The growing family of miRNAs regulate a diverse range of cellular processes and can impact on the progression of various diseases, through RNA-mediated gene-silencing mechanisms. Many current studies focus on regulatory functions of miRNAs, few are directed towards their transcriptional regulation. Although work into defining the structural features of miRNA promoters is limited, characterisation of miRNA promoter regions has gone some way to predicting miRNA transcription start sites.
Advancements in computational and biochemical methods [349-351] have revealed that many miRNAs use their own transcription start sites (TSS), whether they are located within non-protein coding DNA between genes (intergenic) or embedded within the introns of protein coding genes (intronic). However, some intragenic (within introns or exons) miRNAs use host gene TSS [352]. Recent studies of miRNA transcription have elucidated that intragenic miRNAs are co-transcribed by RNA polymerase II, or independently transcribed from their own RNA polymerase II or III initiation sites [353, 354]. It is, therefore, important when determining the TSS for miRNA, to take into consideration RNA polymerase II and III initiation sites within the promoter sequence.
Modifications to histones on promoter regions can provide some indication of where putative TSSs begin, such as trimethylation of lysine-4 of histone-3 (H3K4me3) and acetylation of lysine-9/14 of histone-3 (H3K9/14Ac). These have proven to be valuable markers of transcriptionally active promoters [355-358]. TSS identification has also been hampered by difficulty in determining start sites proximal to the mature miRNA sequence, especially when pri-miRNAs can have large and variable lengths [359, 360]. Despite this, an additional characteristic of transcriptionally active genes was observed, they were found to be
nucleosome-free within 100-130bp surrounding their TSS [361, 362]. A combination of these observations with reoccurring sequences commonly found within known TSS and at the 5’ end of mRNA (TSS and cap analysis gene expression (CAGE) tags respectively), has led to the development of search engine software to help identify putative TSS of pri-miRNAs [352]. These techniques will be used in this chapter to help elucidate putative TSS for miR-7. Several lines of evidence have recently emerged to suggest that miRNAs participate in self- regulatory loops, modulating their own expression. Recently, miR-145 was identified to be dependent on TP53 activation, which in turn could stimulate miR-145 expression [363]. It was also reported that several miRNAs regulate the expression of receptors and are themselves regulated by those same receptors in cancer cell-lines [364]. Since many miRNAs can have multiple mRNA targets, further study of the interaction between miRNAs and their regulatory factors may help illustrate their role in normal function and disease progression. It has been previously indicated that miR-7 was a key mediator of EGFR signalling in lung cancer cells [332, 333]. It was found that c-Myc bound to the miR-7 promoter and enhanced its activity; and this was dependent on the activation of the EGFR-regulated Ras/ERK/c-Myc pathway [332]. In addition, miR-7 has been reported to target Akt and blocking the EGFR- mediated PI3K/Akt pathway also attenuated miR-7 expression [365]. These studies highlight two self-regulatory loops for miR-7 expression. However, in the cellular ageing of fibroblasts, the expression of EGFR is lost as highlighted in previous studies [159, 160] and the results of the previous Chapter, therefore suggesting the activity of Ras/ERK/c-Myc and the EGFR- dependent PI3K/Akt pathway would also be diminished. Therefore, the upregulated expression of miR-7 in aged fibroblasts is likely to be through an alternative receptor or signalling pathway, that is persistently activated through cellular ageing and can potentially activate c-Myc, Akt or additional enhancers of miR-7 expression. This results Chapter will further investigate the promoter region for miR-7 and any changes in transcription factors in
response to cytokine treatments, to help describe an alternative mechanism of miR-7 upregulation.
5.1.2 17β-Estradiol (E2)
17β-Estradiol (E2) is a derivative of the sex hormone estrogen and has the greatest potency of the estrogen by-products [366, 367]. Although E2 has greater serum abundance in females, it is also present in males as an active metabolic product of testosterone. However, the serum levels of E2 in elderly men (20-60pmol/L) are roughly comparable to those of elderly women (<35pmol/L). Therefore, in old age there is little discrepancy between gender and E2 levels [368]. E2 in vivo is interconvertible with estrone [367] and its main role is in reproduction. However, it also plays an important role in other organ systems and bone metabolism [369- 371].
During the reproductive years, most E2 in women is produced by the granulosa cells of the ovaries, through estrone to E2 conversion by 17β-hydroxysteroid dehydrogenase [367]. Smaller amounts of E2 are also produced by the adrenal cortex and in men, by the testes. In both sexes, testosterone is converted to E2 by aromatisation [372, 373] and adipose tissues can produce active precursors of E2 [374].
E2 enters cells freely and interacts with cytoplasmic target cell receptors, estrogen receptor-α (ERα) and estrogen receptor-β (ERβ). The ER-complex can enter the nucleus of the target cell [375]; and regulate gene transcription through the modulation of additional transcription factors and binding to specific DNA sequences. This ultimately leads to modulation of gene expression and mRNA transcription, DNA replication and cellular proliferation. In plasma, E2 is largely bound to globulin or albumin and only a fraction (2.21% ± 0.04%) is free and biologically active [376].
Changes in the body shape affecting bones, joints, fat structure and deposition; and skin composition, are modified by E2 [374, 377-380]. The development and progression of cancers such as breast cancer, ovarian cancer and endometrial cancer, have also been cited as E2 driven [381-383]. Interestingly, it has also been noted that E2 treatments protect against cellular senescence [384].