Guanine content of precursor microRNA’s terminal loop and its association with cancer
Review Article

Guanine content of precursor microRNA’s terminal loop and its association with cancer

Amit Cohen1, Mario Alberto Burgos-Aceves2, Yoav Smith1

1Genomic Data Analysis Unit, The Hebrew University of Jerusalem-Hadassah Medical School, Jerusalem, Israel; 2Departament of Chemistry and Biology, University of Salerno, via Giovanni Paolo II, Fisciano, Italy

Contributions: (I) Conception and design: A Cohen; (II) Administrative support: MA Burgos-Aceves; (III) Provision of study materials or patients: A Cohen, MA Burgos-Aceves; (IV) Collection and assembly of data: All authors; (V) Data analysis and interpretation: Y Smith, A Cohen; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.

Correspondence to: Mario Alberto Burgos-Aceves. Laboratorio di Chimica Biologica, Dipartimento di Chimica e Biologia, Università degli Studi di Salerno, Via Giovanni Paolo II, 132, 84084 Fisciano, SA, Italia. Email: mburgosaceves@unisa.it; marioburgos21@hotmail.com.

Abstract: MicroRNAs (miRNAs) are small noncoding segments of RNA that negatively regulate gene expression at the post-transcriptional level and fine-tune gene functions. Several lines of evidence suggest that terminal loops (TLs) of miRNAs are important features determining their processing efficiency. A global repression in miRNAs expression in different types of human tumors, after exposure to cigarette-smoke (CS), or to the hormone estrogen, was shown to be associated with guanine (G) enrichment in the TLs of their precursors. In this review we summarize the results that show the relation of TLs G content to the regulation of miRNA maturation and function, suggest a new G-dependent miRNA-related model of carcinogenesis, and specify several dietary phytochemicals that can be used for its prevention.

Keywords: Guanine; microRNA (miRNA); cancer; estrogen; cigarette-smoke (CS); c-Myc; DNA adducts; cruciferous vegetables; Phenethyl Isothiocyanate; Indole-3-Carbinol; Sulforaphane; Resveratrol


Received: 03 June 2018; Accepted: 12 September 2018; Published: 18 September 2018.

doi: 10.21037/jxym.2018.09.01


Background

MicroRNAs (miRNAs) are endogenous ~22-nucleotides (nt) RNA molecules, that negatively regulate gene expression at the post-transcriptional level and have a role in networking and fine-tuning gene expression in the cell (1). The miRNA maturation process begins with the primary long transcript (pri-miRNA), which is first processed by an RNase, termed Drosha, that cuts it into ~70-nt stem-loop (SL) precursor (pre-miRNA), containing the mature miRNA sequence in one of its arms and the less abundant partially complementary miRNA mature form in the other arm (2,3). After the first processing step, pre-miRNA is actively transported by exportin-5 (XPO5) from the nucleus to the cytoplasm, where it is processed by another RNase, termed Dicer (4,5). The result of this processing event is a double stranded RNA, where one of its strands is incorporated into the argonaute (Ago) protein of the RNA-induced silencing complex (RISC) that targets it to a 3’ untranslated region (3’UTR) of a specific mRNA and leads to its degradation (1).

A comprehensive reduction in miRNA was commonly observed in human cancers, where miRNAs showed lower expression levels in tumors and cancer cell lines compared with normal tissues (6-9). A widespread repression of miRNA expression has also been reported after exposure to cigarette-smoke (CS) (10-12), treatment with the hormone estrogen (13-15), and c-Myc activation (16), and the observed global downregulation of miRNAs was also inversely correlated with their predicted targets. These aforementioned alterations in miRNA expression can occur as a result of affecting the transcription of miRNA genes (16), miRNA export from the nucleus (17), or at any stage of the miRNA maturation process by modulation of key regulators or components of the miRNA biogenesis pathway, including the microprocessor complex Drosha-DGCR8, and Dicer (18).


