30 minute read
A comparative review of miR-21-5p and miR-27a as potential bioindicators for treatment of Mycobacterium tuberculosis
A comparative review of miR-21-5p and miR-27a as potential bioindicators for treatment of Mycobacterium tuberculosis
Noah Kentmann
Normanhurst Boys High School
Abstract
Tuberculosis (TB) infects over 10 million people each year, and there is a need to develop novel TB treatments that bypass the growing issue of drug-resistant TB. This review aimed to differentiate between the viability of miR-21-5p and miR-27a as potential bioindicators for the treatment of mycobacterium tuberculosis (MTB) infection, with the goal of defining one of the two as a potential focus for future research. Through quantitative real-time polymerase chain reaction (qRT-PCR) analysis of MTB-challenged macrophages, miRNA-21-5p was found to be upregulated, while miR27a was found to be downregulated. Through ELISA, the concentration of interleukin (IL)-β, IL-6 and tumour necrosis factor (TNF)-α in macrophages after infection with MTB and transfection with miRNA-mimics, and miRNA-inhibitors revealed that the respective upregulation and downregulation of miR-21-5p and miR-27a leads to host immunosuppression and increased intracellular survival of MTB. A t-test demonstrated that miR-21-5p has a higher mean correlation of relative expression in relation to MTB infection than miR-27a. These findings indicate that miRNA plays a key role in the immune response to MTB infection, and that miR-21-5p may be an effective bioindicator for future treatment of TB.
Literature review
In 2021, approximately 1.6 million people died as a result of TB infection, caused by the obligate intracellular pathogen MTB. 1 Globally, 25% of the population is infected by latent TB, which becomes active in 5-10% of these individuals. 2 The Bacille Calmette–Guérin (BCG) vaccine, is currently the only licensed vaccine for TB. 3 Whilst the vaccine prevents severe forms of TB in infants and young children it does not prevent primary infection, the reactivation of latent pulmonary infection or sufficiently protect adolescents and adults, who make up almost 90% of TB transmissions globally. 4 Additionally, the most common method for treating active TB, a regimen of the antibiotic isoniazid (INH), paired with three other drugs rifampicin, pyrazinamide and ethambutol, 5 while effective is lengthy, expensive, and can promote the propagation of antibiotic-resistant strains of TB. 6 Consequently, multidrug-resistant (MDR)-TB is becoming increasingly prevalent, and was present in 3.9% of cases in 2021. 7 MDR-TB is problematic as its treatment necessitates second-line TB drugs which are less effective, resulting in longer, more expensive treatment and a greater number of severe side effects for the patient. 8
MTB has several immunosuppressive mechanisms that enable its survival within the host environment, for example, causing cell apoptosis in macrophages, 9 innate immune cells responsible for first-line management and killing of MTB during infection. These mechanisms are generally epigenetic, meaning they involve modifications to gene expression in a stable, heritable manner, without altering gene sequencing itself. 10 One example of such epigenetic changes are miRNA modifications which by binding with mRNA in the cytoplasm cause it to either be recycled into smaller components or preserved and translated later. 11
As these miRNA modifications are critical for the survival of MTB following the infective phase of pathogenesis, they present an opportunity for host-directed therapies that would function by reverting the changes caused by MTB, thereby limiting its survival capability within the host. Host-directed treatments are particularly valuable within the context of MTB as they may improve host response to infection by enabling both the innate and immune systems, whilst concurrently bypassing the risks of both the development of drug resistance and drug resistance itself. 12 Although epigenetic therapies may offer a way to speed up and improve the effectiveness of current treatment, they are novel and have not yet been used to treat infectious diseases in human patients, however, they have been successfully used to treat highly methylated cancers such as some breast cancers. 13 Thus far, several miRNA have been identified as having therapeutic potential against MTB. 14 Recently identified in two review articles, miR21-5p and miR-27a have been noted as facilitators of host-immunosuppression during MTB pathogenesis. 15 16 During infection miR21-5p attenuates the secretion of IL-β, IL-6 and TNF-α in THP-1 macrophages by targeting B-cell lymphoma 2 (Bcl-2) and toll-like receptor 4 (TLR4), indirectly mediating several immune responses and cell survival. 17 Similarly, miR-27a attenuates the secretion of the same cytokines with the addition of interferon-γ (IFN-γ), by targeting interleukin-1 receptor-associated kinase 4 (IRAK4). 18
As an emerging field, little has been done within epigenetics to establish conformance or reproducibility standards for biomarker studies regarding miRNA. As such, a majority of studies contain heterogenous methodological designs, characterised by inconsistent results. 19 To negate this fact, this comparative review examined many peerreviewed articles in order to find those which were reliable, comparable and highly relevant. The Paper Experimental Methods was then developed from those established by Zhao, et al., and Wang et al., in 2019 and 2017 respectively, which were found to meet all criteria established in the Systematic Review. 20
The next frontier in the epigenetic field is to further research potential biomarkers for future treatments. The object of this paper is to then identify which, out of miR-21-5p and miR-27a, is the most promising via analysis of their presence in MTB affected THP-1 cells.
1 Geneva: World Health Organisation, ‘Global Tuberculosis Report 2021’, 2021.
2 Centers for Disease Control and Prevention, ‘The Difference Between Latent TB Infection and TB Disease’, 2014.
3 Geneva: World Health Organisation, ‘Global Tuberculosis Report 2021’, 2021.
4 Ibid.
5 American Lung Association, ‘Treating and Managing Tuberculosis’, 2022.
6 Centers for Disease Control and Prevention, ‘Multidrug-Resistant Tuberculosis (MDR TB) Fact Sheet’, 2016.
7 Geneva: World Health Organisation, ‘Global Tuberculosis Report 2021’, 2021.
8 Rendon, A., Tiberi, S., Scardigli, A., D’Ambrosio, L., Centis, R., Caminero, J.A., Battista Migliori, G., ‘Classification of drugs to treat multidrug-resistant tuberculosis (MDR-TB): evidence and perspectives’, 2016.
