Transcriptome and Gene Fusion Analysis of Synchronous Lesions Reveals lncMRPS31P5 as a Novel Transcript Involved in Colorectal Cancer
Abstract
:1. Introduction
2. Results
2.1. Identification of Chimeric RNAs by EricScript and ChimeraScan Algorithms
2.2. Fusion Junction Validation by Reverse Transcription-PCR (RT-PCR)
2.3. Fusion Junction Validation by Sanger Sequencing
2.4. Differential Gene Expression and Functional Enrichment Analyses
2.5. ceRNA Analysis and RNA-RNA Interactions
3. Discussion
4. Materials and Methods
4.1. Clinical Samples and RNA Extraction
4.2. Whole Transcriptome Sequencing (RNA-Seq)
4.3. Fusion Detection
4.4. Fusion Gene Validation by RT-PCR and Sanger Sequencing
4.5. Fusion Gene Validation and Functional Enrichment Analysis by Targeted RNA-Seq
4.6. Competing Endogenous RNA Analysis
4.7. Study of RNA-RNA and RNA-Protein Interactions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
- Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R.L.; Torre, L.A.; Jemal, A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2018, 68, 394–424. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Siegel, R.L.; Fedewa, S.A.; Anderson, W.F.; Miller, K.D.; Ma, J.; Rosenberg, P.S.; Jemal, A. Colorectal Cancer Incidence Patterns in the United States, 1974–2013. J. Natl. Cancer Inst. 2017, 109. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Jasperson, K.W.; Tuohy, T.M.; Neklason, D.W.; Burt, R. Hereditary and Familial Colon Cancer. Gastroenterology 2010, 138, 2044–2058. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Latournerie, M.; Jooste, V.; Cottet, V.; Lepage, C.; Faivre, J.; Bouvier, A.-M. Epidemiology and prognosis of synchronous colorectal cancers. Br. J. Surg. 2008, 95, 1528–1533. [Google Scholar] [CrossRef]
- Yang, J.; Peng, J.-Y.; Chen, W. Synchronous Colorectal Cancers: A Review of Clinical Features, Diagnosis, Treatment, and Prognosis. Dig. Surg. 2011, 28, 379–385. [Google Scholar] [CrossRef] [PubMed]
- Carethers, J.M.; Jung, B.H. Genetics and Genetic Biomarkers in Sporadic Colorectal Cancer. Gastroenterology 2015, 149, 1177–1190.e3. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Winawer, S.J.; Zauber, A.G.; Ho, M.N.; O’Brien, M.J.; Gottlieb, L.S.; Sternberg, S.S.; Waye, J.D.; Schapiro, M.; Bond, J.H.; Panish, J.F.; et al. Prevention of Colorectal Cancer by Colonoscopic Polypectomy. N. Engl. J. Med. 1993, 329, 1977–1981. [Google Scholar] [CrossRef]
- O’Connell, J.B.; Maggard, M.A.; Ko, C.Y. Colon Cancer Survival Rates With the New American Joint Committee on Cancer Sixth Edition Staging. J. Natl. Cancer Inst. 2004, 96, 1420–1425. [Google Scholar] [CrossRef]
- Medves, S.; Demoulin, J.-B. Tyrosine kinase gene fusions in cancer: Translating mechanisms into targeted therapies. J. Cell. Mol. Med. 2012, 16, 237–248. [Google Scholar] [CrossRef] [Green Version]
- Jung, G.; Hernández-Illán, E.; Moreira, L.; Balaguer, F.; Goel, A. Epigenetics of colorectal cancer: Biomarker and therapeutic potential. Nat. Rev. Gastroenterol. Hepatol. 2020, 17, 111–130. [Google Scholar] [CrossRef]
