Verification of miRNAs in ginseng decoction by high-throughput sequencing and quantitative real-time PCR

Abstract

Panax ginseng C. A. Meyer is a precious traditional Chinese medicine that has been clinically used for over thousands of years. In general, ginseng needs to be prepared to ginseng decoction before taking it. MicroRNAs are a class of small (18–24 nt), single-stranded molecules that regulate gene expression at the post-transcriptional level. Considering that ginseng miRNAs may be bioactive compounds, we used Illumina high-throughput sequencing and quantitative real-time PCR (qRT-PCR) to validate the existence of miRNAs in fresh ginseng decoction which have been boiled at high temperature. Our previous studies have demonstrated that there are several miRNAs in fresh ginseng. The roots of fresh Panax ginseng were prepared according to routine methods, from which miRNAs were extracted and sequenced. A total of 43 miRNAs were identified from water decoction by Illumina high-throughput sequencing, belonging to 71 miRNA families. The target genes of these miRNAs were predicted by sequencing, and were annotated by GO, KEGG and Nr databases. The functions of these target genes mainly included plant hormone signal transduction, transcription regulation, macromolecular metabolism and auxin signaling. Nine highly expressed miRNAs (miR159, miR167, miR396, miR166, miR168, miR156, miR165, miR162 and miR394) were verified by qRT-PCR, and the results of Illumina high-throughput sequencing and qRT-PCR were consistent. Results from this study indicate that miRNAs remained stable in P. ginseng after high-temperature boiling. Additionally, Illumina high-throughput sequencing was superior in the acquisition of higher amount of small RNAs.

Evidence of Cross-Kingdom Gene Regulation by Plant MicroRNAs and Possible Reasons for Inconsistencies

“…18,20,22 Many plant miRNAs have been detected in a variety of herbal decoctions by high-throughput sequencing and reverse transcription quantitative PCR (RT-qPCR), demonstrating that plant miRNAs remain stable in decoctions after hightemperature boiling. 18,20,26,27 Recently, Jiang C. et al 26 conducted a large-scale analysis of the plant miRNA atlases in 245 herbal decoctions. In the present study, a total of 21 757 plant miRNAs were identified; these findings are highly consistent with those of plant miRNAs in miRbase.…”

“…50 Apart from the absorption rate and absorption selectivity of plant miRNAs, variants of a plant miRNA from different plant species may also lead to inconsistencies, as indicated by the finding that three variants of plant miR-168a from rice, tomato, and apple exhibit differing silencing efficacy for LDLRAP1. 78 Considering the absorption rate and absorption selectivity, the expression levels of EV-borne or herbal decoction-borne plant miRNAs, synthetic plant miRNAs mimics and extracted plant miRNAs are generally relatively higher than those of dietary plant miRNAs; moreover, because EV-borne 16,66 or decoction-borne plant miRNAs 18,20,26,27 and extracted plant miRNAs 20 from a certain plant constitute a cocktail of many types of plant miRNAs, some types of plant miRNAs can be selectively absorbed and reach functional levels in the body. In our opinion, this fact is why EV-borne or herbal decoctionborne plant miRNAs, synthetic plant miRNA mimics, and extracted plant miRNAs can effectively mediate cross-kingdom gene regulation (see Tables 1−3).…”

“…Many studies have shown that herbal decoctions can affect gene expression in animals by modifying miRNA profiles, which can be easily observed from the results obtained using the search strategies “decoction” and “microRNA” in the online PubMed database; however, only five of these studies aimed to further determine the origin (i.e., animal or decoction) of the modified miRNAs. ,,,, Nonetheless, these five experiments provide much interesting knowledge and consistent results about the cross-kingdom gene regulation by herbal decoction-borne plant miRNAs via the gastrointestinal tract. ,, …”

“…Over the past decade, a significant amount of research has focused on validating this cross-species gene regulation phenomenon, but a consensus has not yet been reached. , This study aims to determine whether cross-species gene regulation by orally consumed plant miRNAs can indeed occur. The study classified plant miRNAs into various categories, including miRNAs carried by extracellular vesicle (EV), − extracted plant miRNAs, − miRNAs borne by herbal decoctions, ,,,, synthetic plant miRNAs mimics, ,,,,,,− and miRNAs present in plant tissues/juices ,,,,,,− (Figure ). For each category of plant miRNAs, we systematically collected and reviewed the evidence for cross-species gene regulation.…”

Identification of key genes and active anti-inflammatory ingredients in Panax medicinal plants by climate-regulated callus culture combined with gene-component-efficacy grey correlation analysis

“…Liquid chromatography-mass spectrometry [19][20] is an effective method for analyzing and identifying bioactive substances quantitative reverse-transcription polymerase chain reaction (qRT-PCR) [21] is the conventional method used to identify and characterize key genes and is widely used in quantitative gene expression detection. Gray correlation analysis (GCA) [22] is a powerful method for studying the effective material basis of Chinese herbal medicines (CHMs).…”