9 Kundu, M., Basu, J., ‘The Role of microRNAs and Long Non-Coding RNAs in the Regulation of the Immune Response to Mycobacterium tuberculosis Infection’, 2021.
10 Centers for Disease Control and Prevention, ‘What is Epigenetics?’, 2022.
11 O’Brien, J., Hayder, H., Zayed, Y., Peng, C., ‘Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation’, 2018.
12 Tobin, D.M., ‘Host-Directed Therapies for Tuberculosis’, 2015.
13 Mack G.S., ‘Epigenetic Cancer Therapy Makes Headway’, 2006.
14 See footnotes 15 and 16.
15 Sinigaglia, A., Peta, E., Riccetti, S., Venkateswaran, S., Manganelli, R., Barzon, S., ‘Tuberculosis-Associated MicroRNAs: From Pathogenesis to Disease Biomarkers’, 2020.
16 Kundu, M., Basu, J., ‘The Role of microRNAs and Long Non-Coding RNAs in the Regulation of the Immune Response to Mycobacterium tuberculosis Infection’, 2021.
17 Zhao, Z., Hao, J., Li, X., Chen, Y., Qi, X., ‘MiR-21-5p regulates mycobacterial survival and inflammatory responses by targeting Bcl-2 and TLR4 in Mycobacterium tuberculosis-infected macrophages’, 2019.
18 Wang, J., Jia, Z., Wei, B., Zhou, Y., Niu, C., Bai, S., Tong, C., Song, J., Li, Y., ‘MicroRNA-27a restrains the immune response to mycobacterium tuberculosis by targeting IRAK4, a promoter of the NF-κB pathway’, 2017.
19 Ueberberg B., Kohns, M., Mayatepek, E., Jacobsen, M., ‘Are microRNAs suitable biomarkers of immunity to tuberculosis?’, 2014.
20 See reference list for selected papers.
Methodology
to then identify which, out of miR-21-5p and miR-27a, is the most promising via analysis of their presence in MTB affected THP-1 cells.
Scientific research question
Would miR-21-5p or miR-27a be a more effective biomarker to inform future epigenetic studies for the eventual creation of epigenetic treatments for MTB?
Scientific hypothesis
The mean correlation of relative expression of miR-21-5p will be significantly greater than that of miR-27a in THP-1 cells when infected with MTB strain H37Rv. This assertion is supported by existing literature, within which, statistics illustrate a greater increase in the relative expression of miR-21-5p within MTB-challenged macrophages when compared to the decrease in the relative expression of miR-27a in the same cells.
Systematic Review
Secondary data was collated through a systematic review. It was carried out with the intention of determining whether miR-21-5p or miR-27a would be a more effective biomarker in informing future epigenetic treatments for MTB infection. Secondary data was found on the following electronic, web-based databases: Google Scholar, PubMed, PubMed Central, Science Direct, JSTOR, and the New England Journal for Medicine. Non-English articles were omitted. The following terms were used to generate the respective searches: ‘epigenetics’, ‘RNA modification’, ‘miRNA’, ‘miR-27a’, ‘miR-21-5p’ ‘mycobacterium tuberculosis’, and ‘therapies’.
Once data was collected it was reviewed through a classification process regarding the cell chosen for transfection, for example THP1.The resultant papers were then reviewed on an individual basis and consequently grouped relative to their methodology and statistical results to ensure all data was relevant and analogous. Finally, the two selected papers were chosen as their methodologies were discernibly identical and no such links were established among other papers.
Statistical Analysis Methods
All statistics were generated using data from the selected papers. The scatter plots in figures 1 and 2 were generated in Microsoft Excel. The column graphs in figures 3, 4, and 5 were generated in Microsoft Excel. The ttest in figure 6 was generated using the Pearson Correlation Constant for the relative expression of miRNA regarding both MOI and Time-Post Infection (TPI), which were established in the statistical analysis carried out during the creation of figures 1 and 2. The correlation coefficients were then utilised to form a synthetic data set that was consequently bootstrapped in RStudio. Bootstrapping was repeated 1000 times to form a larger, more consistent data that controlled variance within the data set, such that it could be analysed precisely via a t-test.
Results
MTB infection causes upregulation of miRNA-21-5p and downregulation of miR27a
To investigate the expression of the given miRNA, THP-1 cells were infected with H37Rv at an MOI of 0 (the PBS control group), 1, 5, and 10 for 24 hours. The consequent levels of expression were detected by qRT-PCR analysis and are illustrated in Figure 1 and Figure 2 respectively. As shown the expression of miR-21-5p was significantly upregulated relative to both dosage and time, evident in the positive correlation of both figures. In contrast, the expression of miR-27a was significantly downregulated relative both to dosage and time, as indicated by the negative correlation in both figures. These results, particularly considering Figure 1, and the consequent increase or decrease following the complete stability of the PBS control group, indicate that MTB infection of THP-1 cells greatly increased the expression of miR-21-5p and decreased the expression of miR-27a. As such, these findings suggest a potential correlation between MTB infection and the given miRNA.
MiR-21-5p and miR-27a promote MTB survival in MTB-infected THP-1 cells
To explore the biological effects of the given miRNA on the immune response, the concentration of IL-6, IL-β, and TNF-α were determined after MTB infection and transfection with either miR-21-5p mimics or miR-27a mimics. Consequently, these results were compared with those from the transfection of miR-21-5p mimics negative control (NC) and miR-27a mimics NC. As shown in figures 3-5, transfection with miR-215p mimics and miR-27a mimics decreased the concentration of IL-6, IL-β and TNF-α. The concentration of IL-6, IL-β and TNF-α were determined after infection with MTB and transfection with both miR-21-5p and miR-27a inhibitors, which resulted in the promotion of cytokine secretions in both miR-21-5p and miR-27a inhibitor-transfected THP-1 cells.