- Latysheva, N.S.; Babu, M.M. Discovering and understanding oncogenic gene fusions through data intensive computational approaches. Nucleic Acids Res. 2016, 44, 4487–4503. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Barr, F.G. Fusion genes in solid tumors: The possibilities and the pitfalls. Expert Rev. Mol. Diagn. 2016, 16, 921–923. [Google Scholar] [CrossRef] [PubMed]
- Zhu, D.; Singh, S.; Chen, X.; Zheng, Z.; Huang, J.; Lin, T.; Li, H. The landscape of chimeric RNAs in bladder urothelial carcinoma. Int. J. Biochem. Cell Biol. 2019, 110, 50–58. [Google Scholar] [CrossRef] [PubMed]
- Bass, A.J.; Lawrence, M.S.; Brace, L.E.; Ramos, A.H.; Drier, Y.; Cibulskis, K.; Sougnez, C.; Voet, D.; Saksena, G.; Sivachenko, A.; et al. Genomic sequencing of colorectal adenocarcinomas identifies a recurrent VTI1A-TCF7L2 fusion. Nat. Genet. 2011, 43, 964–968. [Google Scholar] [CrossRef] [PubMed]
- Nome, T.; Hoff, A.M.; Bakken, A.C.; Rognum, T.O.; Nesbakken, A.; Skotheim, R.I. High Frequency of Fusion Transcripts Involving TCF7L2 in Colorectal Cancer: Novel Fusion Partner and Splice Variants. PLoS ONE 2014, 9, e91264. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Seshagiri, S.; Stawiski, E.W.; Durinck, S.; Modrusan, Z.; Storm, E.E.; Conboy, C.B.; Chaudhuri, S.; Guan, Y.; Janakiraman, V.; Jaiswal, B.S.; et al. Recurrent R-spondin fusions in colon cancer. Nature 2012, 488, 660–664. [Google Scholar] [CrossRef] [PubMed]
- Storm, E.E.; Durinck, S.; Melo, F.D.S.E.; Tremayne, J.; Kljavin, N.; Tan, C.; Ye, X.; Chiu, C.; Pham, T.; Hongo, J.-A.; et al. Targeting PTPRK-RSPO3 colon tumours promotes differentiation and loss of stem-cell function. Nature 2015, 529, 97–100. [Google Scholar] [CrossRef]
- Yu, J.; Wu, W.K.K.; Liang, Q.; Zhang, N.; He, J.; Li, X.-X.; Zhang, X.; Xu, L.; Chan, M.T.; Ng, S.S.M.; et al. Disruption of NCOA2 by recurrent fusion with LACTB2 in colorectal cancer. Oncogene 2015, 35, 187–195. [Google Scholar] [CrossRef] [Green Version]
- Kim, S.Y.; Oh, S.O.; Kim, K.; Lee, J.; Kang, S.; Kim, K.-M.; Lee, W.; Kim, S.T.; Nam, D.N. NCOA4-RET fusion in colorectal cancer: Therapeutic challenge using patient-derived tumor cell lines. J. Cancer 2018, 9, 3032–3037. [Google Scholar] [CrossRef] [Green Version]
- Kloosterman, W.P.; Braak, R.R.J.C.V.D.; Pieterse, M.; Van Roosmalen, M.J.; Sieuwerts, A.M.; Stangl, C.; Brunekreef, R.; Lalmahomed, Z.S.; Ooft, S.N.; Van Galen, A.; et al. A Systematic Analysis of Oncogenic Gene Fusions in Primary Colon Cancer. Cancer Res. 2017, 77, 3814–3822. [Google Scholar] [CrossRef] [Green Version]
- Yang, Y.; Junjie, P.; Sanjun, C.; Ma, Y. Long non-coding RNAs in Colorectal Cancer: Progression and Future Directions. J. Cancer 2017, 8, 3212–3225. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Pan, H.; Pan, J.; Song, S.; Ji, L.; Lv, H.; Yang, Z. Identification and development of long non-coding RNA-associated regulatory network in colorectal cancer. J. Cell. Mol. Med. 2019, 23, 5200–5210. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Wang, L.; Cho, K.B.; Li, Y.; Tao, G.; Xie, Z.; Guo, B. Long Noncoding RNA (lncRNA)-Mediated Competing Endogenous RNA Networks Provide Novel Potential Biomarkers and Therapeutic Targets for Colorectal Cancer. Int. J. Mol. Sci. 2019, 20, 5758. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Carrara, M.; Beccuti, M.; Lazzarato, F.; Cavallo, F.; Cordero, F.; Donatelli, S.; Calogero, R.A. State-of-the-Art Fusion-Finder Algorithms Sensitivity and Specificity. BioMed Res. Int. 2013, 2013, 1–6. [Google Scholar] [CrossRef] [PubMed]
