Identification of microRNAs from Medicinal Plant Murraya koenigii by High-Throughput Sequencing and Their Functional Implications in Secondary Metabolite Biosynthesis

MicroRNAs (miRNAs) are small noncoding RNA molecules that play crucial post-transcriptional regulatory roles in plants, including development and stress-response signaling. However, information about their involvement in secondary metabolism is still limited. Murraya koenigii is a popular medicinal plant, better known as curry leaves, that possesses pharmaceutically active secondary metabolites. The present study utilized high-throughput sequencing technology to investigate the miRNA profile of M. koenigii and their association with secondary metabolite biosynthesis. A total of 343,505 unique reads with lengths ranging from 16 to 40 nt were obtained from the sequencing data, among which 142 miRNAs were identified as conserved and 7 as novel miRNAs. Moreover, 6078 corresponding potential target genes of M. koenigii miRNAs were recognized in this study. Interestingly, several conserved and novel miRNAs of M. koenigii were found to target key enzymes of the terpenoid backbone and the flavonoid biosynthesis pathways. Furthermore, to validate the sequencing results, the relative expression of eight randomly selected miRNAs was determined by qPCR. To the best of our knowledge, this is the first report of the M. koenigii miRNA profile that may provide useful information for further elucidation of the involvement of miRNAs in secondary metabolism. These findings might be crucial in the future to generate artificial-miRNA-based, genetically engineered M. koenigii plants for the overproduction of medicinally highly valuable secondary metabolites.


Introduction
Murraya koenigii (L.) Spreng (Rutaceae) is a subtropical medicinal plant native to Asia and distributed throughout the subcontinent of India [1]. This plant has a great commercial value since it produces several important therapeutic compounds, including carbazole alkaloids [2] (only a few plant species can produce carbazole alkaloids, and the most significant one is M. koenigii). Different parts of the M. koenigii plant are widely used for treating a variety of ailments such as fever, diarrhea, diabetes, skin eruptions, venomous bites, renal pain, inflammation, and cholesterol management, among others, and have been used in Ayurvedic, Unani, and homeopathic medicine for centuries [3,4]. However, the leaf (curry leaf) of M. koenigii, which has long been used in Indian cuisine, is Due to a significant association of several miRNAs in plant secondary metabolite biosynthesis, plant biotechnologists recently have begun to exploit and/or manipulate different relevant microRNAs as a useful tool for controlling secondary metabolites biosynthetic pathways not only in model plants, but also in economically/medicinally important ones [26][27][28][29]. For example, an artificial inhibition of Sm-miR408 expression in the traditional herb Salvia miltiorrhiza was found to enhance the root accumulation of salvianolic acid B, a chemical compound that has hepatoprotective, cardioprotective, and anticancer properties [30]. Similarly, the production of the commercially used plantderived sweet compounds steviol glycosides from Stevia rebaudiana was increased 24.5 and 51% by anti-miR319g and miRStv_11 coexpression, which target genes related to its biosynthetic pathway [31]. Furthermore, the synthesis of guaianolides, byproducts of chicory (Cichorium intybus L.), was reduced by an artificial miRNA that targeted germacrene A synthase genes, aiming to improve the extraction of inulin, a commercial compound used in the food industry as a prebiotic and sweetener [32].
Currently, a complete miRNA profile and their expression pattern can be investigated authentically using next-generation sequencing (NGS) technology in medicinal plants [33][34][35], which in turn allows elucidating the functions of their potential target transcripts and their relationship with secondary metabolites biosynthesis, strengthening transgenic research. It is also well established that secondary metabolite production can be enhanced in planta by manipulating target transcripts with artificial miRNAs (amiRNAs). However, in order to exploit them in the metabolic engineering process, a better understanding of their modes of action is required. Thus, the present study aimed to generate for the first time an NGS-based miRNA profile of M. koenigii, and to predict their potential targets associated with the secondary metabolism biosynthesis pathways.

Sequence Analysis of M. koenigii Small RNAs
In this study, utilizing high-throughput Illumina sequencing technology, a total of 8,186,145 raw reads were obtained from the M. koenigii leaf tissue samples. The raw data of small RNA sequencing were uploaded to the NCBI SRA database (Accession number: SRR16796893). After removing the adaptors, low-quality reads, and other small RNAs such as rRNA (156,700), snoRNA (1483), snRNA (1315), and tRNA (18) (Table 1), a total of 343,505 unique reads with lengths ranging from 16 to 40 nt were attained. The size distribution of unique M. koenigii reads showed that 24 nt represented the most abundant one (9.52%) followed by 21 nt (4.94%), 22 nt (4.92%), and 23 nt (4.79%) ( Figure 1).