MiR-21-5p has a higher mean correlation of relative expression than miR-27a
To determine which miRNA should be the focus of future research, the mean correlation of relative expression of miR-21-5p and miR27a relative to MTB infection in THP-1 cells was determined through a t-test. The t-test indicated that miR-21-5p has a higher average correlation of relative expression (0.96298205), compared to miR-27a (0.95196439). The t-stat of -28.093397 was less than the t-Critical one-tail value of 1.64568263, indicating that this was a significant result.
Discussion
Considering the annually growing number of global TB cases and the increasing threat of drug resistance, this comparative review aimed to identify potential biomarkers for novel, host-directed epigenetic treatments. It was shown that MTB infection correlates with the upregulation of miR-21-5p and the downregulation of miR-27a. The effects of which were explored through measurement of the secretion of the inflammatory cytokines IL-6, TNF-α and IL-β. It was revealed that miR21-5p had on average a higher relative expression than miR-27a in MTB-infected macrophages.
The results of this study demonstrated that there is a high likelihood that MTB infection within THP-1 cells causes an increase in the expression of miR-21-5p and a decrease in the expression of miR-27a. In figures 1 and 2, miR27p was significantly downregulated in both a time- and dose-dependent manner, indicated by the negative correlation between its relative expression and the MOI or TPI. Conversely, miR-21-5p was significantly upregulated in both a time- and dosedependent manner, indicated by the positive correlation between its relative expression and the MOI or TPI.
The respective upregulation and downregulation of miRNA, consequently attenuated the concentration of cytokines IL6, TNF-α and IL-β. This is evident in the lower values of the miRNA mimic transfected THPcells in figures 3-5 when compared to the miRNA mimic negative control values, which were significantly higher. Similarly, the increase in cytokine concentration brought about by the transfection of miRNA inhibitors indicated that miR-21-5p and miR-27a were responsible for the drops in concentration, as their blocking resulted in higher concentrations of all cytokines. As noted in a 2017 study by Zhao et al. miR-21-5p mediates this process by targeting Bcl-2 and TLR4, 21 while Wang et al. in a 2019 study discovered that miR-27a mediates this same process by targeting IRAK4. 22 Both studies reinforce the utility of miR-21-5p and miR-27a as the existence of mediating pathways present more accessible targets for future epigenetic therapies. These findings indicate that miR-215p and miR-27a are actively manipulated by MTB to ensure survival within macrophages by regulating the inflammatory response through the reduction in concentration of the inflammatory cytokines. Thus resulting in accelerated macrophage apoptosis, exaggerated inflammation, tissue injury and increased intracellular survival of MTB.
While upon this basis it may be concluded that both miR-21-5p and miR-27a are suitable biomarkers for potential treatment of MTB as they are both regulators of pathogenesis and have clear mediating pathways to serve as targets, in the interest of directing future research this study then decided to compare the viability of both targets directly. The results of the t-test progressed scientific understanding of potential miRNA bioindicators by highlighting the higher mean correlation of relative expression of miR-215p in MTB-challenged THP-1 cells when compared to miR-27a, suggesting that miR21-5p should be the focus of future research into epigenetic treatments for MTB.
Whilst the study does well to investigate and compare the potential biomarkers miR-21-5p and miR-27a, there were several limitations. Secondary data was extracted only from what was available within the given publications, which had already been collated and statistically processed. Access to the raw data would have allowed for stronger statistical comparisons to have been made. Nonetheless, all the data gathered from the selected papers was valid as the papers were peer reviewed and congruent with existing literature. More specifically, the assertions that miR-21-5p was upregulated and that miR27a was downregulated during MTB-infection of macrophages, alongside the observations regarding cytokine concentrations, were harmonious with other similar studies found. 23, 24, 25, 26, 27 Additionally, all data that was collected was from experiments that had been repeated independently three times to ensure its reliability. However, it is difficult to verify the precision of the selected papers as there is no mention of systematic or random errors, which may have altered the results, however this possibility seems unlikely given the fact that the experiments had been repeated to similar effects by several studies. Regarding the statistical analysis, figures 1-5 are largely identical to those found within the selected papers, just adjusted to enable easier comparison, and thus do not necessitate greater consideration. However, figure 6 and the related t-test are notably limited by their construction. The t-test was generated from a data set of less than twenty figures, which may have led to a lack of precision within the data set. However, this issue was combated through the construction of a synthetic data set, which was consequently bootstrapped to generate a larger, more reliable data set. While it is possible this process may have caused a discrepancy in the results, this seems unlikely, as the three repeated tests all generated the same result with little variance.
Therefore, this study provides reasonable grounds for future epigenetic research into MTB treatments that can be used to combat the rising issue of drug-resistant strains which limit the efficacy of first-line drugs. 28 In doing so, the first process would be to better understand the mechanisms through which the pathogen overrides general miR-21-5p function and thus alters the epigenome to increase intracellular survival. Following which, Bcl-2 and TFR4, as mediators through which miR-21-5p carries out its biological processes, should become targets for treatments. If a means of reversing or inhibiting these biological processes may be developed, then treatments may be rolled out once appropriate testing has been conducted.
However, it is worthwhile to note that the propagation of epigenetic treatments is likely to raise confidentiality issues. In a similar manner to genetic screenings, epigenetic screenings could provide substantial information about an individual’s health and lifestyle which, when uploaded into comprehensive electronic health records may be disclosed non-consensually, leading to loss of privacy and potential discrimination and reidentification. As such the management of health records will need to be developed alongside epigenetic technology. Additionally, epigenetic experimentation, which often uses knockout mice for testing, must be held to the same strict standards as other fields of biotechnology that test on animals to ensure that they are treated humanely. 29 Further, once the production of epigenetic drugs ensues, production must be carried out ethically such that products may be recycled or otherwise disposed of without causing damage to the environment. Similarly, it is important that these treatments are sold at a relatively reasonable price to ensure equity in accessibility.
21 Zhao, Z., Hao, J., Li, X., Chen, Y., Qi, X., ‘MiR-21-5p regulates mycobacterial survival and inflammatory responses by targeting Bcl-2 and TLR4 in Mycobacterium tuberculosis-infected macrophages’, 2019.