Guanine enrichment in TL sequences of cancer/estrogen/Myc-repressed miRNAs

There are several indications for the importance of guanine (G) content in miRNAs TL sequences to the regulation of their biogenesis and function. Izzotti and Pulliero showed in their study that the G content of the TLs of miRNAs which are involved in stress response, is higher than the G content of the other miRNAs (19). We have recently found an association between the widespread miRNAs reduction that is observed in human cancers and a high TL G content in their precursors (20,21). Using bioinformatic analysis of zebrafish, mouse, and human breast cancer cell lines, we also showed that similar G enrichment exists in TLs of downregulated miRNAs after estrogen (17β-estradiol; E2) exposure (22), and most striking was the observation that of the different G combinations in TL sequences of both cancer and E2-repressed miRNAs, the relative enrichment of double G (GG) and triple G (GGG) was especially dominant (21). Remarkably, this phenomenon is also observed when looking at the ten most c-Myc-repressed miRNAs of a human B cells model (16), where six of them are also common to cancer-repressed miRNAs (miR-15a, miR-24, miR-29a, miR-29c, miR-125b, miR-195) (20), and the other four to E2-repressed miRNAs (miR-23a, miR-23b, miR-26a, miR-27a) (22). The transcription factor c-Myc physically interacts with estrogen receptor alpha (ERα) and is recruited to estrogen-responsive genes (23). Indeed, estrogen can cause cellular growth, proliferation and cancer by inducing oncogenes such as c-Myc (24). Therefore, part of the global miRNA downregulation that is observed after exposure to estrogen, might be attributed to c-Myc regulation. It is interesting to note, however, that c-Myc activation was also shown to be associated with an altered estrogen metabolism (25). The potential carcinogenic activity of estrogen involves the oxidative metabolism of estrogens to catechol estrogens and the reactive quinone metabolites, that form specific DNA adducts at the N-7 G (26,27), and it was shown that miRNAs are even more sensitive than DNA to the formation of G-adducts (19). These adducts generate apurinic sites that can be converted into mutations by error-prone repair, which in turn may initiate tumorigenesis (28). Also, oxidative metabolites of estrogens can react with DNA to form 8-oxo-dG (8-Oxo-2’-deoxyguanosine); the most frequent DNA oxidative damage, which eventually leads to carcinogenesis (29), because G has lower oxidation potential and is most easily oxidized among the four DNA bases (30). Most interestingly, experimental studies have shown that sequences with repeated G bases (GG or GGG) show higher reactivity toward oxidation than isolated G bases (31).


Alterations of G nucleotides in pre-miRNA TL affect miRNA expression level

Several successive studies, conducted by the Chen’s group, have shown that G substitution in pre-miRNAs TLs disrupt their maturation process. In their study, Liu et al. demonstrated that pre-miRNA loop nucleotides play an important role in controlling the biological activity of miRNAs (32). Specifically, substitution of nucleotides GG to CC in the pre-miRNA loop of miR-181a-1 reduced the activity of mir-181a-1 on T cell development by 70%, and cells transfected with this mutant expressed significantly less mature miR-181a (32). Similarly, using a GG to CC loop mutant of let-7 pre-miRNA resulted in a significant reduction in mature miRNA expression levels and in the activity of target gene repression (33,34). Together, the results of these studies show that terminal loop (TL) mutagenesis of GG affects miRNA level and function, which seems to be caused by alterations of loop sequence and/or structure.


G-enriched motifs in miRNA TL sequences affect miRNA processing

Findings suggest that the miRNA TL is an important platform for different RNA-binding proteins (RBPs) that act as activators or repressors of Drosha and Dicer processing, and selectivity regulate miRNAs by binding to G-enriched motifs in the RNA TLs of their precursors (35). It was shown that miRNAs with the tetra-nucleotide sequence motif GGAG in their TL were regulated through binding of the RBP Lin28, which interferes with Dicer processing (36), and that the sequence AGGGU in the TL mediates regulation of miRNA biogenesis by the KSRP RBP (37). In their study, García-Mayoral et al. described the complete analysis of the RNA-binding potential of the four KH domains of KSRP and showed that the KH3 domain can recognize a G-rich sequence (38). Insertion of an isolated G led to a 5-fold increase in KH3 affinity, whereas insertion of a GG element led to a further 4-fold increase (38). Interestingly, KH3 binding docks KSRP to the GGG-containing TL of a subset of miRNAs and promotes their maturation (37). Of note, our results revealed a high enrichment for the sequence motif GGAG in TLs of cancer and E2-repressed miRNAs (20,22). Moreover, also a significant enrichment of the GGG motif in TLs of these miRNAs was observed (21). Therefore, G-adducts formation that disrupts binding of KSRP to the TL might be a possible cause for the observed cancer and E2-repressed miRNAs. Indeed, it was recently shown that modification of KSRP resulting in the downregulation of a subset of TL G-rich miRNAs and promoting tumorigenesis (39).


