Reverse Transcription-qPCR-Based Investigation of Antiviral miRNAs in Medicinal Plants

Abstract

MicroRNAs (miRNAs) are endogenous regulators of gene expression that enable high adaptation to the living conditions of organisms. While plant antiviral miRNAs are effective in combating their viral pathogens, some plant miRNAs exhibit cross-kingdom interactions when targeting animal viruses. Current studies have reported that the miRNA contents of medicinal plants used in treating viral diseases are directly effective in combating the disease. Along with this study, the presence and expression levels of five plant miRNAs (miRNA765, miRNA954, miRNA1086, miRNA1328, and miRNA2911), known for their antiviral effects against human diseases, were analyzed in eight medicinal plants using the reverse transcription-quantitative real-time PCR (RT-qPCR) method. One of the test group medicinal plants was used as dry material for comparison purposes. Triticum aestivum L. fruits that contain high starch were preferred as the calibrator plant sample, and miRNA161 was used as the endogenous miRNA control. cDNAs were synthesized using stem-loop primers and amplified by RT-qPCR with SYBR green. Expression levels of antiviral miRNAs were analyzed using the fold change (Fc) and the relative quantification (RQ) data. At the end of this study, antiviral miRNAs were found in some medicinal plants and detected at high levels in preserved dried plant samples, such as Viscum album leaves.

