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/lp/wolters-kluwer-health/a-66-amino-acid-micro-peptide-encoded-by-long-non-coding-rna-rp11-DnO9hkr46n
- Publisher
- Wolters Kluwer Health
- Copyright
- Copyright © 2022 The Chinese Medical Association, Published by Wolters Kluwer Health, Inc.
- ISSN
- 2577-3585
- eISSN
- 2096-5672
- DOI
- 10.1097/jbr.0000000000000132
- Publisher site
- See Article on Publisher Site
Abstract
Objective: Emerging data have shown that non-coding RNAs (ncRNAs) can encode micro-peptides (≤100 amino acids) that play an important role in regulating physiological and pathological processes. Herein, we explored ncRNAs that may encode micro-peptides that are involved in the development of hepatocellular carcinoma (HCC). Methods: High-throughput sequencing of ribosomal protein S6 (RPS6) was performed in four cancer cell lines using RNA- immunoprecipitation (RIP). UCSC databases obtained the full length of the gene sequences and quantitative polymerase chain reaction (qPCR) was used to evaluate expression levels of ncRNAs of interest. The coding activity of ncRNA was assessed in vitro by co-immunoprecipitation, plasmid transfection, western blot, immunofluorescence and RNA fluorescence in situ hybridization. Mass spectrometry was performed to explore the potential functions of candidate micro-peptide in HCC. This study involving human tissue specimens was conducted in accordance with Declaration of Helsinki and approved by the Institutional Review Board of Changhai Hospital, Naval Military Medical University, China (approval No. CHEC2020-081) on June 6, 2020. Results: We performed RIP assay using primary antibodies for RPS6 and high-throughput sequencing. A total of 223 overlapping genes were captured by RPS6-RIP. Venn diagram analysis revealed that 60 overlapping genes were detected in four cancer cell lines. QRT-PCR showed that six of the candidate genes (RP11-298J20.4, RP11-4O1.2, RP11-119F7.5, RP11-448G15.3, HCP5, RP11-517B11.7) were expressed in Huh7 and Hep3B cells. Further analysis of these six candidate genes and found that five (RP11-298J20.4, RP11-4O1.2, RP11-119F7.5, RP11-448G15.3, RP11-517B11.7) displayed higher expression levels in HCC cell lines (Huh7, Hep3B) and tumor tissues than in liver cell lines (L-02, QSG-7701) and non-tumor tissues, respectively. Performed additional RIP assays and confirmed that four of the genes (RP11-4O1.2, RP11-119F7.5, RP11-448G15.3, RP11- 517B11.7) bound RPS6. We obtained the full length of the four gene sequences from the UCSC database and analyzed the open reading frames by ORF Finder; to determine the translation potential of the four candidate small open reading frames (smORFs), we subcloned a FLAG epitope tag into the C-terminal of the four selected smORFs before the stop codon, and the fusion sequences were then cloned into three different plasmid vectors (pSPT19, pcDNA3.1, and PEGFP-N1). We performed coupled transcription and translation reactions and found that the pSPT19 plasmids encoded small peptides in vitro. After then transfected the pcDNA3.1 constructs into Huh7 cells, and a single 7.2 kDa micro-peptide was encoded from the candidate smORF of RP11.119F7.5. We transfected the recombinant pEGFP-N1 plasmids with smORFs in HCC cells, and western blot analysis revealed a band above GFP in the RP11.119F7.5 recombinant plasmid lane. The coding potential of the RP11-119F7.5 vector was also confirmed by immunofluorescence assay. Fluorescence in situ hybridization assay revealed that RP11-119F7.5 was localized in the cytoplasm and nucleoplasm of HCC cells. Gene ontology enrichment analysis showed that the micro-peptide– interacting proteins were mainly involved in extracellular exosomes. We also found the identified proteins were involved in several biological functions like protein binding, poly(A) RNA binding, translational initiation, and the nuclear-transcribed mRNA catabolic process. