A 66 amino acid micro-peptide encoded by long non-coding RNA RP11-119F7.5 was identified in hepatocellular carcinoma
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 hepato...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Wolters Kluwer Health
2022-12-01
|
| Series: | Journal of Bio-X Research |
| Online Access: | http://journals.lww.com/10.1097/JBR.0000000000000132 |
| _version_ | 1797947130205175808 |
|---|---|
| author | Chengwen He Zhiyong Liu Yanan Pang Yin Jia Qin Qin Ruijiao Kong Hui Zhang Shanrong Liu |
| author_facet | Chengwen He Zhiyong Liu Yanan Pang Yin Jia Qin Qin Ruijiao Kong Hui Zhang Shanrong Liu |
| author_sort | Chengwen He |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-10T21:21:52Z |
| format | Article |
| id | doaj.art-8b975ade1d524834bfc3465658c71a23 |
| institution | Directory Open Access Journal |
| issn | 2096-5672 |
| language | English |
| last_indexed | 2024-04-10T21:21:52Z |
| publishDate | 2022-12-01 |
| publisher | Wolters Kluwer Health |
| record_format | Article |
| series | Journal of Bio-X Research |
| spelling | doaj.art-8b975ade1d524834bfc3465658c71a232023-01-20T02:41:34ZengWolters Kluwer HealthJournal of Bio-X Research2096-56722022-12-015416317010.1097/JBR.0000000000000132202212000-00004A 66 amino acid micro-peptide encoded by long non-coding RNA RP11-119F7.5 was identified in hepatocellular carcinomaChengwen He0Zhiyong Liu1Yanan Pang2Yin Jia3Qin Qin4Ruijiao Kong5Hui Zhang6Shanrong Liu7Department of Laboratory Diagnostic, Changhai Hospital, Naval Military Medical University, Shanghai, ChinaDepartment of Laboratory Diagnostic, Changhai Hospital, Naval Military Medical University, Shanghai, ChinaDepartment of Laboratory Diagnostic, Changhai Hospital, Naval Military Medical University, Shanghai, ChinaDepartment of Laboratory Diagnostic, Changhai Hospital, Naval Military Medical University, Shanghai, ChinaDepartment of Laboratory Diagnostic, Changhai Hospital, Naval Military Medical University, Shanghai, ChinaDepartment of Laboratory Diagnostic, Changhai Hospital, Naval Military Medical University, Shanghai, ChinaDepartment of Laboratory Diagnostic, Changhai Hospital, Naval Military Medical University, Shanghai, ChinaDepartment of Laboratory Diagnostic, Changhai Hospital, Naval Military Medical University, Shanghai, ChinaObjective:. 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.http://journals.lww.com/10.1097/JBR.0000000000000132 |
| spellingShingle | Chengwen He Zhiyong Liu Yanan Pang Yin Jia Qin Qin Ruijiao Kong Hui Zhang Shanrong Liu A 66 amino acid micro-peptide encoded by long non-coding RNA RP11-119F7.5 was identified in hepatocellular carcinoma Journal of Bio-X Research |
| title | A 66 amino acid micro-peptide encoded by long non-coding RNA RP11-119F7.5 was identified in hepatocellular carcinoma |
| title_full | A 66 amino acid micro-peptide encoded by long non-coding RNA RP11-119F7.5 was identified in hepatocellular carcinoma |
| title_fullStr | A 66 amino acid micro-peptide encoded by long non-coding RNA RP11-119F7.5 was identified in hepatocellular carcinoma |
| title_full_unstemmed | A 66 amino acid micro-peptide encoded by long non-coding RNA RP11-119F7.5 was identified in hepatocellular carcinoma |
| title_short | A 66 amino acid micro-peptide encoded by long non-coding RNA RP11-119F7.5 was identified in hepatocellular carcinoma |
| title_sort | 66 amino acid micro peptide encoded by long non coding rna rp11 119f7 5 was identified in hepatocellular carcinoma |
| url | http://journals.lww.com/10.1097/JBR.0000000000000132 |
| work_keys_str_mv | AT chengwenhe a66aminoacidmicropeptideencodedbylongnoncodingrnarp11119f75wasidentifiedinhepatocellularcarcinoma AT zhiyongliu a66aminoacidmicropeptideencodedbylongnoncodingrnarp11119f75wasidentifiedinhepatocellularcarcinoma AT yananpang a66aminoacidmicropeptideencodedbylongnoncodingrnarp11119f75wasidentifiedinhepatocellularcarcinoma AT yinjia a66aminoacidmicropeptideencodedbylongnoncodingrnarp11119f75wasidentifiedinhepatocellularcarcinoma AT qinqin a66aminoacidmicropeptideencodedbylongnoncodingrnarp11119f75wasidentifiedinhepatocellularcarcinoma AT ruijiaokong a66aminoacidmicropeptideencodedbylongnoncodingrnarp11119f75wasidentifiedinhepatocellularcarcinoma AT huizhang a66aminoacidmicropeptideencodedbylongnoncodingrnarp11119f75wasidentifiedinhepatocellularcarcinoma AT shanrongliu a66aminoacidmicropeptideencodedbylongnoncodingrnarp11119f75wasidentifiedinhepatocellularcarcinoma AT chengwenhe 66aminoacidmicropeptideencodedbylongnoncodingrnarp11119f75wasidentifiedinhepatocellularcarcinoma AT zhiyongliu 66aminoacidmicropeptideencodedbylongnoncodingrnarp11119f75wasidentifiedinhepatocellularcarcinoma AT yananpang 66aminoacidmicropeptideencodedbylongnoncodingrnarp11119f75wasidentifiedinhepatocellularcarcinoma AT yinjia 66aminoacidmicropeptideencodedbylongnoncodingrnarp11119f75wasidentifiedinhepatocellularcarcinoma AT qinqin 66aminoacidmicropeptideencodedbylongnoncodingrnarp11119f75wasidentifiedinhepatocellularcarcinoma AT ruijiaokong 66aminoacidmicropeptideencodedbylongnoncodingrnarp11119f75wasidentifiedinhepatocellularcarcinoma AT huizhang 66aminoacidmicropeptideencodedbylongnoncodingrnarp11119f75wasidentifiedinhepatocellularcarcinoma AT shanrongliu 66aminoacidmicropeptideencodedbylongnoncodingrnarp11119f75wasidentifiedinhepatocellularcarcinoma |