Research & Reviews: Journal of Microbiology and Biotechnology
MenuE- ISSN: 2320 - 3528
P- ISSN: 2347 - 2286
E- ISSN: 2320 - 3528
P- ISSN: 2347 - 2286
Zixuan Zhang1, Gaoli Liang1, Shengkai Zhou1, Tianhang Li2,3*
1 Department of Biotechnology, Nanjing University, Nanjing, China
2 Department of Urology, Southeast University, Nanjing, China
3 Department of Pharmacy, Southeast University, Nanjing, China
Received: 04-Jan-2024, Manuscript No. JMB-24-124524; Editor assigned: 08-Jan-2024, PreQC No. JMB-24-124524(PQ); Reviewed: 22-Jan-2024, QC No. JMB-24-124524; Revised: 29-Jan-2024, Manuscript No. JMB-24-124524(R); Published: 05-Feb-2024, DOI: 10.4172/2320-3528.12.4.003.
Citation: Zhang Z, et al. Transfer RNA-Derived Small RNAs (tsRNAs): A New Insight into Liver Cancer. J Microbiol Biotechnol. 2024;12:003.
Copyright: © 2024 Zhang Z, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Current research has highlighted links between tsRNAs and numerous organisms as well as various diseases. However, the exploration of tsRNAs in liver cancer remains in its nascent stage, leaving much about their pivotal role in this field unknown. Considering the existing reports on tsRNAs potential biological functions in liver cancer, they manifest immense research potential in addressing the challenges faced in liver cancer diagnosis and treatment. This study initially describes the biological background, biogenesis, and biological functions of tsRNAs. Then, it discusses the relationship between liver cancer and tsRNAs in three perspectives: (1) tsRNAs involve in pre-tumor microenvironmental (pTME) construction; (2) tsRNAs play their role in cancer progression; (3) the relationship between tsRNAs and liver cancer related microRNA by analogy, thus examining the interplay between liver cancer and tsRNAs. Finally, by comprehensively summarize existing researches, we discuss the potential future of tsRNAs as diagnostic biomarkers and therapeutic targets in liver cancer.
tsRNAs; Biogenesis; Classification; Liver cancer; Biomarker; Therapy strategy
Transfer RNA-derived small RNA (tsRNA) derived from transfer RNA (tRNA), is the most ancient non-coding RNA (ncRNA) that exist in all domains of life with conservative sequence [1]. It is the second most abundant ncRNA after microRNA which is well-studied and is the most abundant ncRNA. Moreover, tsRNAs possess various secondary structure modifications like m1A, m1G modifications [2], rendering it more stable compared to other ncRNAs thus ensuring that tsRNAs are widely and substantial present in various bodily fluids (urine, serum, et al.) cells, and tissues in not easy degraded model. Initially, tsRNAs are considered as "junk" by researchers, so their biological functions remained largely unexplored. Nevertheless, technological advancements revealed that tsRNA not only presents abnormal expression levels associated with pathological processes in many diseases, but also plays an important role in the process of disease.
Liver cancer represents a challenging and globally prevalent malignant disease, Hepatocellular Carcinoma (HCC) is the most common type, accounting more than 90%. Presently, diagnosing and treating liver cancer continue to confront numerous challenges. Liver cancer have multifaceted etiology which imped the discovery of a unified biomarker. It onset stems from diverse factors such as viral infections, chemical inductions, aflatoxins, and unhealthy lifestyle habits like late nights and alcohol abuse, all contributing to its occurrence. Additionally, various liver-related diseases such as viral hepatitis, fatty liver disease, alcoholic liver disease, and cirrhosis hold the potential to cancerization in advanced stage.
Challenge also arises from the character of liver cancer, including the difficulty in early detection, high metastatic potential, poor prognosis in patient, and the absence of effective treatment strategies. Early HCC symptoms are often indiscernible, lacking a highly specific, sensitive, and minimally invasive screening method, resulting in late-stage diagnoses. Although patients are diagnosed with liver cancer at an early stage and resection is the best treatment of choice, there is a risk of incomplete cure and some patients with poor physical condition cannot undergo resection. In advanced stages, the rapid progression of the disease limits treatment options, leading to poorer outcomes when patients undergo resection at this phase. Chemotherapy's efficacy in treating HCC is limited by tumor resistance and drug tolerance. Moreover, the variability in response to immunotherapy among patients with similar disease phenotypes underscores diverse molecular etiologies.
