In this study, we reported the expression profiles of tsRNAs in the rat hippocampus after nicotine exposure for the first time. The sequencing data revealed that there were 26 differentially expressed tsRNAs, involving 7 up-regulated and 19 down-regulated tsRNAs, in the rat hippocampus after nicotine exposure. Subsequently, 8 DEtsRNAs were chosen to validate by RT-qPCR and among them, 5 candidate DEtsRNAs for further bioinformatic predictions. The GO and KEGG pathway analysis demonstrated that the potential targeted genes and the putative pathways of the 5 DEtsRNAs mainly focused on several critical signaling channels, which played important roles in nicotine’ actions. Taken together, our findings suggest that these dysregulated tsRNAs may play regulatory roles on how nicotine functions.
There is a consensus that nicotine exposure can induce changes in pain perception and body weight in animal models and human studies[26-28]. Recent research indicated that nAChRs agonists (such as nicotine, epibatidine, choline) might make up a novel class of analgesics for pain management[29-31]. In our previous research, we found that short-term use of nicotine could produce analgesic effects, but long-term use or withdrawal led to hypersensitivity, and further investigation indicated that nicotine could alter pain sensitivity by affecting the expression of the pain related factor. In this study, when the rats were continuously injected with nicotine, the paw withdrawal threshold increased and the body weight gains decreased, which was in accordance with our previous study.
Nicotine exerts its functions largely through the widespread nAChRs. Substantial researches suggested that nicotine exposure could upregulate nAChRs by multiple processes, including changes in receptor assembly, trafficking, and degradation[32-34]. In recent years, genetic factors is becoming the research focus about nAChRs, and variants in the CHRNA5-CHRNA3-CHRNB4 gene cluster are the most studied associated with nicotine’ actions[35, 36]. Besides, Cameli et al pointed out that genetic variations in CHRNA7 and CHRFAM7A were related to nicotine addiction.In our study, we found that tRF-5c-Glu-CTC-1, tRF-5c-Glu-CTC-3, tRF-5c-Glu-TTC-4 might be involved in the cholinergic synapse pathway, and in turn influence the excitability of neurons and glial cells. Further analysis indicated that the putative genes of these above tsRNAs included CHRM1, CHRM3, CHRNA3, CHRNA4 and CHRNA7, which encoded variant subunits of nAChRs. So we presumed that tRF-5c-Glu-CTC-1, tRF-5c-Glu-CTC-3, tRF-5c-Glu-TTC-4 might function in nicotine’ actions by modifying the genes expression of nAChRs’ subunits, thus influencing the structure and activity of nAChRs.
In addition to nAChRs, there exited some other possible mechanisms involved in nicotine’ actions. On the one hand, nicotine exposure can produce widespread neuroadaptations in nervous system, including dopaminergic (DA) synapses, GABAergic circuitry, glutamatergic synapses, and so on. On the other hand, these neuroadaptations were in turn to participate in the nicotine’ actions[37-39]. Recent studies shown that genetic variations in DA, GABA could regulate nicotine’ actions. For instance, Bühler and his colleagues summarized that apart from nAChRs genes, variations in ANKK1, DRD2 and GABA were likewise associated with nicotine-related phenotypes. Liu et al constructed the networks of candidate genes associated with nicotine addiction, and concluded that cholinergic receptors (CHRNA1, CHRNA4, CHRNA7), dopamine receptors (DRD1, DRD2, DRD3) and GABA receptors (GABRA1, GABRA2, GABRA4) were involved in diverse biological functions. Other researches about genetic modulations in DA (COMT, GCH1, and DRD2) and GABA indicated these changes could affect the modulation of DA in pain pathway[41, 42]. In our study, we predicted the target genes of these five candidate tsRNAs, and found that tRF-5c-Glu-CTC-1 might regulate the expressions of COMT, DRD2, GABRA4, whereas tRF-5c-Glu-CTC-3 might regulate DA related genes COMT and DRD2. The KEGG pathway analysis indicated that tRF-5c-Glu-CTC-1 probably participated in the dopaminergic synapse, GABAergic synapses and glutamatergic synapses pathways, while tRF-5c-Glu-CTC-3 might merely modulate the dopaminergic synapse and glutamatergic synapses pathways, not GABAergic synapses.
