underline in tRFdb means the type (tRF-5, tRF-3 or tRF-1).
Another interesting phenomenon was found that the functional pattern was distinct from that of canonical miRNA. TRF5-GluCTC, a type of 5'-tRFs, plays a gene-silencing role on the target mRNA with complementary target sequence. Regulation studies showed the 5'-portion of miRNAs is the key determinant in target recognition, while this suggested the 3'-portion of tRF5-GluCTC is critical for its gene silencing function through a trans-silencing mechanism (3).
Compared with miRNAs and piRNAs, tRFs and tiRNAs may directly regulate cellular translation, suggesting that these small RNA molecules have more complicated regulatory potentials to maintain various biological function and thus more important roles. Figure 5. 5'-tiRNA could promotethe formation of stress granules (SGs). 5'-tiRNA may replace eIF4F, which is an eukaryotic translation initiation factor, at the mRNA m7 GTP position, to inhibit translation initiation and produce multiple mRNA protein complexes (mRNPs).Those tiRNA may further bind to the cold shock domain (CSD) of the YBX-1 protein (RNA binding protein) to form 5'-TOG-tiRNA-protein complex and then stimulate production of the stress granules (SGs). SGs, stress granules; YBX-1, Y-Box Binding Protein 1; mRNPs, mRNA protein complexes.

Roles of tiRNAs and tRFs in the cellular biology of DT tumor
TRFs and tiRNAs have important roles in tumor development and progression. However, the study on the correlation between tRFs/tiRNAs and tumor remains at an early stage. The expression levels of tRFs and tiRNAs were suggested to be abnormal in many tumor cells (47,48) and correlated with tumor cell proliferation, migration and transformation (6,48). Recent studies on DT tumor (rectal cancer) showed that tRF/miR-1280 expression levels were decreased(5). Furthermore, tRF/miR-1280 inhibits the 3' UTR of JAG2, reducing JAG2 biosynthesis to inhibit the Notch pathway and directly inhibit proliferation, migration and epithelial-mesenchymal transformation (EMT) of rectal cancer cells (5,49,50). TRF/miR-1280 also decreases the expression of CD133+stem cell marker in CRC cells, reduces their activity and migration, and prevents formation of the microenvironment of rectal cancer cell metastasis (5). Moreover, Ts-53 and ts-101 might have important roles in the early transformation stage of rectal cancer (48). Ts-40 might be oncogenic during rectal cancer development (48). Ts-36, correlated with cellular proliferation, may exert roles in the final stage of metastatic transformation of rectal cancer (48). A research applying tiRNA sequencing was performed in 30 HCC patients (including 10 with HBV and 20 with HCV) and 9 controls, and tiRNAs were found to be the most abundant small RNAs in chronically infected liver and the abundance of 5'-tiRNAs was reduced in liver cancer. In addition, in hepatitis B-associated HCC, 5'-tiRNAs abundance was correlated with the expression of tRNA-cleaving ribonuclease, angiogenin (51). Therefore, tRFs and tiRNAs are critical for the biological behavior of DT tumor cells. In-depth investigations would benefit understanding how to exploit these molecules to interfere with tumor progression and how to select molecular targets for therapy.

Correlation between tiRNAs/tRFs and the clinicopathological characteristics of DT tumor
In clinical case, distant metastasis of DT tumor is a major obstacle to optimal patient prognosis(52). Thus, researchers have been exploring the correlation of tiRNAs/tRFs with the cellular invasion and distant metastasis of DT tumor. Recent studies showed that colorectal cancer had elevated ANG levels, increased the levels of 5'-tiRNA-Val, 5'-tiRNA-Cys and 5'-tiRNA-Ala, then promoted cancer cell invasion and migration without affecting cellular proliferation (52). Additionally, 5'-tiRNA-Val levels are positively correlated with lymph node metastasis and distant metastasis of colorectal cancer cells (52). However, the mechanism remains unclear. Further investigations would benefit the early detection of distant tumor metastasis, providing new ideas for the treatment of metastatic tumors and improving prognosis.