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed the peptide-interacting proteins might participate in several critical pathways including ribosome, biosynthesis of amino acids, carbon metabolism, biosynthesis of antibiotics, glycolysis and gluconeogenesis, pathogenic Escherichia coli infection and influenza A. Conclusion: Our study revealed a novel micro-peptide translated by ncRNA RP11-119F7.5, highlighting the coding ability and potential role of ncRNAs in HCC. Keywords: carcinoma, hepatocellular, micro-peptide, ncRNAs, translation, identification Supplemental Digital Content is available for this article. under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download CH and ZL contributed equally to the writing of the article. and share the work provided it is properly cited. The work cannot be Department of Laboratory Diagnostic, Changhai Hospital, Naval Military Medical changed in any way or used commercially without permission from the University, Shanghai, China journal. *Corresponding author: Shanrong Liu, Department of Laboratory Diagnostic, Journal of Bio-X Research (2022) 5:163–170 Changhai Hospital, Naval Military Medical University, Shanghai, China. E-mail: Received: 13 December 2021; Accepted: 6 August 2022 liushanrong01@126.com http://dx.doi.org/10.1097/JBR.0000000000000132 Copyright © 2022 The Chinese Medical Association, Published by Wolters Kluwer Health, Inc. This is an open-access article distributed 163 RESEARCH ARTICLE Journal of Bio-X Research Introduction Human clinical samples Primary liver cancer is one of the most common malignant To compare overlap gene expression differences between cancer tumors in the world. Hepatocellular carcinoma (HCC) is the tissues and adjacent tissues in clinical HCC samples. We col- [1,2] main pathological type of primary liver cancer. Despite the lected the samples of HCC tissue and paired para-cancer tissue continuous development of technology and treatment strategies, (>2 cm from tumor margin) from HCC patients (n=6) under- the successful treatment of liver cancer remains challenging, with going tumor resection in Changhai Hospital, Naval Military obstacles in early detection, metastasis, disease recurrence, and Medical University. All samples were immediately frozen in liq- [3–5] poor prognosis. The overall survival rate for HCC patients uid nitrogen before subsequent analysis. This study followed the [6,7] is only 10% to 25% in most countries. The progression of principles established in the Declaration of Helsinki and was liver cancer is a multi-step process. The evolution from normal approved by the Changhai Internal Review and Ethics Boards at hepatocytes to HCC goes through many pathological stages and Naval Military Medical University (approval No. CHEC2020- involves many molecular events. 081) on June 6, 2020. Informed consent was obtained from all Protein-coding genes account for 2% of the overall human patients. [8] genome sequence. A genome, nevertheless, was transcribed prin- cipally into non-coding RNAs (ncRNAs), which including small Plasmid construction and cell transfection [9–11] nucleolar RNA, microRNAs, circular RNA and lncRNA. It is worth noting that, with the extensive and in-depth studies on To validate the translation potential of candidates, we con- ncRNA in recent years, it has been found that ncRNA plays a structed flag and GFP fusion sequences with smORF by poly- very important role in the maintenance of genome stability, and merase chain reaction (PCR) amplification. The flag fusion could directly participate in the regulation of pathophysiologi- sequences were subcloned into pcDNA3.1 and pSPT19 vec- cal processes as a functional molecule. Long non-coding RNAs tors (Invitrogen, Carlsbad, CA). The GFP fusion sequence was (lncRNAs) are mRNA-like transcripts over 200 nucleotides in subcloned into PEGFP-N1 vector (Invitrogen). Lipofectamine [12,13] length that lack protein-coding ability. LncRNAs are involved 3000 (Invitrogen) was used to transfect the pcDNA3.1 and in the regulation of many biological processes. Multiple studies PEGFP-N1 subcloned plasmids into Huh7 HCC cells in accor- have shown that lncRNAs are also involved in the occurrence and dance with the manufacturer’s protocol. Primer sequences are [14–16] [17–19] development of numerous diseases, including cancer. shown in Additional Table 1, http://links.lww.com/JR9/A42. Notably, in recent years, some research groups have identified sev- eral biologically relevant micro-peptides encoded by small open In vitro translation assay reading frames (smORFs) in lncRNA transcripts from a variety [20] of species. In zebrafish, for example, lncRNA Loc100506013 The smORF-flag fusion subcloned pSPT19 