22 Wang, J., Jia, Z., Wei, B., Zhou, Y., Niu, C., Bai, S., Tong, C., Song, J., Li, Y., ‘MicroRNA-27a restrains the immune response to mycobacterium tuberculosis by targeting IRAK4, a promoter of the NF-κB pathway’, 2017.
23 Hacket, E.E., Charles-Messance, H., O’Leary, S.M., Gleeson, L.E., Muñoz-Wolf, N., Case, S., Wedderburn, A., Johnston, D.G.W., Williams, M.A., Smyth, A., Oiumet, M., Moore, K.J., Lavelle, E.C., Corr, S.C., Gordon, S.V., Keane, J., Sheedy, F.J., ‘Mycobacterium tuberculosis Limits Host Glycolysis and IL-1β by restriction of PFK-M via MicroRNA-21’, 2020.
24 Meng, Q.L., Liu, F., Yang, X.Y., Liu, X.M., Zhang, X., Zhang, C., Zhang, Z.D., ‘Identification of latent tuberculosis infection-related microRNAs in human U937 macrophages expressing Mycobacterium tuberculosis Hsp16.3’, 2014.
25 Furci, L., Schena, E., Miotto, P., Cirillo, D.M., ‘Alteration of human macrophages microRNA expression profile upon infection with Mycobacterium tuberculosis’, 2013.
26 Wang, J., Xion, Y., Fu, B., Guo, D., Zaky, M.Y., Lin, X., Wu, H., ‘MicroRNAs as immune regulators and biomarkers in tuberculosis’, 2022.
27 Sinigaglia, A., Peta, E., Riccetti, S., Venkateswaran, S., Manganelli, R., Barzon, S., ‘Tuberculosis-Associated MicroRNAs: From Pathogenesis to Disease Biomarkers’, 2020.
28 Geneva: World Health Organisation, ‘Global Tuberculosis Report 2021’, 2021.
29 Ormandy, E.H., Dale, J., Griffin, G., ‘Genetic engineering of animals: Ethical issues, including welfare concerns’, 2011.
Conclusion
This study aimed to determine whether miR21-5p or miR-27a would serve as a more viable bioindicator for future epigenetic treatment of MTB infection. Through a review it compared the expression of the miRNA in macrophages following MTB infection and consequently compared the effects on inflammatory cytokines. Through further testing it was concluded that, due to its higher correlation of relative expression miR-21-5p would serve as a more viable bioindicator for future treatments. However, the data upon which this conclusion subsists is small in sample size, somewhat limiting the credibility of this conclusion despite its seeming reliability, precision, and validity. Nonetheless, this experiment suggests that MTB research turns its attention towards miR-21-5p and its therapeutic potential for future studies, considering this new evidence regarding its high correlation of expression with MTBinfection in macrophages.
Acknowledgements
Thank you to Nicole Batten of Sydney University for critiquing my work and providing me with constructive feedback to both improve this project, and my writing. To my science teacher, Ms Ritu Bhamra, thank you for your continued support and feedback on my project. Thank you also to my peers who listened to my ideas regarding my project and gave me the opportunity to bounce my ideas around.
Reference list
Reviewed Papers
Wang, J., Jia, Z., Wei, B., Zhou, Y., Niu, C., Bai, S., Tong, C., Song, J., Li, Y., ‘MicroRNA-27a restrains the immune response to mycobacterium tuberculosis by targeting IRAK4, a promoter of the NF-κB pathway’, International Journal of Clinical and Experimental Pathology, 2017, https://www.ncbi.nlm.nih.gov/pmc/articles/PM C6965936/, (accessed 23 May, 2023).
Zhao, Z., Hao, J., Li, X., Chen, Y., Qi, X., ‘MiR-215p regulates mycobacterial survival and inflammatory responses by targeting Bcl-2 and TLR4 in Mycobacterium tuberculosisinfected macrophages’, FEBS Letters, Vol. 593, Issue 12, 2019, https://febs.onlinelibrary.wiley.com/doi/full/10 .1002/1873-3468.13438, (accessed 2 June, 2023).
Additional References
American Lung Association, ‘Treating and Managing Tuberculosis’, American Lung Association, 2022, https://www.lung.org/lunghealth-diseases/lung-diseaselookup/tuberculosis/treating-andmanaging#:~:text=How%20Is%20Active%20T B%20Treated, rifampin%2C%20pyrazinamide%20and%20et hambutol, (accessed 27 Jan, 2023).
Bierne, H., Hamon, M., Cossart, P., ‘Epigenetics and Bacterial Infections’, Cold Spring Harbor Perspectives in Medicine, 2012, https://www.ncbi.nlm.nih.gov/pmc/articles/PM C3543073/, (accessed 8 Jan, 2023).
Budzik, J.M., Swaney, D.L., Jiminez-Morales, D., Johnson, J.R., Garelis, N.E., Repasy, T., Roberts, A.W., Popov, L.M., Parry, T.J., Pratt, D., Ideker, T., Krogan, N.J. and Cox, J.S., , ‘Dynamic posttranslational modification profiling of Mycobacterium tuberculosis-infected primary macrophages’, eLife, 2020, https://elifesciences.org/articles/51461.pdf, (accessed 3 Jan, 2023).
Cannell, I.G., Wen Kong, Y., Bushell, M., ‘How do microRNAs regulate gene expression?’, Biochem Society Transactions, 2008, https://portlandpress.com/biochemsoctrans/a rticle-abstract/36/6/1224/66536/How-domicroRNAs-regulate-geneexpression?redirectedFrom=fulltext, (accessed 27 Jan, 2023).
Centers for Disease Control and Prevention, ‘Multidrug-Resistant Tuberculosis (MDR TB) Fact Sheet’, Centers for Disease Control and Prevention, 2016, https://www.cdc.gov/tb/publications/factshee ts/drtb/mdrtb.htm, (accessed 23 Jan, 2023).
Centers for Disease Control and Prevention, ‘The Difference Between Latent TB Infection and TB Disease’, Centers for Disease Control and Prevention, 2014, https://www.cdc.gov/tb/publications/factshee ts/general/ltbiandactivetb.htm, (accessed 23 Jan, 2023).