Correlation between tiRNAs/tRFs and DT tumor diagnosis
Seminal exosomes were found to contain more tRFs than miRNAs, suggesting that tRFs could be stably expressed and were abundant in body fluids (53). That makes them to be potential markers for early tumor diagnosis. Recent studies showed that 5'tRF-GluCTC and 5'tRF-ValCAC levels were decreased in colorectal cancer cells, indicating that they were involved in the development and progression of colorectal cancer and could serve as potential diagnostic markers (2). Moreover, tiRNA-5034-GluTTC-2 was significantly decreased in gastric cancer (GC) tissue, neoplasm and GC cells, and low levels of tiRNA-5034-GluTTC-2 in GC tissue were correlated with tumor size (54). TiRNA-5034-GluTTC-2 in GC tissue had higher sensitivity and specificity with the large area under receiver operating characteristics (ROC) curve as a tumor marker. All these findings were statistically significant, suggesting its potential as a marker of early GC (54). Recently, it has been shown that the expression of tsRNAs in plasma exosomes of liver cancer patients is significantly higher than that in healthy controls (55). Four tsRNAs were up-regulated including tRNA-ValTAC-3, tRNA-GlyTCC-5, tRNA-ValAAC-5 and tRNA-GluCTC-5, suggesting that these differentially expressed tsRNAs have the potential to be novel diagnostic markers of liver cancer (55). Exploration on the correlation between tRFs/tiRNAs and DT cancer development would benefit identifying markers for the early diagnosis of DT cancer and optimize detection.

Correlation of tiRNAs/tRFs with DT tumor treatment and prognosis
Investigating the role of tiRNAs/tRFs in tumor progression would improve treatment protocols and disease prognosis. Previous reports demonstrated that elevated ANG in rectal cancer increased 5'-tiRNA-Val levels, and thus established an ANG-tiRNAs-cell invasion/metastasis regulatory axis (52). Such findings provided insights on the mechanism of rectal cancer development and revealed new therapeutic targets for colorectal cancer. TRF/miR-1280 could inhibit the Notch pathway and subsequent EMT and distant metastasis of colorectal cancer(5), suggesting that it could affect cancer prognosis. Quantitative PCR (qPCR) illustrated that tRFs and tiRNAs (tRF-3-LeuAAG-1-1[AS-tDR-000064], tRF-3-GlnCTG-1-1[AS-tDR-000069], tRF-3-AlaCGC-1-1[AS-tDR-000102] and tiRNA-5-ProCGG-1-1[AS-tDR-001391]) had abnormally elevated expression in cancerous pancreatic cells, but not normal pancreatic cells (56). KEGG and GO pathway analyses showed that all the four tRFs and tiRNAs were significantly enriched in cancer-related pathways (including RAS signaling pathway, cancer pathways, axon guidance and PI3K/AKT signaling pathway). Therefore, they might be able to serve as markers for early pancreatic cancer diagnosis and provide clues for pancreatic cancer development (56). Furthermore, the survival rate of patients with low expression of tiRNA-5034-GluTTC-2 was much higher than that of individuals with higher expression levels, indicating that tiRNA-5034-GluTTC-2 could be a potential prognostic marker of GC (54). All the findings suggested that tRFs and tiRNAs were crucial in DT cancer treatment and prognosis.

Summary and future directions
Several limitations should be acknowledged. First, although the sequencing has identified many tRFs and tiRNAs, bioinformatic methodology needs to be improved for more identification. Second, current study only focused on the basic biological function of tRFs and tiRNAs without the correlations with diseases, especially DT tumor. Third, the naming roles of molecules remain not unified. Fourth, we urgently need a tiRNA database and a database to integrate the tRFs (tRFdb) and tiRNAs.
In summary, tRFs and tiRNAs have important roles in gene expression, gene translation, and the development and progression of various DT tumors (17). They affect tumor cell proliferation, migration and invasion (48,57). To date, the origin and function of tRFs and tiRNAs remain to be fully elucidated, and their roles in DT tumor progression also require further investigation. In-depth studies would benefit early detection, prognosis evaluation and DT tumor therapy, further improving prognosis and survival.