vector was applied encoded a micro-peptide called Toddler functions as an activator to explore the translation ability of smORFs in vitro. Four of APJ/Apelin receptor signaling and promotes gastrula forma- micrograms of subcloned vector was added in a clear tube and [21] tion. In humans, the lncRNA LINC01116-encoded peptide incubated 2 hours at 37°C, then put the tube on ice for 5 min- [22] contributes to neuronal function and disease. Identification of utes to stop the reaction. The production was analyzed using translated smORFs remains technically challenging and therefore SDS-PAGE electrophoresis. the mechanisms and modes of action of these micro-peptides are [23–25] poorly understood. RNA-immunoprecipitation and RIP-sequencing This study aimed to identify a novel micro-peptide translated by ncRNA RP11-119F7 and attempted to predict its role in HCC. Ribosomal protein S6 (RPS6) is a critical component of the 40S ribosomal subunit and interacts with the 5ʹ-m GpppG cap-binding complex to regulate mRNA translation initia- Materials and methods [26,27] tion. To obtain the signature of ribosome-binding RNAs, we performed RNA-immunoprecipitation (RIP) assay using Cell culture and treatments primary antibody for RPS6 (ab70227, Abcam, Cambridge, HCC cell lines Hep3B (HB-8064) and Huh7 (JCRB0403), UK) and high-throughput sequencing in four cancer cell lines human prostate cancer cell line DU145 (HTB-81), human pan- (Hep3B, DU145, HCT116, and SW1990) using the Magna creatic cancer cell line SW1990 (CRL-2172), human colon RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, cancer cell line HCT116 (CCL-247) were purchased from Bedford, MA) following the manufacturer’s protocol. Each cell American Type Culture Collection (ATCC, Manassas, VA). Two lines have input and anti-RPS6 samples for analysis. The cDNA human immortalized hepatic stellate cell lines (QSG7701, L-02) libraries were generated by Biotechnology Company (Shanghai, were purchased from Chinese Academy of Sciences Cell Bank China) using RNA. High-throughput RNA-seq was performed (Shanghai, China). DU145 cells were cultured in RPMI 1640 using an Illumina HiSeq 2500 sequencer (Illumina, San Diego, medium (Gibco, Carlsbad, CA) containing 10% fetal bovine CA). RIP-sequencing (RIP-seq) reads were aligned to the human serum (FBS) and penicillin (100U/mL)/streptomycin (100μg/mL). reference genome version 19 using the TopHat algorithm. For HCT116 cells were cultured in McCoy’s 5A medium (Gibco, expression analysis, densities of genes were determined by CA) containing 10% FBS and penicillin (100U/mL)/streptomy- the value of reads per kb per million reads. We analyzed the cin (100µg/mL). The other cell lines were cultured in DMEM sequencing results sand explore the common and critical RNAs (Hyclone, Logan, UT) containing 10% FBS and penicillin in four different tumor cells by Venn diagram analysis. (100U/mL)/streptomycin (100µg/mL). All cells were cultured in a 37°C humidified incubator with 5% CO . The four cell lines smORF identification (Hep3B, DU145, SW1990, and HCT116) were used to detect overlapping genes. Huh7 and Hep3 were used to screen candi- First, we obtain the full sequence of interested RNA from date genes. UCSC database (http://genome.ucsc.edu/). Then, the nucleotide 164 Journal of Bio-X Research RESEARCH ARTICLE sequence of RNA was input in ORF Finder tool of NCBI RNA fluorescence in situ hybridization (https://www.ncbi.nlm.nih.gov/orffinder/) to identify the open fluorescence in situ hybridization (FISH) assay were conducted reading frames (ORFs) for potential protein encoding segments. to determine the cellular location of interested RNA. Wild-type HCC cells were seeded on 24-well glass coverslips. When they reached a density of 60% to 70%, the cells were washed with Quantitative reverse transcription-PCR PBS twice, fixed with 4% paraformaldehyde for 15 minutes Quantitative reverse transcription (qRT-PCR) was performed and permeated with cold PBS containing 0.5% Triton X-100. to analyze the expression levels of interested RNA. Total RNA The cells were washed with wash buffer and incubated with was extracted from HCC cells and clinical tissues using TRIzol pre-hybridization buffer at 37°C for 30 minutes. Linear RNA reagent (Thermo Fisher Scientific, Waltham, MA). cDNA was probes were