Centers for Disease Control and Prevention, ‘What is Epigenetics?’, Centers for Disease Control and Prevention, 2022, https://www.cdc.gov/genomics/disease/epige netics.htm, (accessed 6 Jan, 2023).
Chuang, J.C., Jones, P.A., ‘Epigenetics and MicroRNAs’, Pediatric Research, 2007, https://www.nature.com/articles/pr2007127, (accessed 5 Feb, 2023).
Cole, J., Morris, P., Dickman, M.J., Dockrell, D.H., ‘The therapeutic potential of epigenetic manipulation during infectious disease’, Pharmacology & Therapeutics, pp. 85-99, 2016, https://www.ncbi.nlm.nih.gov/pmc/articles/PM C5109899/#, (accessed 2 Jan, 2023).
Delogu, G., Sali, M., Fadda, G., ‘The Biology of Mycobacterium Tuberculosis Infection’, Mediterranean Journal of Hematology and Infectious Diseases, 2013, https://www.ncbi.nlm.nih.gov/pmc/articles/PM C3867229/, (accessed 2 Jan, 2023).
Ehrt, S., Schnappinger, D., Bekiranov, S., Drenkow, J., Shi, S., Gingeras, T.R., Gaasterland, T., Schoolnik, G., Nathan, C., ‘Reprogramming of the Macrophage Transcriptome in Response to Interferon-γ and Mycobacterium tuberculosis’, Journal of Experimental Medicine, 2001, https://www.ncbi.nlm.nih.gov/pmc/articles/PM C2193509/, (accessed 17 April, 2023).
Etna, M.P., Sinigaglia, A., Grassi, A., Giacomini, E., Romagnoli, A., Pardini, M., Severa, M., Cruciani, M., Rizzo, F., Anastasiadou, E., Di Camillo, B., Barzon, L., Maria Fimia, G., Manganelli, R., Coccia, E.M., ‘Mycobacterium tuberculosis-induced miR-155 subverts autophagy by targeting ATG3 in human dendritic cells’, Plos Pathogens, 2018, https://journals.plos.org/plospathogens/articl e?id=10.1371/journal.ppat.1006790, (accessed 4 June, 2023).
Fatima, S., Kumari, A., Agarwal, M., Pahuja, I., Yadav, V., Prakash Dwivedi, V., Bhaskar, A., ‘Epigenetic code during mycobacterial infections: therapeutic implications for tuberculosis’, The FEBS Journal, Vol. 289, Issue 14, pp. 4172-4191, 2021, https://febs.onlinelibrary.wiley.com/doi/full/10 .1111/febs.16170, (accessed 5 Feb, 2023).
Fischer, N., ‘Infection-induced epigenetic changes and their impact on the pathogenesis of diseases’, Seminars in Immunopathology, 2020, https://www.ncbi.nlm.nih.gov/pmc/articles/PM C7174260/, (accessed 2 Jan, 2023).
Fol, M., Włodarczyk, M., Druszczyńska, M., ‘Host Epigenetics in Intracellular Pathogen Infections’, International Journal of Molecular Science, 2020, https://www.ncbi.nlm.nih.gov/pmc/articles/PM C7369821/#B15-ijms-21-04573, (accessed 18 Jan, 2023).
Fu, Y., Shen, J., Liu, F., Zhang, H., Zheng, Y., Jiang, X., ‘Andrographolide Suppresses Pyroptosis in Mycobacterium tuberculosisInfected Macrophages via the microRNA155/Nrf2 Axis’, Oxidative Medicine and Cellular Longevity, 2022, https://www.hindawi.com/journals/omcl/2022/ 1885066/, (accessed 24 June, 2023).
Fu, Y., Yi, Z., Li, J., Li, R., ‘Deregulated microRNAs in CD4+T cells from individuals with latent tuberculosis versus active tuberculosis’, Journal of Cellular and Molecular Medicine, Vol. 18, Issue 3, pp. 503-513, 2013, https://onlinelibrary.wiley.com/doi/10.1111/jcm m.12205, (accessed 27 May, 2023).
Furci, L., Schena, E., Miotto, P., Cirillo, D.M., ‘Alteration of human macrophages microRNA expression profile upon infection with Mycobacterium tuberculosis’, Divisions of Immunity, Transplantation and Infectious Diseases, Emerging Bacterial Pathogens Unit, San Raffaele Scientific Institute, 2013, https://journals.lww.com/ijmy/_layouts/15/oak s.journals/downloadpdf.aspx?an=01929415201302030-00003, (accessed 28 May, 2023).
Geneva: World Health Organisation, ‘Global Tuberculosis Report 2021’, World Health Organisation, 2021, https://www.who.int/publications/i/item/9789240037021, (accessed 6 Jan, 2023).
Geneva: World Health Organisation, ‘Global Tuberculosis Report 2022’, World Health Organisation, 2022, https://www.who.int/teams/globaltuberculosis-programme/tb-reports/global-tuberculosis-report-2022, (accessed 23 Jan, 2023).
Ghorpade, D.S., Leyland, R., KurowskaStolarska, M., Patil, S.A., Balaji, K.N., ‘MicroRNA-155 is required for Mycobacterium bovis BCG-mediated apoptosis of macrophages’, Molecular and Cellular Biology, 2012, https://pubmed.ncbi.nlm.nih.gov/22473996/, (accessed 28 May, 2023).
Grosjean, H., ‘RNA modification: the Golden Period 1995-2015’, RNA, 2015, https://www.ncbi.nlm.nih.gov/pmc/articles/PM C4371308/, (accessed 14 April, 2023).