Recent studies have highlighted the role of tsRNAs as biomarkers in liver cancer diagnosis and their involvement in tumorigenesis and maintenance of liver cancer. Overall, potential exhibited by tsRNAs in HCC presents a new avenue for both diagnosis and treatment strategies in addressing the complexities of this disease.
tsRNAs biological information and biogenesis
Discovery and development: tsRNAs are approximately 18 to 40 nucleotides in length. They are pervasive across the evolutionary spectrum, functioning in both eukaryotes and prokaryotes that have been identified in viruses, bacteria, archaea, plants, mice, and humans [3]. tsRNA initially discovered in human urine in 1970, perceived as non-functional. However, Qin et al. demonstrated their turnover during tRNA degradation in tumor tissues [3], while Wang et al. proposed their potential as clinical cancer biomarkers [4]. Latterly, the rapid advancement of high- throughput research technologies: ARM-seq, multiplex small RNA-seq library preparation methods (MSR-seq), CPA-seq, PANDORA-seq, AQRNA-seq, cP-RNA-seq, and 5’XP sRNA-seq [2], makes us to find more tsRNA’s function and has unveiled a myriad of functions performed by them. Those researches altered the perception of tsRNAs from being considered mere "junk" or degradation products to multifunctional mediators.
Classification: Based on the size and cleavage position of tsRNAs, they can be categorized into six types: tRF1, tRF3 (encompassing tRF-3a and tRF-3b), tRF5 (including tRF-5a, tRF-5b, and tRF-5c), i-tRF, 5‘tiRNA, and 3’tiRNA. Some scholars prefer to classify tsRNAs as tRNA-derived fragments (tRFs) and tRNA halves (tiRNAs), or stress-induced tRNA fragments. The former includes tRF1, tRF3, tRF5, and i-tRF, while the latter comprises 5‘tiRNA and 3’tiRNA. Others categorize tsRNAs as type I (including tRF3 and tRF5), type II (tRF-1), internal tsRNA (i-tRF), and tiRNA (5’tiRNA, 3’tiRNA). The nomenclature of tsRNAs is summarized in Figure 1. To establish a standardized nomenclature, which is lacking now, is a universal consensus [4,5].
Figure 1: tsRNAs classification with different classification methods of tsRNAs have been reported, as well as the length of each type of tsRNAs.
Biogenesis
In the generation process of tsRNAs, the involvement of key enzymes and the tRNA modification system plays a crucial role. Studies have reported the involvement of enzymes such as Dicer, Angiogenases, RNaseT2, RNasesZ, ElaC domain protein 2 (ELAC2), Rnylp, in the formation of tsRNAs [1,6]. The generation of tsRNAs involves both Dicer-dependent and Dicer-independent modes, likely attributed to differences in cell types, tissue types, and species. Knockout of Dicer has shown varied impacts on tsRNA expression levels in different samples. Angiogenin and RNaseT2 participate in the formation of type 1 tsRNAs. RNase Z, an endonuclease, is involved in the maturation of 3‘tsRNAs, contributing to the generation of tRF-1001 (the first reported tsRNA). Additionally, RNasesZ plays an important role in tRNA processing and maintaining balance in tRFs. Both RNasesZ and Elac2 are involved in the generation process of type 2 tsRNAs [1,7-9].
Apart from enzymatic actions, tRNA modification system also involved in tsRNA’s generation. The tRNA-methyltransferase Trm9 catalyzes the 5-methylcarboxylmethyl modification of uridine, conferring resistance to tRNA cleavage in yeast [3]. Dnmt2-mediated 5-methylcytosine modifications suppress the specific cleavage of the tRNA anticodon loop regulated by Angiogenin, subsequently decreasing tsRNA synthesis within cells [9,10]. Interestingly, scientist reported that the generation of tsRNA occurs entirely independent of tRNA. Knocking out leucyl-tRNA synthetases does not affect mature tRNA production but reduces LeuCAG 3‘tsRNA generation [11].