mixed with hybridization buffer at a concentration synthesized using the Hifair II 1st Strand cDNA Synthesis of 500 nM and the samples were added to cells and incubated SuperMix kit (Yeasen Biotech, Shanghai, China). qPCR analy- overnight at 37°C. After 12 hours, washing cells twice using 4× sis was conducted using the Hieff QPCR SYBR Green Master SSC contain 0.1% Tween-20 at 42°C for 5 minutes, and then Mix kit (Yeasen Biotech) in QuantStudio™ (Thermo Fisher washing cells twice in 2× SSC and 1× SSC, respectively. Cells Scientific). qPCR were performed as following thermocycling were incubated with 4ʹ,6-diamino-2-phenylindole for 10 min- conditions: 95°C for 5 minutes, followed by 40 cycles at 95°C utes. Cells were analyzed using a fluorescence microscope. Anti- for 10 seconds, 60°C for 30 seconds and elongation at 72°C for sense and sense oligonucleotides for the RP11-119F7.5, 18S 2 minutes. Relative expression was determined using the com- and U6 probes were designed by RIBO Biotechnology Co., Ltd. −ΔCq parative 2 method. β-actin was used as an endogenous con- (Guangzhou, China). trol. Primer sequences are shown in Additional Table 1, http:// links.lww.com/JR9/A42. Co-immunoprecipitation and mass spectrometry To further analysis the proteins interacted with smORF encoded Western blot assay peptide, co-immunoprecipitation (Co-IP) and mass spectrome- To determine the translation ability of smORFs in vivo, we try (MS; OEbiotech, Shanghai, China) were conducted. Huh7 explored the flag and GFP protein expression of recombi- cells (5×10 ) transfected with smORF plasmids were lysed nant plasmids using western blotting. The transfected Huh7 with protein lysis buffer. Co-IP was performed as previously cells were lysed with RIPA buffer (Beyotime Biotechnology, [20] reported using anti-Flag antibody (Cell Signaling Technology, Shanghai, China) containing protease inhibitor cocktail (MCE, Danvers, MA), the Dynabeads Protein G Immunoprecipitation NJ). The bicinchoninic acid method (Beyotime Biotechnology) Kit (Thermo Fisher Scientific) and a silver staining kit (Beyotime was used to determine the protein concentration of cell lysates. Biotechnology) following the manufacturers’ protocols. The Equal amounts of protein (30µg) were separated by 10% SDS- protein bands of interest were cut out of the gel and analyzed PAGE and transferred onto 0.45-µm PVDF membranes (Bio- by MS. The identified potential proteins interacted with smORF Rad Laboratories, CA). The membranes were blocked using encoded peptide were further analyzed by Search Tool for the 5% non-fat milk solution at room temperature for 1 hour Retrieval of Interacting Genes/Proteins (STRING) database and incubated with primary antibodies at 4°C overnight. The (https://string-db.org/). And the protein–protein interaction net- following primary antibodies were used: flag (1:1000 dilu- work was constructed using Cytoscape software (version 3.4.0; tion, AE063, ABclonal, Wuhan, Hubei Province, China), GFP National Resource for Network Biology). To explore the bio- (1:1000 dilution, AE011, ABclonal), GAPDH (1:1000 dilution, logical functions and signaling pathways associated with the A19056, ABclonal). The membranes were then incubated with identified proteins, we further performed Gene ontology (GO) horseradish peroxidase–conjugated goat anti-rabbit IgG (H + enrichment and Kyoto Encyclopedia of Genes and Genomes L) (1:10,000 dilution, AS014, ABclonal) secondary antibody (KEGG) pathway analyses using the Database for Annotation, at room temperature for 1 hour. Protein bands were visual- Visualization and Integrated Discovery (DAVID) (https://david. ized using Super ECL Detection Reagent (Yeasen Biotech) in ncifcrf.gov). ImageQuant LAS4000 (GE Healthcare). Protein levels were normalized to that of GAPDH. Statistical analysis Statistical analyses were performed using GraphPad Prism 5.0 Immunofluorescence (GraphPad Software, San Diego, CA, www.graphpad.com). We performed immunofluorescence assay to further confirm Data that conformed to a normal distribution were expressed the smORF translation by detecting flag expression in HCC as the mean±standard deviation (SD). Comparisons between cells. Cells transfected with recombinant plasmids were fixed parametric data were analyzed by two-tailed Student t-tests. with 