Hacket, E.E., Charles-Messance, H., O’Leary, S.M., Gleeson, L.E., Muñoz-Wolf, N., Case, S., Wedderburn, A., Johnston, D.G.W., Williams, M.A., Smyth, A., Oiumet, M., Moore, K.J., Lavelle, E.C., Corr, S.C., Gordon, S.V., Keane, J., Sheedy, F.J., ‘Mycobacterium tuberculosis Limits Host Glycolysis and IL-1β by restriction of PFK-M via MicroRNA-21’, Cell Reports, Vol. 30, Issue 1, pp. 124-136, 2020, https://www.sciencedirect.com/science/articl e/pii/S2211124719316651?pes=vor, (accessed 8 June, 2023).
Hussain, T., Zhao, D., Zahid Ali Shah, S., Wang, J., Yue, R., Liao, Y., Sabir, N., Yang, L., Zhou, X., ‘MicroRNA 27a-3p Regulates Antimicrobial Responses of Murine Macrophages Infected by Mycobacterium avium subspecies paratuberculosis by Targeting Interleukin-10 and TGF-β-Activated Protein Kinase 1 Binding Protein 2’, Frontiers in Immunology, 2017, Vol. 8, https://www.frontiersin.org/articles/10.3389/fi mmu.2017.01915/full, (accessed 9 May, 2023).
Jang, D., Lee, A.H., Shin, H.Y., Song, H.R., Park, J.H., Kang, T.B., Lee, S.R., Yang, S.H., ‘The Role of Tumor Necrosis Factor Alpha (TNF-α) in Autoimmune Disease and Current TNF-α Inhibitors in Therapeutics’, International Journal of Molecular Sciences, 2021, https://www.ncbi.nlm.nih.gov/pmc/articles/PM C7962638/#:~:text=Tumor%20necrosis%20factor%20alpha%20(TNF%2Dα)%20is%20a%2 0cytokine,and%20autoimmune%20diseases% 20%5B1%5D., (accessed 19 July, 2023).
Kulkarni, S., Arumugam, T., Chuturgoon, A., An, P. and Ramsuran, V., ‘Editorial: Epigenetics of Infectious Diseases’, Frontiers, 2022, https://www.frontiersin.org/articles/10.3389/fimmu.2022.1054151/full, (accessed 2 Jan 2023).
Kumar, R., Halder, P., Sahu, S.K., Kumar, M., Kumari, M., Jana, K., Ghosh, Z., Sharma, P., Kundu, M., Basu, J., ‘Identification of a novel role of ESAT-6-dependent miR-155 induction during identification of macrophages with Mycobacterium tuberculosis’, Cellular Microbiology, Vol. 14, Issue 10, pp. 1620-1631, 2012, https://onlinelibrary.wiley.com/doi/full/10.1111/ j.1462-5822.2012.01827.x, (accessed 24 June, 2023).
Kundu, M., Basu, J., ‘The Role of microRNAs and Long Non-Coding RNAs in the Regulation of the Immune Response to Mycobacterium tuberculosis Infection’, Frontiers Microbial Immunology, Vol. 12, 2021, https://www.frontiersin.org/articles/10.3389/fi mmu.2021.687962/full#h8, (accessed 18 May, 2023).
Khadela, A., Chavda V.P., Postwala, H., Shah, Y., Mistry, P., ‘Epigenetics in Tuberculosis: Immunomodulation of Host Immune Response’, Vaccines (Basel), 2022, https://www.ncbi.nlm.nih.gov/pmc/articles/PM C9611989/, (accessed 4 Jan, 2023).
Liang, S., Song, Z., Wu, Y., Gao, Y., Gao, M., Liu, F., Wang, F., Zhang, Y., ‘MicroRNA-27b Modulates Inflammatory Response and Apoptosis during Mycobacterium tuberculosis Infection’, The Journal of Immunology, 2018, https://journals.aai.org/jimmunol/article/200/1 0/3506/106606/MicroRNA-27b-ModulatesInflammatory-Response-and, (accessed 5 Feb, 2023).
Liu, F., Chen, J., Wang, P., Li, H., Zhou, Y., Liu, H., Liu, Z., Zheng, R., Wang, L., Yang, H., Cui, Z., Wang, F., Huang, X., Wang, J., Sha, W., Xiao, H., Ge, B., ‘MicroRNA-27a controls the intracellular survival of Mycobacterium tuberculosis by regulating calcium-associated autophagy’, Nature Communications, 2018, https://www.nature.com/articles/s41467-01806836-4, (accessed 27 July, 2023).
Looney, M., Lorenc, R., Halushka, M.K., Karakousis, P.C., ‘Key Macrophage Responses to Infection with Mycobacterium tuberculosis Are Co-Regulated by microRNAs and DNA Methylation’, Frontiers in Immunology, Vol. 12, 2021, https://www.frontiersin.org/articles/10.3389/fi mmu.2021.685237/full, (accessed 8 July, 2023).
Lopez-Castejon, G., Brough, D., ‘Understanding the mechanism of IL-1β secretion’, Cytokine & Growth Factor Reviews, 2011, https://www.ncbi.nlm.nih.gov/pmc/articles/PM C3714593/#:~:text=The%20cytokine%20inter leukin%2D1β%20., (accessed 19 July, 2023).
Mack G.S., ‘Epigenetic Cancer Therapy Makes Headway’, JNCI: Journal of the National Cancer Institute, Vol. 98, Issue 20, 2006, https://academic.oup.com/jnci/article/98/20/1 443/2521877, (accessed 18 Jan, 2023).
Madden, K., Chu Liang, Y., Rajabalee, N., Alvarez, G., Sun, J., ‘Surveying the Epigenetic Landscape of Tuberculosis in Alveolar Macrophages’, Infection and Immunity, 2022, https://www.ncbi.nlm.nih.gov/pmc/articles/PM C9119088/, (accessed 15 Jan, 2023).
McLeod, D.V., Wild, G., Ubeda, F., ‘Epigenetic memories and the evolution of infectious diseases’, Nature Communications, 2021, https://www.nature.com/articles/s41467-02124580-0, (accessed 7 Jan, 2023).