Keywords

Antiviral miRNAs - medicinal plants - RT-qPCR - stem-loop primers

Publication History

Received: 24 February 2025

Accepted after revision: 27 June 2025

Article published online:
05 August 2025

• References

• 1 Sihag N, Singh T, Sheoran S, Singh O, Malik R, Kumar L, Singh J. Role of RNA interference in drought stress management: physiological, biochemical and molecular approach. Crop Pasture Sci 2024; 75
• 2 Asadi M, Millar AA. Plant microRNAs in pathogen defense: A panacea or a piece of the puzzle?. Plant Sci 2024; 341: 111993
• 3 Bishwal SC, Niranjan R. MicroRNAs and dengue viral disease: MicroRNA in Human Infectious Diseases. Academic Press 2024; 123-133
• 4 Wang J, Li Y. Current advances in antiviral RNA interference in mammals. FEBS J 2024; 291: 208-216
• 5 Lukasik A, Zielenkiewicz P. Plant MicroRNAs-novel players in natural medicine?. Int J Mol Sci 2016; 18: 9
• 6 Sun M, Xu S, Mei Y, Li J, Gu Y, Zhang W, Wang J. MicroRNAs in medicinal plants. Int J Mol Sci 2022; 23: 10477
• 7 Ding T, Li W, Li F, Ren M, Wang W. MicroRNAs: Key regulators in plant responses to abiotic and biotic stresses via endogenous and cross-kingdom mechanisms. Int J Mol Sci 2024; 25: 1154
• 8 Akram M, Tahir IM, Shah SMA, Mahmood Z, Altaf A, Ahmad K, Munir N, Daniyal M, Nasir S, Mehboob H. Antiviral potential of medicinal plants against HIV, HSV, influenza, hepatitis, and coxsackievirus: A systematic review. Phytother Res 2018; 32: 811-822
• 9 Zhou Z, Li X, Liu J, Dong L, Chen Q, Liu J, Kong H, Zhang Q, Qi X, Hou D, Zhang L, Zhang G, Liu Y, Zhang Y, Li J, Wang J, Chen X, Wang H, Zhang J, Chen H, Zen K, Zhang CY. Honeysuckle-encoded atypical microRNA2911 directly targets influenza A viruses. Cell Res 2015; 25: 39-49
• 10 Chi Y, Shi L, Lu S, Cui H, Zha W, Shan L, Shen Y. Inhibitory effect of Lonicera japonica-derived exosomal miR2911 on human papilloma virus. J Ethnopharmacol 2024; 318: 116969
• 11 Xie W, Adolf J, Melzig MF. Identification of Viscum album L. miRNAs and prediction of their medicinal values. PLoS One 2017; 12: e0187776
• 12 Mohammed FS, Uysal I, Sevindik M. A review on antiviral plants effective against different virus types. Prospect Pharm Sci 2023; 21: 1-21
• 13 Abou Baker DH, Amarowicz R, Kandeil A, Ali MA, Ibrahim EA. Antiviral activity of Lavandula angustifolia L. and Salvia officinalis L. essential oils against avian influenza H5N1 virus. J Agric Food Res 2021; 4: 100135
• 14 Habán M, Korczyk-Szabó J, Čerteková S, Ražná K. Lavandula species, their bioactive phytochemicals, and their biosynthetic regulation. Int J Mol Sci 2023; 24: 8831
• 15 Yanik H, Turktas M, Dundar E, Hernandez P, Dorado G, Unver T. Genome-wide identification of alternate bearing-associated microRNAs (miRNAs) in olive (Olea europaea L.). BMC Plant Biol 2013; 13: 1-22
• 16 Aydin M, Tombuloglu H, Hernandez P, Dorado G, Unver T. Olive-tree genome sequencing: towards a better understanding of oil biosynthesis. In: Oil Crop Genomics 2021; 75 – 87.
• 17 Zhang C, Song C, Chen L, Ma H, Zhang Y, Guo D, Guo L, Hou X. Selection and validation of miRNA reference genes by quantitative real-time PCR analysis in Paeonia suffruticosa . Horticulturae 2023; 9: 148
• 18 Zhao Y, Kao H, Chen X, Wang T, Zhang Y, Sun Y, Xiao H, Dong S, Cheng J. Identification of novel and salt-stress-regulated miRNAs from root of Melilotus officinalis (L.) Pall. by qRT-PCR. Res Sq 2023; 1-11
• 19 Jamshidi-Kia F, Lorigooini Z, Amini-Khoei H. Medicinal plants: Past history and future perspective. J HerbMed Pharmacol 2017; 7: 1-7
• 20 Miraldi E, Baini G. Medicinal plants and health in human history: From empirical use to modern phytotherapy. JSAS 2018; 10
• 21 Mourenza Á, Lorente-Torres B, Durante E, Llano-Verdeja J, Aparicio JF, Fernández-López A, Gil JA, Mateos LM, Letek M. Understanding microRNAs in the context of infection to find new treatments against human bacterial pathogens. Antibiotics 2022; 11: 356
• 22 Jin Y, Zhao JH, Guo HS. Recent advances in understanding plant antiviral RNAi and viral suppressors of RNAi. Curr Opin Virol 2021; 46: 65-72