This independence in tsRNA generation from tRNA implies the involvement of tsRNAs in various biological processes. Besides, external stress environments such as nutrient starvation, heat shock, hypoxia, oxidative stress, UV-irradiation, toxicity, ischemia, and acute injury, induce tiRNAs generation [1,3]. However, studies have also detected the generation of ti-RNAs in situations without external environmental stimuli.
The mechanisms underlying tsRNAs generation warrant further investigation. However, it's evident that the generation of tsRNAs is highly diverse. A single enzyme, such as Dicer, can be an indispensable step in this process, yet it might also be an overlooked aspect. Notably, some of reported function in key enzymes are associated with cancerization. The secretion of Angiogenin is typically associated with hypoxic conditions in cancer cells and is considered a critical factor in stress response. Elac2, a prostate cancer susceptibility gene, might participate in the generation of tRF-1.
Functions
Unlike "junk," tsRNAs not only exhibit a rich array of generation methods but also have substantial reports showcasing their significant functional roles. tsRNAs play a role in various cellular activities such as proliferation, differentiation, migration, cell cycle, and apoptosis. tsRNAs participate in different stages of the central dogma, including DNA transcription to RNA, post-transcriptional RNA modifications, as well as protein translation processes, and exert their functions in different forms throughout these processes.
tsRNAs respond to DNA damage [12]. They can modulate gene expression by directly binding to coding sequence (CDS) regions, Untranslated Regions (UTRs), akin to microRNAs by binding to Argonautes, or akin to piRNAs by binding to PiwiL2 protein, ultimately affecting gene expression [9,13]. tsRNAs can also influence the translation process, globally inhibiting it, affecting translation activation phenomena, or directly binding to ribosomes to impact protein expression [1,3]. Besides, they can affect RNA stability, mediate epigenetics, participate in transposition and retro transposition phenomena, and interfere with viral replication [14] and reverse transcription processes [7].
Moreover, expression profile variations of tsRNAs have been observed across various human tumors such as laryngeal squamous cell carcinoma, breast cancer, hepatocellular carcinoma, pancreatic cancer, papillary thyroid carcinoma, lung cancer, gastric cancer, ovarian cancer, colorectal cancer, chronic lymphocytic leukemia, oral squamous cell carcinoma, bladder cancer, prostate cancer, leukemia, renal cell carcinoma, among others [3,7,8]. These abnormal expression patterns are closely associated with disease progression. We hypothesized that different microenvironments would exist in cell under different disease states, leading to the diversity of tsRNAs generation modes in different cancer type.
Potential biomarker
Studies conducted on serum, plasma, cancer cells, and tissues of liver cancer patients have identified significantly aberrant tsRNA expression profiles compared to control groups. This highlights the enormous potential of tsRNAs in tumor diagnosis.
Zhan et al. observed abnormal expression of free serum tsRNAs in the serum of liver cancer patients, analysing the serum tsRNA expression profiles of liver cancer patients and healthy controls [14]. Among the notably differential expressions, the mitochondrial tsRNA tRF-Gln-TTG-006 exhibited potential as a predictive biomarker for liver cancer diagnosis. Furthermore, tRF-Gln-TTG-006 was found to exert regulatory functions during liver cancer development. They postulated that liver cancer cells might release tRF-Gln-TTG-006 into the serum to reduce its cellular damage, thereby promoting tumor progression.
Zhu et al. detected a significant abundance of tsRNAs in extracellular vesicles from the blood plasma of liver cancer patients, observing elevated levels of tRNA-ValTAC-3, tRNA-GlyTCC-5, tRNA-ValAAC-5, and tRNA-GluCTC-5 compared to healthy controls, suggesting the potential of these tsRNAs as diagnostic biomarkers for liver cancer [15]. Gly-tRF demonstrated high expression in hepatocellular carcinoma patients [16].