Meng, Q.L., Liu, F., Yang, X.Y., Liu, X.M., Zhang, X., Zhang, C., Zhang, Z.D., ‘Identification of latent tuberculosis infection-related microRNAs in human U937 macrophages expressing Mycobacterium tuberculosis Hsp16.3’, BMC Microbiology, 2014, https://bmcmicrobiol.biomedcentral.com/artic les/10.1186/1471-2180-14-37, (accessed 18 May, 2023).
Nehme, Z., Pasquereau, S., Herbein, G., ‘Control of viral infections by epigenetictargeted therapy’, Clinical Epigenetics, 2019, https://clinicalepigeneticsjournal.biomedcentr al.com/articles/10.1186/s13148-019-0654-9, (accessed 8 Jan, 2023).
O’Brien, J., Hayder, H., Zayed, Y., Peng, C., ‘Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation’, Frontiers in Endocrinology, 2018, https://www.ncbi.nlm.nih.gov/pmc/articles/PM C6085463/, (accessed 23 Jan, 2023).
Ormandy, E.H., Dale, J., Griffin, G., ‘Genetic engineering of animals: Ethical issues, including welfare concerns’, Canadian Veterinary Journal, 2011, https://www.ncbi.nlm.nih.gov/pmc/articles/PM C3078015/, (accessed 17 August, 2023).
Pereira, J. M., Hamon, M.A., Cossart, P., ‘A Lasting Impression: Epigenetic Memory of Bacterial Infections?’, Cell Host & Microbe, Vol. 19, Issue 5, 2016, https://www.cell.com/cell-hostmicrobe/fulltext/S1931-3128(16)301482?_returnURL=https%3A%2F%2Flinkinghub. elsevier.com%2Fretrieve%2Fpii%2FS1931312 816301482%3Fshowall%3Dtrue, (accessed 4 Jan 2023).
Rendon, A., Tiberi, S., Scardigli, A., D’Ambrosio, L., Centis, R., Caminero, J.A., Battista Migliori, G., ‘Classification of drugs to treat multidrug-resistant tuberculosis (MDRTB): evidence and perspectives’, Journal of Thoracic Disease, 2016, https://www.ncbi.nlm.nih.gov/pmc/articles/PM C5107456/, (accessed 27 Jan, 2023).
Rothchild, A.C., Sissons, J.R., Shafiani, S., Plaisier, C., Min, D., Mai, D., Gilchrist, M., Peschon, J., Larson, R.P., Bergthaler, A., Baliga, N.S., Urdahl, K.B., Aderem., ‘MiR-155–regulated molecular network orchestrates cell fate in the innate and adaptive response to Mycobacterium tuberculosis’, Proceedings of the National Academy of Sciences of the United States of America, 2016,
Rothstein, M.A., Cai, Y., Marchant, G.E., ‘Ethical implications of epigenetics research’, Nature Reviews Genetics, 2009, https://www.nature.com/articles/nrg2562, (accessed 7 April, 2023).
Sato, F., Tsuchiya, S., Meltzer, S.J., Shimizu, K., ‘MicroRNAs and Epigenetics’, The FEBS Journal, Vol. 278, Issue 10, pp. 1598-1609, 2011, https://febs.onlinelibrary.wiley.com/doi/full/10 .1111/j.1742-4658.2011.08089.x, (accessed 24 Feb, 2023).
Seung, K.J., Keshavjee, S., Rich, M.L., ‘Multidrug-Resistant Tuberculosis and Extensively Drug-Resistant Tuberculosis’, Cold Spring Harbor Perspectives in Medicine, 2015, https://www.ncbi.nlm.nih.gov/pmc/articles/PM C4561400/, (accessed 24 Feb, 2023).
Sinigaglia, A., Peta, E., Riccetti, S., Venkateswaran, S., Manganelli, R., Barzon, S., ‘Tuberculosis-Associated MicroRNAs: From Pathogenesis to Disease Biomarkers’, Cells, 2020, https://www.mdpi.com/20734409/9/10/2160, (accessed 23 Jan, 2023).
Sui, J., Qiao, W., Xiang, X., Luo, Y., ‘Epigenetic changes in Mycobacterium tuberculosis and its host provide potential targets or biomarkers for drug discovery and clinical diagnosis’, Pharmacological Research, Vol. 179, 2022, https://www.sciencedirect.com/science/articl e/abs/pii/S1043661822001402?via%3Dihub, (accessed 24 Feb, 2023).
Tanaka, T., Narazaki, M., Kishimoto, T., ‘IL-6 in Inflammation, Immunity, and Disease’, Cold Spring Harbor Perspectives in Biology, 2014, https://www.ncbi.nlm.nih.gov/pmc/articles/PM C4176007/#:~:text=Interleukin%206%20(IL% 2D6),%2C%20hematopoiesis%2C%20and%2 0immune%20reactions., (accessed 19 July, 2023).
Tobin, D.M., ‘Host-Directed Therapies for Tuberculosis’, Cold Spring Harbor Perspectives in Medicine, 2015, https://www.ncbi.nlm.nih.gov/pmc/articles/PM C4588138/, (accessed 14 March, 2023).
Ueberberg B., Kohns, M., Mayatepek, E., Jacobsen, M., ‘Are microRNAs suitable biomarkers of immunity to tuberculosis?’, Molecular and Cellular Pediatrics, 2014, https://molcellped.springeropen.com/articles/ 10.1186/s40348-014-0008-9, (accessed 5 Feb, 2023).
Valeria, M., Daniela, D., Francesca, M., Nicola, P., and Luigi, A., ‘Epigenetics and Infectious Disease: State-of-the-Art and Perspectives in New Generation Therapies’, Epigenetic Mechanisms in Health and Disease, vol. 2, no. 4, 2018, https://www.lidsen.com/journals/genetics/gen etics-02-04-048, (accessed 2 Jan, 2023).
Wang, J., Yang, K., Zhou, L., Wu, M., Wu. Y., Zhu, M., Lai, X., Chen, T., Feng, L., Li, M., Huang, C., Zhong, Q., Huang, X., ‘MicroRNA155 Promotes Autophagy to Eliminate Intracellular Mycobacteria by Targeting Rheb’, Plos Pathogens, 2013, https://journals.plos.org/plospathogens/articl e?id=10.1371/journal.ppat.1003697#s4, (accessed 4 June, 2023).