Identification of Conserved and Novel miRNAs in M. koenigii
To identify conserved miRNAs in M. koenigii, the unique reads were aligned against miRbase-22 using the BLASTn tool, and a total of 142 conserved miRNAs, representing 34 miRNA families, were identified ( Table 2). All the conserved miRNAs showed significant homology (no more than one mismatch) with their respective homologs, and their frequency varied widely between families. The most abundant miRNA families were miR166, miR159, miR396, and miR167 with 16, 12, 12, and 10 members, respectively (Figure 2). The read counts of the miRNA families varied from 1 to 2795, where the miR166 family had the highest number of reads (2795), followed by the miR396 (1598) and miR159 (1364) families.

Identification of Conserved and Novel miRNAs in M. koenigii
To identify conserved miRNAs in M. koenigii, the unique reads were aligned against miRbase-22 using the BLASTn tool, and a total of 142 conserved miRNAs, representing 34 miRNA families, were identified ( Table 2). All the conserved miRNAs showed significant homology (no more than one mismatch) with their respective homologs, and their frequency varied widely between families. The most abundant miRNA families were miR166, miR159, miR396, and miR167 with 16, 12, 12, and 10 members, respectively ( Figure 2). The read counts of the miRNA families varied from 1 to 2795, where the miR166 family had the highest number of reads (2795), followed by the miR396 (1598) and miR159 (1364) families.  Following the identification of conserved miRNAs, the remaining unaligned 42,658 reads were subjected to predict novel M. koenigii miRNA candidates by applying strict filtering criteria. A total of seven novel miRNAs were identified in this study (Table 3), and their secondary hairpin structures were deduced ( Figure 3). The read counts of these novel miRNA candidates varied from 5 to 93. The mko-miRN7-3p displayed the highest number of reads (93), followed by mko-miRN4-5p (15) and mko-miRN1-3p (14). The precursor sequences of these novel miRNA candidates had high Minimum Folding Free En-   Following the identification of conserved miRNAs, the remaining unaligned 42,658 reads were subjected to predict novel M. koenigii miRNA candidates by applying strict filtering criteria. A total of seven novel miRNAs were identified in this study (Table 3), and their secondary hairpin structures were deduced ( Figure 3). The read counts of these novel miRNA candidates varied from 5 to 93. The mko-miRN7-3p displayed the highest number of reads (93), followed by mko-miRN4-5p (15) and mko-miRN1-3p (14). The precursor sequences of these novel miRNA candidates had high Minimum Folding Free Energy Index (MFEI) values ranging from 0.70 to 0.98 with an average of 0.85 ± 0.11, distinguishing them from other types of RNAs such as tRNAs (0.64), rRNAs (0.59), and mRNAs (0.62-0.66) [36].

Human Target Gene Prediction of M. koenigii miRNAs
Apart from regulating the secondary metabolism and other biological processes, plant miRNAs are also known to regulate human target genes with the potential for treating human diseases [37][38][39]. In this study, the human target prediction analysis indicated that 48 conserved M. koenigii miRNAs targeted 4362 human genes. Specifically, mko-miR160g, mko-miR160e-5p, and mko-miR477 accounted for the highest number of human targets: 516, 476, and 384, respectively. Interestingly, several M. koenigii miRNAs such as mko-miR5082, mko-miR5368, mko-miR482b, mko-miR156q, and mko-miR396a-5p targeted proteins involved in pathways related to inflammation, cancer, and neurological disorders including Alzheimer's, Parkinson's, and Huntington's. Regarding the novel miRNAs of M. koenigii, mko-miRN6-5p targeted the highest number (161) of human genes, followed by mko-miRN3-3p (75) and mko-miRN2-3p (62), which are related to insulin secretion, cardiomyopathy, and autoimmune thyroid disease pathways, respectively. Nevertheless, the precise implications of M. koenigii miRNAs in human health must be elucidated further with relevant experimental approaches.