- Xu, Y.; Deng, Y.; Ji, Z.; Liu, H.; Liu, Y.; Peng, H.; Wu, J.; Fan, J. Identification of Thyroid Carcinoma Related Genes with mRMR and Shortest Path Approaches. PLoS ONE 2014, 9, e94022. [Google Scholar] [CrossRef] [Green Version]
- Mori, M.; Iwatsuki, M.; Mimori, K.; Sato, T.; Toh, H.; Yokobori, T.; Tanaka, F.; Ishikawa, K.; Baba, H. Mori Overexpression of SUGT1 in human colorectal cancer and its clinicopathological significance. Int. J. Oncol. 2010, 36, 569–575. [Google Scholar] [CrossRef] [Green Version]
- Wu, H.; Singh, S.; Shi, X.; Xie, Z.; Lin, E.; Li, X.; Li, H. Functional heritage: The evolution of chimeric RNA into a gene. RNA Biol. 2019, 17, 125–134. [Google Scholar] [CrossRef]
- Lu, Z.; Zhang, Q.C.; Lee, B.; Flynn, R.A.; Smith, M.A.; Robinson, J.T.; Davidovich, C.; Gooding, A.R.; Goodrich, K.J.; Mattick, J.S.; et al. RNA Duplex Map in Living Cells Reveals Higher-Order Transcriptome Structure. Cell 2016, 165, 1267–1279. [Google Scholar] [CrossRef] [Green Version]
- Fearon, E.R.; Vogelstein, B. A genetic model for colorectal tumorigenesis. Cell 1990, 61, 759–767. [Google Scholar] [CrossRef]
- Lichtenstein, P.; Holm, N.V.; Verkasalo, P.K.; Iliadou, A.; Kaprio, J.; Koskenvuo, M.; Pukkala, E.; Skytthe, A.; Hemminki, K. Environmental and heritable factors in the causation of cancer-analyses of cohorts of twins from Sweden, Denmark, and Finland. N. Engl. J. Med. 2000, 343, 78–85. [Google Scholar] [CrossRef]
- Wu, H.; Singh, S.; Xie, Z.; Li, X.; Li, H. Landscape characterization of chimeric RNAs in colorectal cancer. Cancer Lett. 2020, 489, 56–65. [Google Scholar] [CrossRef] [PubMed]
- Kim, K.; Shin, E.A.; Jung, J.H.; Park, J.E.; Kim, D.S.; Shim, B.S.; Kim, S.H. Ursolic Acid Induces Apoptosis in Colorectal Cancer Cells Partially via Upregulation of MicroRNA-4500 and Inhibition of JAK2/STAT3 Phosphorylation. Int. J. Mol. Sci. 2018, 20, 114. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Xicola, R.M.; Bontu, S.; Doyle, B.J.; Rawson, J.; Garre, P.; Lee, E.; De La Hoya, M.; Bessa, X.; Clofent, J.; Bujanda, L.; et al. Association of a let-7 miRNA binding region of TGFBR1 with hereditary mismatch repair proficient colorectal cancer (MSS HNPCC). Carcinogenesis 2016, 37, 751–758. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Zhou, J.; Lin, J.; Zhang, H.; Zhu, F.; Xie, R. LncRNA HAND2-AS1 sponging miR-1275 suppresses colorectal cancer progression by upregulating KLF14. Biochem. Biophys. Res. Commun. 2018, 503, 1848–1853. [Google Scholar] [CrossRef] [PubMed]
- Gilkes, D.M.; Pan, Y.; Coppola, D.; Yeatman, T.; Reuther, G.W.; Chen, J. Regulation of MDMX Expression by Mitogenic Signaling. Mol. Cell. Biol. 2008, 28, 1999–2010. [Google Scholar] [CrossRef] [Green Version]
- Ling, X.; Xu, C.; Fan, C.; Zhong, K.; Li, F.; Wang, X. FL118 induces p53-dependent senescence in colorectal cancer cells by promoting degradation of MdmX. Cancer Res. 2014, 74, 7487–7497. [Google Scholar] [CrossRef] [Green Version]
- Shen, X.; Zuo, X.; Zhang, W.; Bai, Y.; Qin, X.; Hou, N. MiR-370 promotes apoptosis in colon cancer by directly targeting MDM4. Oncol. Lett. 2017, 15, 1673–1679. [Google Scholar] [CrossRef] [Green Version]