Wang, J., Xion, Y., Fu, B., Guo, D., Zaky, M.Y., Lin, X., Wu, H., ‘MicroRNAs as immune regulators and biomarkers in tuberculosis’, Frontiers in Immunology, 2022, https://www.ncbi.nlm.nih.gov/pmc/articles/PM C9647078/, (accessed 4 June, 2023).
Wu, Z., Lu, H., Sheng, J., Li, L., ‘Inductive microRNA-21 impairs anti-mycobacterial responses by targeting IL-12 and Bcl-2’, FEBS Letters, Vol. 586, Issue. 16, pp. 2459-2467, 2012, https://febs.onlinelibrary.wiley.comS./doi/10.1 016/j.febslet.2012.06.004, (accessed 27 July, 2023).
Xiao, M., Yang, S., Zhou, A., Li, T., Liu, J., Chen, Y., Luo, Y., Qian, C., Yang, F., Tang, B., Li, C., Su, N., Li, J., Jiang, M., Yang, S., Lin, H., ‘MiR-27a3p and miR-30b-5p inhibited-vitamin D receptor involved in the progression of tuberculosis’, Frontiers in Microbiology, Vol. 13, 2022, https://www.frontiersin.org/articles/10.3389/f micb.2022.1020542/full, (accessed 27 May, 2023).
Yang, F., Yang, Y., Chen, Y., Li, G., Zhang, G., Chen, L., Zhang, Z., Mai, Q., Zeng, G., ‘MiR-21 Is Remotely Governed by the Commensal Bacteria and Impairs Anti-Tb Immunity by Down-Regulating IFN-γ, Frontiers in Microbiology, 2021, https://www.frontiersin.org/articles/10.3389/f micb.2020.512581/full, (accessed 2 June, 2023).
Yang, S., Li, F., Jia, S., Zhang, K., Jiang, W., Shang, Y., Chang, K., Deng, S., Chen, M., ‘Early Secreted Antigen ESAT-6 of Mycobacterium Tuberculosis Promotes Apoptosis of Macrophages via Targeting the MicroRNA155SOCS1 Interaction’, Cellular Physiology and Biochemistry, 2015, https://karger.com/cpb/article/35/4/1276/722 56/Early-Secreted-Antigen-ESAT-6-ofMycobacterium, (accessed 28 May, 2023).
Ying, H., FengYing, S., YanHong, W., YouMing, H., FaYou, Z., HongXiang, Z., XiaoLei, T., ‘MicroRNA-155 from sputum as noninvasive biomarker for diagnosis of active pulmonary tuberculosis’, Iranian Journal of Basic Medical Sciences, 2020, https://www.ncbi.nlm.nih.gov/pmc/articles/PM C7671418/, (accessed 18 June, 2023).
Zhang, Z., Zhang, A.R., Xu, M., Lou, J., Qiu, W.Q., ‘TLR-4/miRNA-32-5p/FSTL1 signalling regulated mycobacterial survival and inflammatory responses in Mycobacterium tuberculosis-infected macrophages’, Experimental Cell Research, Vol. 352, Issue 2, 2017, https://www.sciencedirect.com/science/articl e/abs/pii/S0014482717300782, (accessed 17 August, 2023).
Zoghbi, H.Y., Beaudet, A.L., ‘Epigenetics and Human Disease’, Cold Spring Harbor Perspectives in Biology, 2016, https://www.ncbi.nlm.nih.gov/pmc/articles/PM C4743078/, (accessed 6 Jan, 2023).
Appendices
Paper Experimental Methods
THP-1 macrophages were obtained from respective culture collections and cultured in a medium containing 10% foetal bovine serum (FBS) and antibiotics, incubated at 37°C in air containing 5% CO2. MTB H37Rv was obtained from respective culture collections and cultured in a medium at 37oC. To up-regulate or down-regulate the expression of miRNA in THP-1 cells, oligonucleotides containing miRNA-mimics were obtained from respective biotechnology companies. Mimics were then transfected into cultured THP-1 cells via Lipofectamine 2000. qRT-PCR analysis was performed to evaluate the effects of cell transfection. It was carried out using the respective PCR kits, the PCR conditions included an initial step of denaturing, followed by annealing and finally extension, all at respective times and temperatures. Human glyceraldehyde-3-phosphate dehydrogenase gene GAPDH was used as an internal control. Relative gene expression levels were calculated using either the 2−ΔCt or 2 ΔΔCt method. The concentration of cytokines IL-β, IL-6, and (TNF-α) were measured to explore the influence of MTB infection on cytokines. MTB-infected macrophages were transfected with mimics and inhibitors to do so.Raw Data Sets
Click here to access a Dropbox file containing the data sets used for this experiment.
R Code
generate_synthetic_data <- function(n, cor_coeff) {
cov_matrix <- matrix(c(1, cor_coeff, cor_coeff, 1), nrow = 2)
synthetic_data <- MASS::mvrnorm(n, c(0, 0), cov_matrix)
return(synthetic_data)
}
data_points <- c(data here, …)
num_bootstrap <- 1000
bootstrap_results
for (i in 1:num_bootstrap) {
synthetic_data <- generate_synthetic_data(n = 100, cor_coeff = data_points[1])
correlation_coefficient <- cor(synthetic_data)
bootstrap_results$Bootstrapped_Correlation[ i]<- correlation_coefficient[1, 2]
}
print(bootstrap_results)
Clean Data set (removes listing numbers)
data <- "1 data here
2 data here
3 data here
... remaining data here"
lines <- strsplit(data, "\n")[[1]]
cleaned_lines <- vector("list", length(lines)) for (i in seq_along(lines)) {
cleaned_lines[[i]] <- gsub("^\\d+\\s+", "", lines[i], perl = TRUE)
}
cleaned_data <- paste(cleaned_lines, collapse = "\n")
cat(cleaned_data)