• 23 Saiyed AN, Vasavada AR, Johar SK. Recent trends in miRNA therapeutics and the application of plant miRNA for prevention and treatment of human diseases. Futur J Pharm Sci 2022; 8: 24
• 24 Li D, Yang J, Yang Y, Liu J, Li H, Li R, Cao C, Shi L, Wu W, He K. A timely review of cross-kingdom regulation of plant-derived MicroRNAs. Front Genet 2021; 12: 613197
• 25 Nazaruk J, Orlikowski P. Phytochemical and pharmacological properties of Viscum album L. Nat Prod Res 2016; 30: 373-385
• 26 Barozai MYK, Din M. Initial screening of plant most conserved microRNAs targeting infectious viruses: HBV and HCV. 14th International Bhurban Conference on Applied Sciences and Technology; 2017; 192 – 196.
• 27 Zhou LK, Zhou Z, Jiang XM, Zheng Y, Chen X, Fu Z, Xiao G, Zhang CY, Zhang LK, Yi Y. Absorbed plant MIR2911 in honeysuckle decoction inhibits SARS-CoV-2 replication and accelerates the negative conversion of infected patients. Cell Discov 2020; 6: 54
• 28 Kalarikkal SP, Sundaram GM. Edible plant-derived exosomal microRNAs: Exploiting a cross-kingdom regulatory mechanism for targeting SARS-CoV-2. Toxicol Appl Pharmacol 2021; 414: 115425
• 29 Regier N, Frey B. Experimental comparison of relative RT-qPCR quantification approaches for gene expression studies in poplar. BMC Mol Biol 2010; 11: 1-8
• 30 Bolha L, Dušanić D, Narat M, Irena O. Comparison of methods for relative quantification of gene expression using real-time PCR. Acta Agric Slov 2012; 100: 97-106
• 31 Martins TF, Souza PF, Alves MS, Silva FDA, Arantes MR, Vasconcelos IM, Oliveira JT. Identification, characterization, and expression analysis of cowpea (Vigna unguiculata [L.] Walp.) miRNAs in response to cowpea severe mosaic virus (CPSMV) challenge. Plant Cell Rep 2020; 39: 1061-1078
• 32 Farooq T, Hussain MD, Wang Y, Kamran A, Umar M, Tang Y, He Z, She X. Enhanced antiviral defense against begomoviral infection in Nicotiana benthamiana through strategic utilization of fluorescent carbon quantum dots to activate plant immunity. J Nanobiotechnology 2024; 22: 707
• 33 Kirsch A, Hajto T. Case reports of sarcoma patients with optimized lectin-oriented mistletoe extract therapy. J Altern Complement Med 2011; 17: 973-979
• 34 Liu C, Xu M, Yan L, Wang Y, Zhou Z, Wang S, Sun Y, Zhang J, Dong L. Honeysuckle-derived microRNA2911 inhibits tumor growth by targeting TGF-β1. Chin Med 2021; 16: 1-9
• 35 Perry EK, Pickering AT, Wang WW, Houghton P, Perry NS. Medicinal plants and Alzheimerʼs disease: Integrating ethnobotanical and contemporary scientific evidence. J Altern Complement Med 1998; 4: 419-428
• 36 Abou Baker DH, Amarowicz R, Kandeil A, Ali MA, Ibrahim EA. Antiviral activity of Lavandula angustifolia L. and Salvia officinalis L. essential oils against avian influenza H5N1 virus. J Agric Food Res 2021; 4: 100135
• 37 Yang J, Xia B, Han Y, Wang Y, Bi Y, Zhang Y. Therapeutic potential of Lonicerae japonicae flos against emerging respiratory viral infections. Pharmacol Res Mod Chin Med 2024; 10: 100362
• 38 Zakay-Rones Z, Thom E, Wollan T, Wadstein J. Randomized study of the efficacy and safety of oral elderberry extract in the treatment of influenza A and B virus infections. Int J Med Res 2015; 33: 171-185
• 39 Tiralongo E, Wee SS, Lea RA. Elderberry supplementation reduces cold duration and severity in air travelers: A randomized, double-blind, placebo-controlled clinical trial. Nutrients 2016; 8: 182
• 40 Torabian G, Valtchev P, Adil Q, Dehghani F. Anti-influenza activity of elderberry (Sambucus nigra) extracts. J Funct Foods 2019; 54: 353-360
• 41 Cicerale S, Lucas LJ, Keast RS. Antimicrobial, antioxidant and anti-inflammatory phenolic activities in extra virgin olive oil. Curr Opin Biotechnol 2012; 23: 129-135
• 42 Ramalakshmana J, Sowmithri C, Babu YR, Padal SB. Phytochemical screening, antioxidant, antimicrobial activities and GC-MS analysis of Ficus benjamina L. and Ficus hispida L. IJIEMR 2023; 12: 100-115
• 43 Varkonyi-Gasic E, Hellens RP. RNAi and Plant Gene Function Analysis, Methods in Molecular Biology: Quantitative Stem Loop RT-PCR for Detection of MicroRNAs. Berlin/Heidelberg: Springer Science+Business Media; 2011: 145-156