4% formaldehyde for 20 minutes and permeated with Comparisons between non-parametric data were analyzed by 0.3% Triton X-100 (Bio-Light, Zhuhai, Guangdong Province, Mann–Whitney U tests. P<0.05 was considered as statistically China) for 10 minutes. After three washes in PBS, the cells significant. were blocked with 10% goat serum for 1 hour and then incu- bated overnight with flag (1:200 dilution, AE063, ABclonal) at 4°C. After washing, cells were incubated with Alexa 488– Results conjugated goat anti-rabbit IgG (Yeasen Biotech) at room Identification of ribosome-binding genes temperature for 1 hour. Next, 4ʹ,6-diamino-2-phenylindole (Yeasen Biotech) was applied to stain the nuclei. Fluorescence Overlapping genes were two or more structural genes that was observed with a fluorescence microscope (Olympus, shared an identical gene sequence and were widely present in [28] Tokyo, Japan). the genome of organisms. Overlapping genes not only enable 165 RESEARCH ARTICLE Journal of Bio-X Research organisms to use limited genetic information to encode more were selected (Fig. 1C). qRT-PCR showed that six of the can- [29] proteins, but also participate in gene expression regulation. didate genes (RP11-298J20.4, RP11-4O1.2, RP11-119F7.5, A total of 223 overlapping genes were captured by RPS6-RIP RP11-448G15.3, HCP5, RP11-517B11.7) were expressed in (Fig. 1A). Venn diagram analysis revealed that 60 overlapping Huh7 and Hep3B cells (Fig. 1D). We performed further anal- genes were detected in four cancer cell lines (Fig. 1B). Among ysis of these six candidate genes and found that five (RP11- the identified genes, 19 overlapping RNA lacking parental genes 298J20.4, RP11-4O1.2, RP11-119F7.5, RP11-448G15.3, Figure 1. Identification of ribosome-binding genes. (A) RNA-immunoprecipitation followed by high-throughput sequencing was used to identify ribosomal-bind- ing RNA molecules in four cancer cell lines (Hep3B, HCT116, SW1990, DU145). A total of 223 overlapping genes were captured by RPS6-RIP. (B) Venn diagram analysis identified 60 overlapping genes binding ribosomes in the four cell lines (HCT116, SW1990, DU145, Hep3B). (C) The expression levels of 19 unknown sequences without parental genes were detected in Huh7 and Hep3B cells. (D) Six candidate genes (P3 RP11-298J20.4, P10 RP11-4O1.2, P11 RP11-119F7.5, P33 RP11-448G15.3, P37 HCP5, P42 RP11-517B11.7) were expressed in HCC cell lines (Huh7 and Hep3B). (E) The expressions of the five candidate genes (P3 RP11-298J20.4, P10 RP11-4O1.2, P11 RP11-119F7.5, P33 RP11-448G15.3, P42 RP11-517B11.7) were higher in HCC cell lines (Huh7 and Hep3B) than in normal liver cell lines (QSG7701, L02) (*P<0.05). (F) The expression levels of the five genes (P3 RP11-298J20.4, P10 RP11-4O1.2, P11 RP11-119F7.5, P33 RP11-448G15.3, P42 RP11-517B11.7) were significantly higher in six pairs of tumor tissues of HCC patients compared with levels in para-tumor control tissues (*P<0.05). (G) RIP assays and qRT-PCR with specific primers of the four candidate genes (P10 RP11-4O1.2, P11 RP11-119F7.5, P33 RP11-448G15.3, P42 RP11-517B11.7) verified that the four genes bound ribosomes (*P<0.05). IgG=negative control, input=positive control, RPS6=target gene. HCC=hepatocellular carcinoma, qRT-PCR=quantitative reverse transcription-polymerase chain reaction, RPS6-RIP=ribosomal protein S6-RNA-immunoprecipitation. 166 Journal of Bio-X Research RESEARCH ARTICLE RP11-517B11.7) displayed higher expression levels in HCC cell Furthermore, we identified a 66 amino acid micro-peptide lines (Huh7, Hep3B) and tumor tissues than in liver cell lines encoded by RP11-119F7.5 in HCC cells. MS data revealed (L-02, QSG-7701) and para-tumor tissues, respectively (Fig. 1E, that various proteins bind the micro-peptide encoded by F). We performed additional RIP assays and confirmed that four RP11-119F7.5. of the genes (RP11-4O1.2, RP11-119F7.5, RP11-448G15.3, Many ncRNAs play an important role in the occurrence [30–32] RP11-517B11.7) bound RPS6 (Fig. 1G). and development of liver cancer. These ncRNAs func- tion by a variety of molecular mechanisms, such as regu- [33,34] lating RNA–DNA–protein interactions. Some ncRNAs RP11-119F7.5 encodes a micro-peptide localize to specific regions of the nucleus and play transcrip- [35,36] We obtained the full length of the four gene sequences from