- Rasmussen, S.L.; Krarup, H.B.; Sunesen, K.G.; Pedersen, I.S.; Madsen, P.H.; Thorlacius-Ussing, O. Hypermethylated DNA as a biomarker for colorectal cancer: A systematic review. Color. Dis. 2016, 18, 549–561. [Google Scholar] [CrossRef]
- Ivashchenko, A.; Berillo, O.; Pyrkova, A.; Niyazova, R. Binding Sites of miR-1273 Family on the mRNA of Target Genes. BioMed Res. Int. 2014, 2014, 620530. [Google Scholar] [CrossRef] [Green Version]
- Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 2014, 30, 2114–2120. [Google Scholar] [CrossRef] [Green Version]
- Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 2011, 17, 10–12. [Google Scholar] [CrossRef]
- Iyer, M.K.; Chinnaiyan, A.M.; Maher, C.A. ChimeraScan: A tool for identifying chimeric transcription in sequencing data. Bioinformatics 2011, 27, 2903–2904. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Benelli, M.; Pescucci, C.; Marseglia, G.; Severgnini, M.; Torricelli, F.; Magi, A. Discovering chimeric transcripts in paired-end RNA-seq data by using EricScript. Bioinformatrics 2012, 28, 3232–3239. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Dobin, A.; Davis, C.A.; Schlesinger, F.; Drenkow, J.; Zaleski, C.; Jha, S.; Batut, P.; Chaisson, M.; Gingeras, T.R. STAR: Ultrafast universal RNA-seq aligner. Bioinformatics 2012, 29, 15–21. [Google Scholar] [CrossRef] [PubMed]
- Love, M.I.; Huber, W.; Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014, 15, 002832. [Google Scholar] [CrossRef] [Green Version]
- Chen, Y.; Wang, X. miRDB: An online database for prediction of functional microRNA targets. Nucleic Acids Res. 2019, 48, D127–D131. [Google Scholar] [CrossRef] [Green Version]
- John, B.; Enright, A.J.; Aravin, A.; Tuschl, T.; Sander, C.; Marks, D.S. Human MicroRNA targets. PLoS Biol. 2004, 2, e363. [Google Scholar] [CrossRef] [Green Version]
- Kozomara, A.; Birgaoanu, M.; Griffiths-Jones, S. miRBase: From microRNA sequences to function. Nucleic Acids Res. 2018, 47, D155–D162. [Google Scholar] [CrossRef]
- Xie, B.; Ding, Q.; Han, H.; Wu, D. miRCancer: A microRNA-cancer association database constructed by text mining on literature. Bioinformatics 2013, 29, 638–644. [Google Scholar] [CrossRef]
Fusion | Gene Name 5p | Gene Name 3p | chr 5p | Breakpoint 1 (End 5p) | Strand 5p | chr 3p | Breakpoint 2 (Start 3p) | strand 3p | Fusiozn Type | JunctionSequence | Score |
---|---|---|---|---|---|---|---|---|---|---|---|
RNF123-STAT3 | RNF123 | STAT3 | 3 | 49,728,680 | + | 17 | 49,728,680 | - | inter-chr | ccgcaagagctataggctgacctcagatgctgagaaatccagggtcacagCTACTCGGGAGGCTGAGGCAGGAGAATCGCTTGAACCTGAGAGGCGGAGG | 0.91586235 |
PLK1-ERN2 | PLK1 | ERN2 | 16 | 23,701,614 | + | 16 | 23,702,074 | - | Cis | gtgggttctacagccttgtccccctccccctcaaccccaccatatgaattGCTGGGTGCAGTGGCTCACACCTGTAATCCCAGCATTTTGGGAGGCTGAG | 0.690112004 |
MRPS31-SUGT1 | MRPS31 | SUGT1 | 13 | 41,323,274 | - | 13 | 53,231,667 | + | intra-chr | gtggacaaaagaggggaaactatgggagttcccaattaacaatgaagcagGAGCTGACTAAGGCTTTGGAACAGAAACCAGATGATGCACAGTATTATTG | 0.734629203 |
LPHN1-SUZ12 | LPHN1 | SUZ12 | 19 | 14,316,797 | - | 17 | 30,267,305 | + | inter-chr | cgagccgcaggagagacacgctgggccgaccccagagaggcgctggacagAGCCAACACAGATCTATAGATTTCTTCGAACTCGGAATCTCATAGCACCA | 0.855475528 |