Identification of M. koenigii miRNA Targets Involved in Plant Secondary Metabolite Biosynthesis
The medicinal properties of M. koenigii can be attributed to the presence of secondary metabolites synthesized by specific enzymes. Therefore, in this study, our principal goal was to identify the M. koenigii miRNAs whose target genes coded for enzymes involved in the secondary metabolite biosynthesis. The results revealed a total of 286 target genes of M. koenigii miRNAs associated with ubiquinone and terpenoid-quinone biosynthesis; tropane, piperidine, and pyridine alkaloid biosynthesis; isoquinoline alkaloid, sesquiterpenoid, and triterpenoid biosynthesis; terpenoid backbone biosynthesis; mevalonate bi-

Human Target Gene Prediction of M. koenigii miRNAs
Apart from regulating the secondary metabolism and other biological processes, plant miRNAs are also known to regulate human target genes with the potential for treating human diseases [37][38][39]. In this study, the human target prediction analysis indicated that 48 conserved M. koenigii miRNAs targeted 4362 human genes. Specifically, mko-miR160g, mko-miR160e-5p, and mko-miR477 accounted for the highest number of human targets: 516, 476, and 384, respectively. Interestingly, several M. koenigii miRNAs such as mko-miR5082, mko-miR5368, mko-miR482b, mko-miR156q, and mko-miR396a-5p targeted proteins involved in pathways related to inflammation, cancer, and neurological disorders including Alzheimer's, Parkinson's, and Huntington's. Regarding the novel miRNAs of M. koenigii, mko-miRN6-5p targeted the highest number (161) of human genes, followed by mko-miRN3-3p (75) and mko-miRN2-3p (62), which are related to insulin secretion, cardiomyopathy, and autoimmune thyroid disease pathways, respectively. Nevertheless, the precise implications of M. koenigii miRNAs in human health must be elucidated further with relevant experimental approaches.

Experimental Validation of M. koenigii miRNAs by qPCR
Four conserved and four novel M. koenigii miRNAs were randomly selected for qPCR analysis to validate the high-throughput sequencing data. The results showed similar expression patterns between the Illumina sequencing and qPCR analysis, except for mko-miRN5-3p. Moreover, the post hoc statistical analysis of the qPCR data indicated that the relative expression of most of the validated miRNAs was similar (as specified by the letters above the bars in Figure 9).

Experimental Validation of M. koenigii miRNAs by qPCR
Four conserved and four novel M. koenigii miRNAs were randomly selected for qPCR analysis to validate the high-throughput sequencing data. The results showed similar expression patterns between the Illumina sequencing and qPCR analysis, except for mko-miRN5-3p. Moreover, the post hoc statistical analysis of the qPCR data indicated that the relative expression of most of the validated miRNAs was similar (as specified by the letters above the bars in Figure 9). lants 2022, 10, x FOR PEER REVIEW 15 of 23 Figure 9. Quantitative PCR analysis of conserved and novel miRNAs of M. koenigii. The relative expression (Log10) of 4 conserved and 4 novel miRNAs was detected by qPCR. U6 was used as an endogenous control. The expression level of miR156a-5p was set as control and taken as 1, and the expression level in all other miRNAs was quantified relative to it. The analysis was performed as triplicates, and the error bars indicate standard deviations. Significant differences between miRNAs at p < 0.05 by ANOVA and Tukey's test are indicated with different letters.

Discussion
Plant secondary metabolites are molecules synthesized under specific situations to interact with the environment and to adapt to biotic and abiotic stress conditions [40]. Their biosynthesis is highly energy-consuming, making it a tightly regulated process [11]. Few studies have demonstrated the important regulatory roles of miRNAs in plant secondary metabolism [11,41]. In the present work, the Illumina small-RNA sequencing technology was employed to explore the miRNA profile of the medicinal plant M. koenigii, aiming to understand their implication in secondary metabolism.
It has been stated that miRNAs can either target the transcripts of the enzymes responsible for the biosynthesis of these secondary metabolites or the transcripts of regulatory proteins that control the expression of the former, such as transcription factors [42]. In this study, the conserved M. koenigii miRNAs were distributed in 34 families, with miR166 as the family with the highest number of members (16) and the highest number of total reads (2795) compared to other families. Kajal and Singh [43] reported that Figure 9. Quantitative PCR analysis of conserved and novel miRNAs of M. koenigii. The relative expression (Log10) of 4 conserved and 4 novel miRNAs was detected by qPCR. U6 was used as an endogenous control. The expression level of miR156a-5p was set as control and taken as 1, and the expression level in all other miRNAs was quantified relative to it. The analysis was performed as triplicates, and the error bars indicate standard deviations. Significant differences between miRNAs at p < 0.05 by ANOVA and Tukey's test are indicated with different letters.