Dietary anti-estrogenic phytochemical compounds used for cancer chemoprevention

It is well known that dietary phytochemicals from vegetables and fruits exhibit chemopreventive activities against various types of cancer (40). Administration of the dietary agents Phenethyl Isothiocyanate (PEITC) and Indole-3-Carbinol (I3C), two major components of cruciferous vegetables, attenuated the cigarette smoke-induced downregulation of miRNA expression (41). The combined treatment with PEITC and I3C had profound effects on almost all CS-downregulated miRNAs and their expression even exceeded the baseline situation (41). PEITC, which has both chemopreventive and chemotherapeutic effects (42), was also the most effective agent in inhibition of CS-related cytogenetic damage, transcriptome alterations, and lung tumorigenesis (43-45). Also, I3C and its condensation product 3,3’-diindolylmethane (DIM) exhibited potent anti-tumor effects with negligible levels of toxicity in a wide range of human cancer cells, including lung cancer (46). Interestingly, both PEITC and I3C have proved to be anti-estrogenic compounds and inhibited ERalpha expression (47-51). Furthermore, PEITC was shown to significantly inhibit the formation of the xenoestrogen bisphenol A (BPA)-induced DNA adducts in mice (52).

Another compound that has anti-estrogenic effects is the dietary polyphenol derived from grapes, Resveratrol (RES). This natural product is known as an antioxidant and antimutagen, with cancer chemopreventive activity (53).

The Cavalieri-Rogan’s groups have described in multiple reports that RES decreases estrogen metabolism, and prevents estrogen-DNA adduct formation (54-57). In these studies, RES was shown to block the oxidation of catechol estrogens to their quinones and their reaction with DNA, and by this way to prevent cancer initiation (27). Both RES and Sulforaphane (SFN), additional isothiocyanate of cruciferous, were shown to induce protective phase II enzymes activity, resulting in reduction of estrogen-induced DNA damage (58).


Conclusions

Taken together, the above results suggest that G content, especially GG and GGG, in miRNA TL sequences, may have some important role in the carcinogenic process induced by estrogen and c-Myc. As mentioned before, the mechanisms of estrogen carcinogenesis include unbalanced estrogen metabolism and the formation of G-adducts, which can also potentially be formed in miRNA TLs. This raises the possibility that G oxidation and/or formation of G-adducts in TLs may lead to the extensive downregulation of tumor suppressor miRNAs, which will then cause induction of their target oncogenes, and commit cells towards carcinogenesis (Figure 1). Indeed, several of the repressed miRNAs were shown to function as tumor suppressors (21), and were upregulated by dietary phytochemical agents such as PEITC, I3C and RES (59).

Figure 1 A model summarizing the miRNA biogenesis pathway and its possible regulation through G enrichment in miRNAs TLs. Disruption of transcription and processing of tumor suppressor miRNAs (denoted by the red crosses) by Estrogen/Cigarette smoke/Myc may lead to carcinogenesis, while phytochemical compounds (PEITC, I3C, SFN, RES) may potentially prevent it.

Acknowledgements

None.


Footnote

Conflicts of Interest: The author has no conflicts of interest to declare.


