Translated Long Non-Coding Ribonucleic Acid ZFAS1 Promotes Cancer Cell Migration by Elevating Reactive Oxygen Species Production in Hepatocellular Carcinoma

Abstract

Micropeptides (≤100 amino acids) are essential regulators of physiological and pathological processes, which can be encoded by small open reading frames (smORFs) derived from long non-coding RNAs (lncRNAs). Recently, lncRNA-encoded micropeptides have been shown to have essential roles in tumorigenesis. Since translated smORF identification remains technically challenging, little is known of their pathological functions in cancer. Therefore, we created classifiers to identify translated smORFs derived from lncRNAs based on ribosome-protected fragment sequencing and machine learning methods. In total, 537 putative translated smORFs were identified and the coding potential of five smORFs was experimentally validated via green fluorescent protein-tagged protein generation and mass spectrometry. After analyzing 11 lncRNA expression profiles of seven cancer types, we identified one validated translated lncRNA, ZFAS1, which was significantly up-regulated in hepatocellular carcinoma (HCC). Functional studies revealed that ZFAS1 can promote cancer cell migration by elevating intracellular reactive oxygen species production by inhibiting nicotinamide adenine dinucleotide dehydrogenase expression, indicating that translated ZFAS1 may be an essential oncogene in the progression of HCC. In this study, we systematically identified translated smORFs derived from lncRNAs and explored their potential pathological functions in cancer to improve our comprehensive understanding of the building blocks of living systems.

Keywords: ZFAS1; hepatocellular carcinoma; reactive oxygen species; ribosome-protected fragment sequencing; translated small open reading frames.

Figure 1

Systematic overview of translated small open reading frame (smORF) prediction. Two sequencing of ribosome-protected mRNA fragments (RPF-Seq) originated from U2OS and HeLa cells were applied to respectively calculate three ribosome features of the positive and negative datasets. Three features of each RPF-Seq combined with one of machine learning models could create one classifier. As four classification models were used, four classifiers were developed to predict the translated smORFs in each RPF-Seq dataset. RPFC, ribosome-protected mRNA fragments coverage; ORFS, ORF score; RRS, ribosome release score.

Figure 2

Features of predicted translated small open reading frames (smORFs) based on U2OS sequencing of ribosome-protected mRNA fragments (RPF-Seq). (A) The performance of the four classifiers based on logistic regression (LR), linear discriminant analysis (LDA), support vector machine (SVM), and random forest models (RF). (B) Ribosome features of different ORFs. The ribosome release score and ORF score values of putative translated smORFs were similar, and were higher than ORFs derived from untranslated regions (UTRs) and long non-coding RNAs (lncRNAs) but lower than annotated ORFs of protein-coding genes. The ribosome-protected mRNA fragment coverage scores of translated smORFs and protein-coding genes were similar, but their distributions differed substantially from UTRs and lncRNAs. (C) Length distribution of micropeptides encoded by putative translated smORFs. ACC, accuracy; SEN, sensitivity; SPE, specificity; RPFC, ribosome-protected mRNA fragments coverage; ORFS, ORF score; RRS ribosome release score.

Figure 3

Constructs generated to validate the protein-coding potential of small open reading frames. The 5′UTR-ORFs of glyceraldehyde 3-phosphate dehydrogenase, ZFAS1, SNHG8, and RP11-879F14.2 were fused to a GFPmut in which the green fluorescent protein start codon ATGGTG was mutated to ATTGTT. Substantial amounts of fusion protein were detected following transfection of the constructs into SK-Hep1 cells. In addition, the fusion protein was abolished after SK-Hep1 cells were transfected with the GFPmut construct.

Figure 4

Expression profiles of ZFAS1 in multiple tumor and normal tissues. (A) ZFAS1 expression levels in 11 cancer-related long non-coding RNA microarray datasets derived from seven cancer types. Y-axis means the signals detected by the microarray. The red asterisk means that the false positive rate (FDR) is less than 0.1. (B) Relative expression levels of ZFAS1 in 32 pairs of hepatocellular carcinoma tumor tissue and corresponding adjacent normal tissue detected by reverse transcription quantitative polymerase chain reaction. ZFAS1 was significantly expressed in tumor tissues (p-value = 1.6e−05, paired Wilcoxon rank sum test, n = 32). (C) Expression profiles of ZFAS1 in 25 normal tissues downloaded from Genotype-Tissue xpression. ZFAS1 was nearly unexpressed in normal liver tissue. N and T represent adjacent normal tissues and tumor tissues. ***P-value < 0.001.

Figure 5

Cell motility changed significantly following ZFAS1 overexpression and knockdown. (A) SK-Hep1 cells transfected with ZFAS1 expression plasmid. The cell motility of SK-Hep1 cells was significantly increased following transfection with the ZFAS1 expression plasmid (p-value = 9e−04, paired Student’s t-test, n = 3). (B) SK-Hep1 cells transfected with small interfering RNAs (siRNAs) of ZFAS1. The cell motility of SK-Hep1 cells decreased significantly following transfection with the two independent siRNAs (siGFP vs. siZFAS1-1: P-value = 7e−04, siGFP vs. siZFAS1-2: P-value = 3.2e−03, paired Student’s t-test, n = 3). Imax means cells exposed to Lipofectamine RNAiMAX but not RNA duplexes. siGFP indicates cells transfected with siRNA of green fluorescent protein. **P-value < 0.01, ***P-value < 0.001.

Figure 6

Change in reactive oxygen species (ROS) production following ZFAS1 knockdown. Cellular ROS production was significantly downregulated following transfection of SK-Hep1 cells with two independent siRNAs (siGFP vs. siZFAS1-1: P-value = 0.022, siGFP vs. siZFAS1-2: P-value = 0.025, paired Student’s t-test, n = 3). Imax means cells exposed to Lipofectamine RNAiMAX but not RNA duplexes. siGFP indicates cells transfected with small interfering RNAs of green fluorescent protein. *P-value < 0.05.