Discussion
Plant secondary metabolites are molecules synthesized under specific situations to interact with the environment and to adapt to biotic and abiotic stress conditions [40]. Their biosynthesis is highly energy-consuming, making it a tightly regulated process [11]. Few studies have demonstrated the important regulatory roles of miRNAs in plant secondary metabolism [11,41]. In the present work, the Illumina small-RNA sequencing technology was employed to explore the miRNA profile of the medicinal plant M. koenigii, aiming to understand their implication in secondary metabolism.
It has been stated that miRNAs can either target the transcripts of the enzymes responsible for the biosynthesis of these secondary metabolites or the transcripts of regulatory proteins that control the expression of the former, such as transcription factors [42]. In this study, the conserved M. koenigii miRNAs were distributed in 34 families, with miR166 as the family with the highest number of members (16) and the highest number of total reads (2795) compared to other families. Kajal and Singh [43] reported that miR166i-3p was involved in regulating sesquiterpenes and triterpenoids targeting the squalene synthase in Chlorophytum borivilianum, which produces a type of saponins known as borivilianosides, with several pharmacological activities such as immunomodulatory, antidiabetic, and androgenic. However, in contrast, it has been reported that the miR166 family from blueberry (Vaccinium ashei) can target the transcription factor squamosa promoter binding protein-like (SPL), which prevents the expression of the biosynthetic genes of anthocyanins flavonoid [42].
Several studies investigated the essential oil composition from the leaves of M. koenigii, which varies depending on the type of oil extraction and the sample origin [44,45]. Nevertheless, they coincided in that it contained C10 monoterpenes and C15 sesquiterpenes. For example, Verma et al. [46] reported that α-pinene, sabinene, (E)-caryophyllene, β-pinene, terpinen-4-ol, γ-terpinene, limonene, α-terpinene, (E)-nerolidol, α-humulene, α-thujene, and β-elemene were the major components in the essential oil of M. koenigii from the Western Himalayas. The backbone structure of these terpenoids was built up with C5 isoprenoid units. Intriguingly, the present study showed that both conserved and novel M. koenigii miRNAs were implicated in the terpenoid backbone biosynthesis, which can be synthesized either by the mevalonate pathway (MVA) in the cytoplasm or by the 2C-methyld-erythritol-4-phosphate pathway (MEP) in the plastids [47]. The former pathway focuses on synthesizing monoterpenes, diterpenes, and tetraterpenes, while the latter focuses on the sesquiterpenes, sterols, and triterpenes [48]. In this context, the target prediction analysis of M. koenigii miRNAs revealed that mko-miR5082 targeted the enzyme acetyl-CoA C-acetyltransferase, responsible for condensing two molecules of acetyl-CoA into acetoacetyl-CoA, a very first step in terpenoid backbone synthesis; while mko-miR396g-5p and the novel mko-miRN7-3p targeted the enzyme hydroxymethylglutaryl-CoA synthase, which condenses the third acetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA. A similar kind of work has also been reported in Rauvolfia serpentina, where miR396 targeted the secologanin synthase, an oxidoreductase involved in synthesizing the monoterpene secologanin [49]. Additionally, mko-miR827b targeted the mevalonate kinase, whereas mko-miR167a targeted the mevalonate diphosphate decarboxylase, two important enzymes acting sequentially and responsible for supplying the C5 prenyl diphosphates for the downstream terpenoid biosynthesis. It was also found that mko-mir156 targeted 1-Deoxy-d-xylulose 5-phosphate synthase, which performs a committed step in the MEP pathway, controlling its flux. Our results corroborated those of Singh et al. [50], who claimed that miR156 targeted the same aforesaid enzyme in Mentha spp. Likewise, another study showed that miR156 targeted SLP9, a transcription factor that modulates the expression of the sesquiterpene synthase gene TPS21 in Pogostemon cablin (patchouli) [51]. Furthermore, mko-mir396c targeted geranyl diphosphate synthase, the principal enzyme responsible for producing geranyl diphosphate, a crucial molecule for monoterpenoid and diterpenoid biosynthesis [48]. Regarding the novel M. koenigii miRNAs, mko-miRN4-5p targeted both the enzyme 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase and the isoprene synthase, with the latter producing isoprene from dimethylallyl diphosphate in the MEP pathway.
Several well-known flavonoids such as rutin, quercetin, myricetin, kaempferol, and catechin have already been extracted from M. koenigii leaves [52,53]. These kinds of molecules are initially synthesized through the phenylpropanoid pathway, in which phenylalanine is first converted into 4-coumaroyl-CoA and then enters into the flavonoid biosynthesis pathway [54]. The current study revealed that the conserved mko-miR168b, mko-miR858, mko-miR8610.1, and mko-miR5082 targeted three important enzymes: chalcone synthase, chalcone isomerase, and shikimate O-hydroxycinnamoyltransferase, associated with the flavonoid biosynthesis pathway. Specifically, chalcone synthase produces, among other chalcones, naringenin chalcone and pinocembrin chalcone, which in turn are converted into naringenin and pinocembrin by chalcone isomerase. Similarly, in blueberry, chalcone synthase is targeted by miR166 and miR390 families, whereas flavonol synthase is targeted by the miR159, miR171_1, and miR845_1 families [42]. Additionally, studies of A. thaliana and Diospyros kaki showed that miR858 targeted MYB transcription factors, which regulate genes of the flavonoid and proanthocyanidin biosynthesis [55,56]. In addition, miR159b-3p targeted caffeoyl-CoA O-methyltransferase, the enzyme responsible for synthesizing feruloyl-CoA from caffeoyl-CoA; and miR167c-5p targeted phlorizin synthase, which converts phloretin into phlorizin, its glucoside form. Nevertheless, further studies are needed to explain the potential role of these enzymes in the flavonoid biosynthesis pathway of M. koenigii leaves.
There is growing evidence of the cross-kingdom miRNA transfer between humans and plants [37]. Moreover, studies suggested that plant miRNAs have the potential to treat human diseases [38,39]. For example, it was reported that miR159 not only inhibited the cell proliferation of the breast cancer cell line MDA-MB-231, but also suppressed the growth of mice xenograft breast tumors [61]. Likewise, Li et al. [62] observed that miR167e-5p inhibited the proliferation of enterocytes (IPEC-J2) and human colon carcinoma (Caco-2) cell lines in vitro. The present study found that several M. koenigii miRNAs, including mko-miR8175, mko-miR5368, and mko-miR156, targeted genes encoding proteins involved in colorectal, prostate, and breast cancers, corroborating the report of Xie et al. [63], who showed that val-miR1086 and val-miR1127 from Viscum album (mistletoe) could regulate the expression level of the p53 tumor suppressor gene. In this context, the predicted human targets of M. koenigii might shed light on its pharmacological properties. Nevertheless, further experimental validation is needed.

