Identification of aberrant tRNA-halves expression patterns in clear cell renal cell carcinoma

Abstract

Small non-coding RNAs (sncRNA; <200 nt) regulate various cellular processes and modify gene expression. Under nutritional, biological or physiochemical stress some mature sncRNAs (e.g. tRNAs) are cleaved into halves (30-50 nt) and smaller fragments (18-22 nt); the significance and functional role of these tRNA fragments is unknown, but their existence has been linked to carcinogenesis. We used small RNA sequencing to determine the expression of sncRNAs. Subsequently the findings were validated for miR-122-5p, miR-142-3p and 5'tRNA4-Val-AAC using qPCR. We identified differential expression of 132 miRNAs (upregulated: 61, downregulated: 71) and 32 tRNAs (upregulated: 13, downregulated: 19). Read length analysis showed that miRNAs mapped in the 20-24 nt fraction, whereas tRNA reads mapped in the 30-36 nt fraction instead the expected size of 73-95 nt thereby indicating cleavage of tRNAs. Overexpression of miR-122-5p and miR-142-3p as well as downregulation of 5'tRNA4-Val-AAC was validated in an independent cohort of 118 ccRCC and 74 normal renal tissues. Furthermore, staging and grading was inversely correlated with the 5'tRNA4-Val-AAC expression. Serum levels of miR-122-5p, miR-142-3p and 5'tRNA4-Val-AAC did not differ in ccRCC and control subjects. In conclusion, 5' cleavage of tRNAs occurs in ccRCC, but the exact functional implication of tRNA-halve deregulation remains to be clarified.

Figure 1. miRNA expression profiles discriminate normal and ccRCC tissue.

(A) A multi dimensional scaling plot demonstrates accurate classification of 18 corresponding normal (green dots) and ccRCC (pink dots) tissue samples based on the miRNA expression profile. Distances between samples are corresponding to leading log2-fold changes between each pair of RNA samples. The leading log-fold-change is the average of the largest absolute log-fold-changes between the corresponding samples. The volcano plots are showing the expression of miRNA (B) and tRNA (C) in normal and ccRCC tissue. miRNAs/tRNAs with an at least 2-fold significant expression difference are indicated with blue dots.

Figure 2. Read length distributions demonstrate the presence of tRNA cleavage.

Read length distributions indicated the presence of two major peaks: a green peak indicates the enrichment of miRNA in the typical 20–24 nt fraction; a pink peak at 30–35 nt indicates the presence of 5′tRNA-halves. Exemplarily, the read length distribution of one corresponding pair of normal renal (A) and ccRCC (B) tissue is shown. It is important to note that the distribution of the read counts vary between the different corresponding samples. (C) the structure of tRNA4-Val-AAC is shown; it may be cleaved at the anticodon loop, resulting in 5′tRNA-halves of 30 to 35 nt, tRNA covariance model fold borrowed from Chan, P.P. & Lowe, T.M. (2009) GtRNAdb: A database of transfer RNA genes detected in genomic sequence. Nucl. Acids Res. 37(Database issue):D93-D97.

Figure 3. Validation of sncRNA expression using quantitative real-time PCR.

The expression levels of 5′tRNA4-Val-AAC, miR-122-5p and miR-142-3p were different in ccRCC (n = 118) compared to normal (n = 74) renal tissue.

Figure 4. 5′tRNA4-Val-AAC expression is associated with adverse pathology in ccRCC.

Correlation of tissue small non-coding RNA levels with clinical-pathological parameters: (A) The expression of miR-122-5p was inversely correlated with M1-stage (p = 0.006), and the expression of 5′tRNA4-Val-AAC was inversely correlated with (B) less differentiated (p = 0.002) and (C) advanced (p = 0.001) ccRCC.

Figure 5. Analysis of sncRNA expression in serum.

The expression levels of (A) 5′tRNA4-Val-AAC, (B) miR-122-5p and (C) miR-142-3p in serum samples are similar in ccRCC patients (n = 30) healthy control subjects (n = 15).