Zuo et al. research supported tsRNAs as novel diagnostic and prognostic biomarkers for liver cancer, significantly enhancing the overall survival prediction of liver cancer patients [17]. tRF-40-EFOK8YR951K36D26, tRF-34-QNR8VP94FQFY1Q, tRF-32-79MP9P9NH57SJ, tRF-31-87R8WP9N1EWJ0, were significantly increase in plasma exosome of HCC patient they can function as diagnostic biomarker [4]. These findings collectively suggest that tsRNAs might serve as standalone diagnostic biomarkers for liver cancer [18].
Moreover, microRNAs are usually considered the most abundant non-coding small RNAs in exosomes. With advancements in high-throughput sequencing techniques such as Pandora sequencing [19], the issue of undetectability of tsRNAs due to their secondary structures during sequencing has been overcome.
Extensive research reveals higher tsRNA content in exosomes during liver cancer and other disease states compared to microRNAs [20]. However, studies also report the highest levels of microRNAs among ncRNAs within exosomes. This discrepancy could be attributed to specific disease states, types, stages, and individual patient characteristics.
tsRNAs in liver cancer
The liver is rich in blood vessels and performs functions related to blood filtration and detoxification. This exposes the liver to high levels of exogenous substances and a diverse range of external stimuli, further increasing the likelihood of inducing tsRNA generation (Figure 2). tRFs account 62% of total reads in Huh7 cell line by deep-sequencing analysis of smRNAs [21]. Additionally, the liver serves as the body's largest metabolic organ, housing a variety of tRNAs and ribonucleases. In instances of liver damage, the metabolic rate of tRNA increases, consequently elevating the probability of tRNA cleavage by ribonucleases, leading to the production of numerous tsRNAs [13].
Figure 2: Schematic diagram of how tsRNAs functions. Existing reports have shown that tsRNA can function by directly binding to ribosomes, affecting DNA structure, directly binding to protein receptors, or encased in extracellular vesicles.
The role in pre-tumor microenvironment construction for liver cancer development
One of the requisites for liver cancer development is the presence of an acute/chronic inflammatory environment. This inflammatory milieu plays a crucial role in inducing liver cancer, as it introduces various stress factors such as oxidative stress, heat/cold shock, ultraviolet irradiation, and endonucleolytic cleavage of tRNA. These stress inducers are also contributory factors in the generation of tsRNAs. Liver-related diseases create multiple stressors within the hepatic environment, serving as precursors for the eventual evolution into cancer. Researchers have detected the significant role played by tsRNAs in some liver-related conditions. Wu et al. identified that under alcoholic liver conditions, downstream products of C3 activation regulate the expression levels of tsRNA (Gly-tRF), thereby promoting hepatosteatosis [22]. Additionally, the use of Gly-tRF inhibitors significantly alleviated disease progression. tRF-Gln-CTG-026 affects the association between TSR1 (pre-rRNA-processing protein TSR1 homolog) and pre-40S ribosomes, inhibits global protein synthesis, thus mitigating liver injury [23]. Those studies highlight the crucial role of tsRNAs in liver injury diseases and underscore the potential value of tsRNAs as a developing therapeutic target. More experimental validation and verification in human samples are necessary to deepen our understanding of tsRNA involvement in liver injury research.
Notably, there remain many unknowns in the process of liver-related diseases transitioning into cancer. Is the progression of chronic inflammation an accumulation process leading to a threshold event, or is it the result of one or multiple key gene regulations? This pre-oncogenic environment holds significant potential to induce tsRNA generation, but why and how tsRNAs are generation? Are there any specific roles that tsRNAs might play in facilitating this disease transition process warrants further investigation?
Functions in liver cancer progression
Studies conducted on animal models and human samples have reported the involvement of tsRNAs in various pathways contributing to liver tumorigenesis and progression.
Kim et al. reported that significantly elevated expression of the 22nt LeuCAG3‘tsRNA in the livers of HCC mice compared to those of normal mice [9]. In vitro cellular models and patient-derived xenograft (PDX) HCC tumor models have demonstrated that inhibiting LeuCAG3‘tsRNA induces apoptosis in tumor cells. This study suggested that LeuCAG3‘tsRNA directly interacts with RPS28 and RPS15, influencing mRNA translation, consequently leading to the loss of 18S rRNA maturation and inducing apoptosis in tumor cells. Zhou et al. reported that overexpression of 5′-tiRNA-Gln in HCC cells impaired the proliferation, migration, and invasion both in vitro and in vivo [16].