References

  1. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell 2009;136:215-33. [Crossref] [PubMed]
  2. Zeng Y, Yi R, Cullen BR. Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha. EMBO J 2005;24:138-48. [Crossref] [PubMed]
  3. Yang JS, Phillips MD, Betel D, et al. Widespread regulatory activity of vertebrate microRNA* species. RNA 2011;17:312-26. [Crossref] [PubMed]
  4. Lund E, Güttinger S, Calado A, et al. Nuclear export of microRNA precursors. Science 2004;303:95-8. [Crossref] [PubMed]
  5. Cullen BR. Transcription and processing of human microRNA precursors. Mol Cell 2004;16:861-5. [Crossref] [PubMed]
  6. Lu J, Getz G, Miska EA, et al. MicroRNA expression profiles classify human cancers. Nature 2005;435:834-8. [Crossref] [PubMed]
  7. Ozen M, Creighton CJ, Ozdemir M, et al. Widespread deregulation of microRNA expression in human prostate cancer. Oncogene 2008;27:1788-93. [Crossref] [PubMed]
  8. Dvinge H, Git A, Gräf S, et al. The shaping and functional consequences of the microRNA landscape in breast cancer. Nature 2013;497:378-82. [Crossref] [PubMed]
  9. Cohen A, Burgos-Aceves MA, Smith Y. Estrogen repression of microRNA as a potential cause of cancer. Biomed Pharmacother 2016;78:234-8. [Crossref] [PubMed]
  10. Izzotti A, Calin GA, Arrigo P, et al. Downregulation of microRNA expression in the lungs of rats exposed to cigarette smoke. FASEB J 2009;23:806-812. [Crossref] [PubMed]
  11. Schembri F, Sridhar S, Perdomo C, et al. MicroRNAs as modulators of smoking-induced gene expression changes in human airway epithelium. Proc Natl Acad Sci U S A 2009;106:2319-24. [Crossref] [PubMed]
  12. Graff JW, Powers LS, Dickson AM, et al. Cigarette smoking decreases global microRNA expression in human alveolar macrophages. PLoS One 2012;7. [Crossref] [PubMed]
  13. Maillot G, Lacroix-Triki M, Pierredon S, et al. Widespread estrogen-dependent repression of micrornas involved in breast tumor cell growth. Cancer Res 2009;69:8332-40. [Crossref] [PubMed]
  14. Yamagata K, Fujiyama S, Ito S, et al. Maturation of microRNA is hormonally regulated by a nuclear receptor. Mol Cell 2009;36:340-7. [Crossref] [PubMed]
  15. Cohen A, Smith Y. Estrogen regulation of microRNAs, target genes, and microRNA expression associated with vitellogenesis in the zebrafish. Zebrafish 2014;11:462-78. [Crossref] [PubMed]
  16. Chang TC, Yu D, Lee YS, et al. Widespread microRNA repression by Myc contributes to tumorigenesis. Nat Genet 2008;40:43-50. [Crossref] [PubMed]
  17. Sun HL, Cui R, Zhou J, et al. ERK Activation Globally Downregulates miRNAs through Phosphorylating Exportin-5. Cancer Cell 2016;30:723-36. [Crossref] [PubMed]
  18. Lin S, Gregory RI. MicroRNA biogenesis pathways in cancer. Nat Rev Cancer 2015;15:321-33. [Crossref] [PubMed]
  19. Izzotti A, Pulliero A. The effects of environmental chemical carcinogens on the microRNA machinery. Int J Hyg Environ Health 2014;217:601-27. [Crossref] [PubMed]
  20. Cohen A, Burgos-Aceves MA, Smith Y. microRNAs downregulation in cancer is associated with guanine enrichment in the terminal loop sequences of their precursors. MicroRNA 2018;7:20-7. [Crossref] [PubMed]
  21. Cohen A, Burgos-Aceves MA, Smith Y. Global microRNA downregulation: all roads lead to estrogen. J Xiangya Med 2017;2:59. [Crossref]
  22. Cohen A, Burgos-Aceves MA, Kahan T, et al. Estrogen repression of microRNAs is associated with high guanine content in the terminal loop sequences of their precursors. Biomedicines 2017;5:3. [Crossref] [PubMed]