tional regulation functions. Other ncRNAs function as the UCSC database and analyzed the open reading frames by competitive endogenous RNA molecules to regulate miR- [37] ORF Finder; the conditions were set as an ATG start and ORF NAs. Previous studies showed that several lncRNAs bind [38,39] length of less than 300 bp. All four genes contain smORFs to ribosomes and have coding potential in cancer cells. In in their RNA sequences that potentially encode putative this study, we identified ribosome-associated lncRNAs in can- micro-peptides of less than 100 amino acids (Fig. 2A). To deter- cer cell lines by RIP-seq using RPS6 antibody. We identified mine the translation potential of the four candidate smORFs, a novel polypeptide produced by the translation of RP11- we subcloned a FLAG epitope tag into the C-terminal of the 119F7.5. Recent studies have found that there are millions of four selected smORFs before the stop codon, and the fusion smORF sequences in eukaryotic genomes that have the abil- [40,41] sequences were then cloned into three different plasmid vectors ity to encode and translate proteins (<100 codons). These (pSPT19, pcDNA3.1, and PEGFP-N1) (Fig. 2B). We performed ncRNAs encode functional peptides that are important in [42] coupled transcription and translation reactions and found that regulating many physiological and pathological processes. the pSPT19 plasmids encoded small peptides in vitro (Fig. 2C). Some reports have indicated that the functional peptides We then transfected the pcDNA3.1 constructs into Huh7 cells, encoded by ncRNAs may play an important role in the devel- [43] and a single 7.2 kDa micro-peptide was encoded from the can- opment of tumors. In our study, we found that RP11- didate smORF of RP11.119F7.5 (Fig. 2D). We transfected the 119F7.5 was highly expressed in HCC tissues compared with recombinant pEGFP-N1 plasmids with smORFs in HCC cells, that in the corresponding para-cancer tissues. At the same and western blot analysis revealed a band above GFP in the time, GO enrichment analysis showed that the micro-pep- RP11.119F7.5 recombinant plasmid lane (Fig. 2E). The coding tide–interacting proteins were mainly located in extracellular potential of the RP11-119F7.5 vector was also confirmed by exosome, past research have shown that in the process of immunofluorescence assay (Fig. 2F). These results suggested tumorigenesis and development, exosomal ncRNA plays a that RP11.119F7.5 contains a smORF and encodes a small role in changing the tumor microenvironment, mediating the peptide in HCC cells. FISH assay revealed that RP11-119F7.5 proliferation, metastasis, and drug resistance of tumor cells, was localized in the cytoplasm and nucleoplasm of HCC cells promoting angiogenesis and mediating hypoxia signals. So (Fig. 2G). it is reasonable to speculate that the micro-peptide encoded by ncRNA might regulate the tumor physiological status in exosomes. We also found that these identified proteins were Identification of proteins potentially binding to the involved in several biological functions like protein binding, micro-peptide poly(A) RNA binding, translational initiation, and the nucle- To explore the molecular mechanism of the small peptide, we ar-transcribed mRNA catabolic process. KEGG pathway performed Co-IP using FLAG antibody in HCC cells trans- analysis revealed that the enriched pathways of the identified fected with the FLAG-tagged construct, and candidate proteins proteins included ribosome, biosynthesis of amino acids, car- that potentially interact with the small peptide were identi- bon metabolism, biosynthesis of antibiotics, glycolysis and fied by silver staining and MS (Fig. 3A). A protein–protein gluconeogenesis, pathogenic Escherichia coli infection, and interaction network of the identified proteins was determined influenza A. Our results identified various proteins that bind (Fig. 3B). GO enrichment analysis showed that the micro-pep- to the micro-peptide encoded by RP11-119F7.5 that may tide–interacting proteins were mainly involved in extracellular be involved in HCC. These findings suggest that micro-pep- exosomes, indicating the small micro-peptide modulates the tides encoded by lncRNAs are not redundant products of the tumor development through exosomes secretion. The identi- translation process