Conversely, knockdown of 5′-tiRNA-Gln resulted in contrasting outcomes. 5′-tiRNA-Gln binds eukaryotic initiation factor 4A-I (EIF4A1), which unwinds complex RNA secondary structures during translation initiation, causing the partial inhibition of translation. Gly-tRF shows significantly increased expression in HCC tissues compared to adjacent tissues [24]. Gly-tRF negatively regulates the expression of NDFIP2 mRNA by binding to it 3’UTR, activating the AKT signaling pathway, promoting Epithelial-Mesenchymal Transition (EMT), and inducing liver cancer cells to acquire Liver Cancer Stem Cell (LCSC)-like properties, thereby facilitating HCC progression [24]. Liu et al. found that high expression level of 5’tRF-Gly in HCC patient positively correlate with tumor size and metastasis [23]. They further confirmed that 5’tRF-Gly directly target carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), which is a tumor suppressor, to promote tumor progression [25].
Research regarding the functional role of tsRNAs in the liver is still in need of further in-depth exploration and expansion. Considering the existing research foundation and the characteristics of tsRNAs, their pivotal role in liver cancer holds immense potential. This holds promise for providing new perspectives and considerations for the treatment of liver cancer.
Analogy discussion of potential functions of tsRNA in hepatocellular carcinoma microRNA
Prior to the emergence of tsRNA research, numerous studies had already reported the relationship between various ncRNAs and liver cancer. Among them, microRNA represents the most extensively studied and comprehensive type of ncRNA. Interestingly, tsRNA shares several commonalities with microRNA. Both share similar lengths, possess non-coding characteristics, and can form complexes with AGO proteins to exert gene-regulatory functions. They target and inhibit the same mRNA to exert regulatory effects and can be found in stable, abundant quantities in various bodily fluids and tissues, such as blood and urine [3,8]. Moreover, it's noteworthy that tsRNAs exhibit a more diverse range of functional mechanisms compared to microRNAs.
MicroRNA research in live cancer
We would like to briefly describe the tight connection between microRNA and liver cancer. Certainly, in the realm of microRNA and liver cancer research, several specific microRNAs have emerged as potential diagnostic biomarkers for HCC. For instance, miR-21, miR-122, and miR-145 have been extensively studied and found to exhibit abnormal expression patterns in individuals with liver cancer [1,26]. The dysregulation of circulating or exosomal microRNAs correlates closely with the development and progression of HCC, thus holding promise as diagnostic indicators [27]. microRNAs play their function in almost all phrases of liver cancer progression. For example, microRNA-15a/16-1 disrupting the communication between kupffer cells and regulatory T cells to prevents HCC [28]. microRNA-26amicroRNA-140-5p, both can suppresses tumor growth and metastasis of human HCC [1,29,30].
tsRNAs, microRNAs, liver cancer
Zuo et al. reported that ts-N84, ts-N102, and ts-N22 exhibit a high correlation with microRNAs in HCC development. ts-N84 shows a strong correlation with multiple microRNAs, with hsa-mir-210 being the most prominent [17]. hsa-mir-210 promotes venous metastasis in HCC and is involved in hepatic ischemic reperfusion injury as part of a negative feedback loop with mothers against decapentaplegic homolog 4 gene(SMAD4) [31,32]. ts-N102 works as proto-oncogene, inhibiting the expression of tumor suppressor has-mir-215 [18]. Elevated expression of ts-N22 in liver cancer patients is associated with higher survival rates. ts-N22 displays a significant correlation with tumor suppressors has-mir-331 and has-mir33a [18,33-35].
Furthermore, tsRNAs and microRNAs are capable of binding to the same mRNA, potentially competing or collaborating at shared sites in disease states. tRF/miR-1280 suppresses cancer stem cells by directly targeting Gata1/3 and miR-200b genes [8]. MicroRNAs originate from mRNA while tsRNAs derive from tRNA; both are cleavage products that do not affect the basic functions of their sources. They both play crucial regulatory roles in maintaining biological homeostasis and influencing disease states. Maybe the relationship between microRNA and tsRNA might parallel the cooperative effects observed between mRNA and tRNA, jointly contributing to specific biological functions.