Apart from the above neurotransmitters, previous researches suggested that nicotine shared the similar functional molecules, signal paths and acting sites with some other addictive drug, such as cocaine, morphine[11, 43, 44]. We found in our study that the candidate tsRNAs could modulate the pathways of cocaine, morphine and amphetamine addiction. To be specific, tRF-5c-Glu-CTC-1 took part in all the three paths of cocaine, morphine and amphetamine addiction, tRF-1-T28-His-GTG-1 and tRF-5c-Glu-CTC-3 took part in cocaine addiction, and tRF-5c-Gly-GCC-2-M2 in morphine addiction. Hence, we speculated that those candidate tsRNAs might likely contribute to nicotine addiction in a similar way. More researches are needed.
According to the KEGG pathway analysis, we also discovered that except the above pathways, the putative genes of the five candidate tsRNAs were also enriched in some other critical paths, such as MAPK signaling pathway, mTOR signaling pathway, neurotrophin signaling pathway, which have been suggested to involve nicotine’ actions in body[45-47]. And further analysis indicated that the majority of the putative pathways of tRF-5c-Glu-CTC-1 were associated with the previously known mechanisms of nicotine’ actions, so we regard tRF-5c-Glu-CTC-1 as the most promising candidate for further study in nicotine’ actions.
Generally, tsRNAs with lengths of 18–40 nucleotides, include two main types based on the length and cleavage sites on tRNA or pre-tRNA: tRNA-derived fragments (tRFs) and tRNA-derived, stress-induced RNAs (tiRNAs). tiRNAs are usually the products of angiogenin cleavage of mature tRNAs at the anticodon site during stress, which contain two subtypes tiRNA-5 and tiRNA-3. While tRFs derive from cleavage on any sites of mature or pre-tRNAs, which contain four subtypes tRF-5, tRF-3, tRF-1, tRF-2[14, 15, 48]. Growing evidence indicated that the functions of tsRNAs depended on their subtype and specific subcellular localization. Some researchers stated that cytosolic and mitochondrial tiRNAs could repress protein translations and be associated with apoptosis initiation[49, 50]. Kumar et al remarked that in HeLa cell line, tRF-5s are mostly nuclear while tRF-3s and -1s are cytoplasmic, and further study in human HEK 293 cells suggested that tRF-5s and tRF-3s are associated with Argonautes 1, 3 and 4, and then target mRNAs in a manner similar to miRNAs. Zhang and his colleagues reported that in monocytes/dendritic Cells, td-piR(Glu) (tRF-5) could interact with PIWI protein and play a role in regulation of chromatin remodeling in somatic cells, just like piRNAs. According to the known literature of tsRNAs as well as our data, the most promising candidate tRF-5c-Glu-CTC-1 belonged to tRF-5, so we postulated that it’s likely that tRF-5c-Glu-CTC-1 might function by interacting with Ago proteins or PIWI protein, and have post-transcriptional regulations similarly to miRNAs or epigenetic modulations like piRNAs. In nicotine exposure model, nicotine exposure could upregulate the expression of nAChRs and downregulate tRF-5c-Glu-CTC-1 whose downstream genes CHRM1, CHRM3, CHRNA4 and CHRNA7 could encode the subunits of nAChRs shown in our data. Given the negative regulatory relationship between tRF-5c-Glu-CTC-1 and its putative genes, we conjecture that nicotine exposure may reduce the biosynthesis of tRF-5c-Glu-CTC-1, then alleviate its suppression on the genes expression of nAChRs subunits, and thereby upregulate nAChRs, which lay the foundation for nicotine’ actions. Further research is needed in future.
There are some limitations about our study. First, we only detect the profiles of tsRNAs on nicotine exposure condition, not nicotine withdrawal, and lack the dynamic detection of tsRNAs at different time point. Secondly, our research is preliminary and further functional research is needed to identify the specific locations and functions of the candidate tsRNAs in vitro and in vivo. Finally, detection of tsRNAs in human trial is hopefully to conduct in future.
To sum up, our study provided the differentially expressed profiles of tsRNAs in rat hippocampus after nicotine exposure for the first time. Further bioinformation analysis revealed that the tsRNAs might be a new class of regulatory molecules in nicotine related researches, which might function by regulating some important signaling pathways, such as the cholinergic synapse, dopaminergic synapse. Among them, tRF-5c-Glu-CTC-1 is the most promising one, and probably act in a manner of miRNAs or piRNAs. In a word, our study paves a new road for research into nicotine’ actions, especially in addiction and nicotine-mediated analgesia, and provides novel modulatory targets for this area.