but may be involved in the occurrence fied proteins were also involved in several biological functions and development of HCC. such as protein binding, poly(A) RNA binding, translational There are some limitations to our study. First, the exact role initiation, and nuclear-transcribed mRNA catabolic process of the micro-peptides in the development and progression of (Fig. 3C). KEGG pathway analysis revealed that the identi- HCC was not identified. Second, our current work has not fied proteins were enriched in pathways including ribosome, examined mechanisms of the micro-peptide in HCC. biosynthesis of amino acids, carbon metabolism, biosynthe- In conclusion, our study found that a 66 amino acid sis of antibiotics, glycolysis and gluconeogenesis, pathogenic micro-peptide encoded by ncRNA RP11-119F7.5 may be Escherichia coli infection, and influenza A (Fig. 3D). involved in the development of HCC. Future studies on the pathological functions of functional peptides or proteins pro- duced by the translation of ncRNAs may open a new door for Discussion research in the cancer field. In this study, we examined possible polyadenylated, ribo- somal-associated lncRNAs in different cancer cell lines. Acknowledgments We found that the lncRNA RP11-119F7.5 is upregulated in HCC tissues compared with that in para-cancer tissues. None. 167 RESEARCH ARTICLE Journal of Bio-X Research Figure 2. Non-coding RNA RP11-119F7.5 encodes a 66 amino acid micro-peptide. (A) Sequence information of the four candidate genes. (B) The full-length smORF sequences with a FLAG tag were cloned into pSPT19, pcDNA3.1, and pEGFP-N1 plasmids. (C) In vitro translation reaction of the PSPT19 plasmids containing the four candidate genes followed by western blot analysis. (D) Huh7 cells were transfected with pcDNA3.1 plasmids containing the four candidate genes, followed by western blot analysis. A single 7.2 kDa peptide was produced from the target smORF of RP11.119F7.5. (E) Huh7 cells were transfected with recombinant pEGFP-N1 plasmid containing the smORFs, and western blot results revealed a band migrating above GFP in the RP11.119F7.5 recombinant plasmid lane. (F) Immunofluorescence assay in Huh7 cells transfected with the plasmid expressing RP11-119F7.5. Green, FLAG; blue, DAPI (scale bar=20 µm). (G) RNA fluorescence in situ hybridization assays showed that RP11-119F7.5 was localized in the cytoplasm and nucleoplasm in Huh7 cells. DAPI=4ʹ,6- diamino-2-phenylindole, ORF=open reading frames, smORF=small open reading frames. 168 Journal of Bio-X Research RESEARCH ARTICLE Figure 3. Identification of proteins that bind with the micro-peptide encoded by RP11-119F7.5 (A) Co-immunoprecipitation and mass spectrometry were used to identify proteins that interact with the peptide. (B) Protein–protein interaction network of the peptide-interacting proteins. (C) GO enrichment analysis showed that the peptide-interacting proteins were mainly involved in the extracellular exosome. (D) KEGG pathways indicated that the identified proteins were enriched in pathways including ribosome, biosynthesis of amino acids, carbon metabolism, biosynthesis of antibiotics, glycolysis and gluconeogenesis, pathogenic Escherichia coli infection and influenza A. GO=Gene ontology, KEGG=Kyoto Encyclopedia of Genes and Genomes. Author contributions The funder did not participate in data collection and analysis, manuscript writing or submission. SL conceived the project and designed the experiments; ZL, YP, YJ, QQ, RK, and HZ performed the experiments and analyzed data. CH designed and wrote the manuscript. SL revised the manuscript. Institutional review board statement All authors approved the final manuscript for publication. This study involving human tissue specimens was conducted in accordance with Declaration of Helsinki and approved by Financial support the Institutional Review Board of Changhai Hospital, Naval This work was supported by the State Key Program of National Military Medical University, China (approval No. CHEC2020- Natural Science Foundation of China (No. 82030073). 081) on June 6, 2020. 169 RESEARCH ARTICLE Journal of Bio-X Research [21] Pauli A, Norris ML, Valen E, et al. 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Journal
Journal of Bio-X Research – Wolters Kluwer Health
Published: Dec 2, 2022