EIF5AL1-MSH3 | EIF5AL1 | MSH3 | 10 | 81,274,508 | + | 5 | 81,274,508 | + | inter-chr | aagactgtgaaaatgaatccagaggtgacccaagcattgaatttaacaatGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGTAGGCAGA | 0.532811583 |
GUCY2C-PLBD1 | GUCY2C | PLBD1 | 12 | 14,765,813 | - | 12 | 14,721,126 | - | Read- Through | accttccactctggaaccttattccagcagttgttccagggagcttctacCTGTGGAGGCCTCTCCAGAAACAGCAGAGGATCCGAGCTGCGTGTAGGCA | 0.896360711 |
HSPE1-MOB4 | HSPE1 | MOB4 | 2 | 198,367,852 | + | 2 | 198,388,348 | + | Read- Through | aagttcttctcccagaatatggaggcaccaaagtagttctagatgacaagGATTTCTATAATTGGCCTGATGAATCCTTTGATGAAATGGACAGTACACT | 0.821493951 |
PDLIM2-CCAR2 | PDLIM2 | CCAR2 | 8 | 22,455,537 | + | 8 | 22,463,248 | + | intra-chr | agagattggctgtgggcctcagtttccccattttataaagttttaaaatctGCCTTTTCCCCACGACTCTGAAAGAGGACAGCGTTCCCAATGTCCCAGTTT | 5 |
HPSE2-HSD11B2 | HPSE2 | HSD11B2 | 10 | 100,995,631 | - | 16 | 67,469,859 | + | inter-chr | tctcttcctactgggtctcgctagtgactaattgtccttatctaaagtgtgGGCCTGTGGGGCCTCGTCAACAACGCAGGCCACAATGAAGTAGTTGCTGAT | 2 |
HDAC1-MARCKSL1 | HDAC1 | MARCKSL1 | 1 | 32,799,223 | + | 1 | 32,799,429 | - | Adjacent_ Converging | agatactattttcatttttgtgagcctctttgtaataaaatggtacatttcTAAAGCACCACTAAAGGGACGACATTTATTCCTTTTCCAAATGTTACAGTA | 2 |
ARSA-TNS4 | ARSA | TNS4 | 22 | 51,066,600 | - | 17 | 38,632,079 | - | inter-chr | gccggtaccgggctgcgggcgcttccgcctcggccccgccccgtgacctgtCTTACTGTTTTGCAAAGACAAACATTTTATTTTTCATGATAGGAGCTGTAG | 4 |
ERBB2-MIEN1 | ERBB2 | MIEN1 | 17 | 37,883,255 | + | 17 | 37,885,408 | - | Adjacent_ Converging | cccgggcgctgggggcatggtccaccacaggcaccgcagctcatctaccagATTAGTGTTTGTAGCGCCACTTTACTGCCAATAGCTGACATTGCCCTGGGT | 4 |
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Panza, A.; Castellana, S.; Biscaglia, G.; Piepoli, A.; Parca, L.; Gentile, A.; Latiano, A.; Mazza, T.; Perri, F.; Andriulli, A.; et al. Transcriptome and Gene Fusion Analysis of Synchronous Lesions Reveals lncMRPS31P5 as a Novel Transcript Involved in Colorectal Cancer. Int. J. Mol. Sci. 2020, 21, 7120. https://doi.org/10.3390/ijms21197120
Panza A, Castellana S, Biscaglia G, Piepoli A, Parca L, Gentile A, Latiano A, Mazza T, Perri F, Andriulli A, et al. Transcriptome and Gene Fusion Analysis of Synchronous Lesions Reveals lncMRPS31P5 as a Novel Transcript Involved in Colorectal Cancer. International Journal of Molecular Sciences. 2020; 21(19):7120. https://doi.org/10.3390/ijms21197120
Chicago/Turabian StylePanza, Anna, Stefano Castellana, Giuseppe Biscaglia, Ada Piepoli, Luca Parca, Annamaria Gentile, Anna Latiano, Tommaso Mazza, Francesco Perri, Angelo Andriulli, and et al. 2020. "Transcriptome and Gene Fusion Analysis of Synchronous Lesions Reveals lncMRPS31P5 as a Novel Transcript Involved in Colorectal Cancer" International Journal of Molecular Sciences 21, no. 19: 7120. https://doi.org/10.3390/ijms21197120
APA StylePanza, A., Castellana, S., Biscaglia, G., Piepoli, A., Parca, L., Gentile, A., Latiano, A., Mazza, T., Perri, F., Andriulli, A., & Palmieri, O. (2020). Transcriptome and Gene Fusion Analysis of Synchronous Lesions Reveals lncMRPS31P5 as a Novel Transcript Involved in Colorectal Cancer. International Journal of Molecular Sciences, 21(19), 7120. https://doi.org/10.3390/ijms21197120