tRF-Leu-CAG promotes cell proliferation and cell cycle in non-small cell lung cancer

Abstract

tRNA-derived RNA fragments (tRFs), non-coding single-stranded RNAs with 14-35 nt in length, were found to play important roles in gene regulation, even in carcinogenesis. In this study, we investigated the expression of tRF-Leu-CAG in human non-small cell lung cancer (NSCLC) and its function in the cell proliferation and cell cycle of NSCLC. The expression level of tRF-Leu-CAG was detected in NSCLC tissues, cell lines, and sera. tRF-Leu-CAG RNA levels were higher in NSCLC tumor tissues than in normal tissues, and also upregulated in NSCLC cell lines. A significant relationship was observed between stage progression and tRF-Leu-CAG in NSCLC sera. We found that in H1299 cells, inhibition of tRF-Leu-CAG suppressed cell proliferation and impeded cell cycle. AURKA was also repressed with the knockdown of tRF-Leu-CAG. Thus, our study revealed that tRF-Leu-CAG may be involved in regulating AURKA and could be a new diagnostic marker and potential therapeutic target in NSCLC.

Keywords: NSCLC; cell cycle; diagnostic biomarker; proliferation; tRF-Leu-CAG.

Figure 1

The reads of tRNA‐Leu‐CAG‐1 and tRNA‐Leu‐CAG‐2 are different in NSCLC tumor tissues and matched normal tissues. (a) The length of tRFs in NSCLC tumor tissues and matched normal tissues mainly ranged from 18 to 24 nt. (b) As two of the most prevalent tRNAs, tRNA‐Leu and tRNA‐Val occupied 37% and 26% of the 27 kinds of tRNAs. (c) The reads of tRNA‐Leu‐CAG‐1 were unregulated in NSCLC tumor tissues as compared with corresponding non‐tumor tissues. (d) The reads of tRNA‐Leu‐CAG‐2 were unregulated in NSCLC tumor tissues as compared with corresponding non‐tumor tissues. ***p < .001 [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

tRF‐Leu‐CAG is a kind of tRNA halves. (a) The product of qRT‐PCR (10 μl) was run on 2% agarose gel, stained with GoldView, and visualized under UV illumination. (b) and (c) tRF‐Leu‐CAG was derived from 5′ ends of tRNA‐Leu‐CAG‐1 and tRNA‐Leu‐CAG‐2 with the length of 34 nt. The cleavage site is located on the anticodon loop [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

tRF‐Leu‐CAG is upregulated in NSCLC tissues, cell lines and sera. (a) The relative expression level of tRF‐Leu‐CAG in corresponding non‐tumor lung tissues (NT) and tumor tissues (T) as obtained by qRT‐PCR. U6 was used for normalization. (b) The relative expression level of tRF‐Leu‐CAG in NSCLC cell lines or a pulmonary epithelial cell line (control), as measured by qRT‐PCR. (c) The relative expression level of tRF‐Leu‐CAG in NSCLC sera as obtained from qRT‐PCR. U6 was used for normalization. (d) According to the analysis of ROC curve, tRF‐Leu‐CAG could be a potential diagnostic marker in stage IV NSCLC cases. *p < .05, **p < .01, ***p < .001 [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4

tRF‐Leu‐CAG can promote cell proliferation and cell cycle progression in NSCLC cell lines. (a) The expression of tRF‐Leu‐CAG was measured by qRT‐PCR in H1299 cells 48 hr after transfection with negative control (NCi) and tRF‐Leu‐CAG inhibitor (tRFi). tRF‐Leu‐CAG was suppressed by tRF‐Leu‐CAG inhibitor. (b) H1299 cells were transfected with NC or tRF‐Leu‐CAG inhibitor, and cell proliferation was determined by CCK‐8. (c‐d) The cell cycle distributions of H1299 cells transfected with NC or tRF‐Leu‐CAG inhibitor as detected by flow cytometry. *p < .05, **p < .01, ***p < .001 [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5

The protein level of AURKA is downregulated in H1299 cells transfected with tRF‐Leu‐CAG inhibitor. (a) The representative images of Western blot analyses for protein levels of several target genes. The protein level of AURKA was quantified by densitometry after normalization to β‐actin. Values are mean ± SEM. **p < .01, ***p < .001. (b) We propose that tRF‐Leu‐CAG plays the role of tumor driver in NSCLC by downregulating AURKA, and may also be closely correlated with other genes related to the cancer process [Colour figure can be viewed at wileyonlinelibrary.com]