  23. Cheng AS, Jin VX, Fan M, Smith LT, et al. Combinatorial analysis of transcription factor partners reveals recruitment of c-MYC to estrogen receptor-alpha responsive promoters. Mol Cell 2006;21:393-404. [Crossref] [PubMed]
  24. Musgrove EA, Sergio CM, Loi S, et al. Identification of functional networks of estrogen- and c-Myc-responsive genes and their relationship to response to tamoxifen therapy in breast cancer. PLoS One 2008;3. [Crossref] [PubMed]
  25. Telang NT, Arcuri F, Granata OM, et al. Alteration of oestradiol metabolism in myc oncogene-transfected mouse mammary epithelial cells. Br J Cancer 1998;77:1549-54. [Crossref] [PubMed]
  26. Boysen G, Pachkowski BF, Nakamura J, et al. The formation and biological significance of N7-guanine adducts. Mutat Res 2009;678:76-94. [Crossref] [PubMed]
  27. Cavalieri EL, Rogan EG. Depurinating estrogen-DNA adducts, generators of cancer initiation: their minimization leads to cancer prevention. Clin Transl Med 2016;5:12. [Crossref] [PubMed]
  28. Cavalieri E, Chakravarti D, Guttenplan J, et al. Catechol estrogen quinones as initiators of breast and other human cancers: implications for biomarkers of susceptibility and cancer prevention. Biochim Biophys Acta 2006;1766:63-78. [PubMed]
  29. Słowikowski BK, Lianeri M, Jagodziński PP. Exploring estrogenic activity in lung cancer. Mol Biol Rep 2017;44:35-50. [Crossref] [PubMed]
  30. Kawanishi S, Hiraku Y, Oikawa S. Mechanism of guanine-specific DNA damage by oxidative stress and its role in carcinogenesis and aging. Mutat Res 2001;488:65-76. [Crossref] [PubMed]
  31. Senthilkumar K, Grozema FC, Guerra CF, et al. Mapping the sites for selective oxidation of guanines in DNA. J Am Chem Soc 2003;125:13658-9. [Crossref] [PubMed]
  32. Liu G, Min H, Yue S, et al. Pre-miRNA loop nucleotides control the distinct activities of mir-181a-1 and mir-181c in early T cell development. PLoS One 2008;3. [Crossref] [PubMed]
  33. Trujillo RD, Yue SB, Tang Y, et al. The potential functions of primary microRNAs in target recognition and repression. EMBO J 2010;29:3272-85. [Crossref] [PubMed]
  34. Yue SB, Trujillo RD, Tang Y, et al. Loop nucleotides control primary and mature miRNA function in target recognition and repression. RNA Biol 2011;8:1115-23. [Crossref] [PubMed]
  35. Libri V, Miesen P, van Rij RP, et al. Regulation of microRNA biogenesis and turnover by animals and their viruses. Cell Mol Life Sci 2013;70:3525-44. [Crossref] [PubMed]
  36. Heo I, Joo C, Kim YK, et al. TUT4 in concert with Lin28 suppresses microRNA biogenesis through pre-microRNA uridylation. Cell 2009;138:696-708. [Crossref] [PubMed]
  37. Trabucchi M, Briata P, Garcia-Mayoral M, et al. The RNA-binding protein KSRP promotes the biogenesis of a subset of microRNAs. Nature 2009;459:1010-4. [Crossref] [PubMed]
  38. García-Mayoral MF, Díaz-Moreno I, Hollingworth D, et al. The sequence selectivity of KSRP explains its flexibility in the recognition of the RNA targets. Nucleic Acids Res 2008;36:5290-6. [Crossref] [PubMed]
  39. Yuan H, Deng R, Zhao X, et al. SUMO1 modification of KHSRP regulates tumorigenesis by preventing the TL-G-Rich miRNA biogenesis. Mol Cancer 2017;16:157. [Crossref] [PubMed]
  40. Surh YJ. Cancer chemoprevention with dietary phytochemicals. Nat Rev Cancer 2003;3:768-80. [Crossref] [PubMed]
  41. Izzotti A, Calin GA, Steele VE, et al. Chemoprevention of cigarette smoke-induced alterations of MicroRNA expression in rat lungs. Cancer Prev Res (Phila) 2010;3:62-72. [Crossref] [PubMed]