Regarding the relationship between tsRNAs and miRNAs, research suggests their potential interaction within the cellular regulatory network. Evidence indicates that some tsRNAs may compete with miRNAs for the same binding sites on AGO proteins, thus influencing miRNA functionality [34]. Additionally, they may participate in gene expression regulation through common pathways. Although the relationship between tsRNAs and miRNAs is under investigation, their interactions within cells likely have intricate effects on gene expression and cellular functions. Presently, the scientific community is striving to deepen our understanding of the interplay between these two small RNAs and their exact roles within the cellular regulatory network. In HCC, the connection between these two entities mediates various processes, including the onset, progression, metastasis, and immune evasion of liver cancer. Thus, we hypothesized the role of tsRNA-microRNA network in liver cancer. By integrating these two molecular entities, could we discover a more precise diagnostic biomarker?
Targeting the potential of tsRNA therapy for liver cancer
Non-coding RNA therapy is undergoing vigorous development, employing various non-coding RNAs like siRNA and microRNA for in vivo delivery to treat multiple diseases. tsRNAs can be generated during the translation process to adjust the ribosomal homeostasis and alter their generation patterns in the pathological environment of cancer [35]. Therefore, these small RNAs hold promise as a novel therapeutic target for cancer. In the translation process, tsRNAs bind to the ribosomal protein S28 mRNA complementary sequence, playing a crucial auxiliary role. Studies report that systemic delivery of specific anti-tsRNA oligonucleotides (a sequence identical in humans and mice) into mice does not induce liver damage in normal mice. However, in mice with liver cancer, injecting the anti-tsRNA sequence induces apoptosis in liver cancer cells. This indicates that tsRNAs could serve as a new pathway in post-transcriptional gene regulation, offering a novel target and treatment option for liver cancer. tRF-LeuCAG-3 can work as potential clinical therapeutic target. Liu et al. employed bioinformatics algorithms to establish a tsRNA expression profile of tumor patients compared to healthy groups, encompassing 15 types of 3' tsRNAs [26]. These tsRNAs exhibited aberrant expression across nine human cancers, suggesting their pivotal role in tumor development by influencing cellular protein synthesis capability. Through various mechanisms, tsRNAs play critical roles in diseases, impacting disease progression. Targeting tsRNAs shows therapeutic potential, indicating the development prospects and future advantages of tsRNAs as a novel therapeutic target [35].
Several microRNA-based therapeutic drugs for treating liver cancer have entered various stages of clinical trials. Some notable candidates include MRX34, a liposomal miR-34 mimic developed by mirna therapeutics (now synlogic) targeting multiple cancers, including liver cancer. MRX34 reached phase I clinical trials but faced temporary setbacks due to immune-related adverse events.
We mentioned the concept of micro-tsRNA network in the previous article. We hypothesized that tsRNA and microRNA share a common binding site based on their similarity in characteristics, and they can perform opposite functions through competition. Therefore, by developing tsRNA-microRNA co-collaborating drugs, it may be possible to slow down the severe immune response in patients when using micro drugs alone. Strategies targeting these microRNAs mainly involve the use of microRNA mimics and inhibitors. MicroRNA mimics can be designed to restore or enhance the function of dysregulated microRNAs in liver cancer, thereby inhibiting tumor growth and metastasis.
Conversely, microRNA inhibitors aim to suppress oncogenic microRNAs associated with HCC to impede tumor progression. Combining the similarity between microRNAs and tsRNAs, the process of microRNA drugs development can provide a reliable reference for the development of tsRNA drugs or therapy strategy in liver cancer.
Presently, liver cancer therapeutic agents based on tsRNA are in various stages of development. The effect can mainly observe in animal experiments. Further clinical research is necessary to validate their safety and efficacy in humans. The progress of these studies is of paramount importance for deeper exploration and application of tsRNAs as diagnostic markers and therapeutic targets in HCC.