  42. Gupta P, Wright SE, Kim SH, et al. Phenethyl isothiocyanate: a comprehensive review of anti-cancer mechanisms. Biochim Biophys Acta 2014;1846:405-24. [PubMed]
  43. Hecht SS, Trushin N, Rigotty J, et al. Complete inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced rat lung tumorigenesis and favorable modification of biomarkers by phenethyl isothiocyanate. Cancer Epidemiol Biomarkers Prev 1996;5:645-52. [PubMed]
  44. Izzotti A, Balansky RM, Dagostini F, et al. Modulation of biomarkers by chemopreventive agents in smoke-exposed rats. Cancer Res 2001;61:2472-9. [PubMed]
  45. Izzotti A, Bagnasco M, Cartiglia C, et al. Modulation of multigene expression and proteome profiles by chemopreventive agents. Mutat Res 2005;591:212-23. [Crossref] [PubMed]
  46. Aggarwal BB, Ichikawa H. Molecular targets and anticancer potential of indole-3-carbinol and its derivatives. Cell Cycle 2005;4:1201-15. [Crossref] [PubMed]
  47. Kang L, Ding L, Wang ZY. Isothiocyanates repress estrogen receptor alpha expression in breast cancer cells. Oncol Rep 2009;21:185-92. [PubMed]
  48. Kang L, Wang ZY. Breast cancer cell growth inhibition by phenethyl isothiocyanate is associated with down-regulation of oestrogen receptor-alpha36. J Cell Mol Med 2010;14:1485-93. [Crossref] [PubMed]
  49. Sundar SN, Kerekatte V, Equinozio CN, et al. Indole-3-carbinol selectively uncouples expression and activity of estrogen receptor subtypes in human breast cancer cells. Mol Endocrinol 2006;20:3070-82. [Crossref] [PubMed]
  50. Meng Q, Yuan F, Goldberg ID, et al. Indole-3-carbinol is a negative regulator of estrogen receptor-alpha signaling in human tumor cells. J Nutr 2000;130:2927-31. [Crossref] [PubMed]
  51. Cohen A, Burgos-Aceves MA, Smith Y. A potential role for estrogen in cigarette smoke-induced microRNA alterations and lung cancer. Transl Lung Cancer Res 2016;5:322-30. [Crossref] [PubMed]
  52. Izzotti A, Kanitz S, D'Agostini F, et al. Formation of adducts by bisphenol A, an endocrine disruptor, in DNA in vitro and in liver and mammary tissue of mice. Mutat Res 2009;679:28-32. [Crossref] [PubMed]
  53. Jang M, Cai L, Udeani GO, et al. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 1997;275:218-20. [Crossref] [PubMed]
  54. Zahid M, Gaikwad NW, Rogan EG, et al. Inhibition of depurinating estrogen-DNA adduct formation by natural compounds. Chem Res Toxicol 2007;20:1947-53. [Crossref] [PubMed]
  55. Lu F, Zahid M, Wang C, et al. Resveratrol prevents estrogen-DNA adduct formation and neoplastic transformation in MCF-10F cells. Cancer Prev Res (Phila) 2008;1:135-45. [Crossref] [PubMed]
  56. Zahid M, Saeed M, Beseler C, et al. Resveratrol and N-acetylcysteine block the cancer-initiating step in MCF-10F cells. Free Radic Biol Med 2011;50:78-85. [Crossref] [PubMed]
  57. Hinrichs B, Zahid M, Saeed M, et al. Formation of diethylstilbestrol-DNA adducts in human breast epithelial cells and inhibition by resveratrol. J Steroid Biochem Mol Biol 2011;127:276-81. [Crossref] [PubMed]
  58. Yager JD. Mechanisms of estrogen carcinogenesis: The role of E2/E1-quinone metabolites suggests new approaches to preventive intervention-A review. Steroids 2015;99:56-60. [Crossref] [PubMed]
  59. Izzotti A, Cartiglia C, Steele VE, et al. MicroRNAs as targets for dietary and pharmacological inhibitors of mutagenesis and carcinogenesis. Mutat Res 2012;751:287-303. [Crossref] [PubMed]
doi: 10.21037/jxym.2018.09.01
Cite this article as: Cohen A, Burgos-Aceves MA, Smith Y. Guanine content of precursor microRNA’s terminal loop and its association with cancer. J Xiangya Med 2018;3:33.