Plant Materials and RNA Extraction
Leaf samples from healthy 5-year-old-M. koenigii plants (grown under natural conditions) were collected, instantly frozen in liquid nitrogen, and subsequently stored at −80 • C until used. Total RNA was isolated from 100 mg of leaf sample using a Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer's instructions. The quality and quantity of the RNA were assessed using Nanodrop2000 (Thermo Scientific, Wilmington, DE, USA), Qubit (Thermo Scientific, Wilmington, DE, USA), and Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA).

Small RNA Library Construction and Sequencing
The small RNA library was constructed using the QIAseq ® miRNA Library Kit (Qiagen, Germantown, MD, USA) protocol. Briefly, 100 ng of total RNA was utilized as initial material, and 3 adapters were ligated to the specific 3 OH group of microRNAs followed by 5 adapters ligation. Consequently, the adapter-ligated fragments were reverse transcribed with Unique Molecular Index (UMI) assignment, and the cDNA was barcoded and amplified in a single step by PCR. The resulting cDNA library was then quantified by Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), and the fragment size distribution was analyzed with an Agilent 2200 Tapestation system. Finally, the sequencing was performed for 75 cycles on an Illumina NextSeq 550 High Output sequencing platform following the manufacturer's protocol.

Small RNA Sequencing Data Analysis
Following the completion of the sequencing run, the Illumina GA raw data were processed for the removal of adaptors and low-quality reads (<Q30) using sRNA-workbench (V3.0_ALPHA). Sequences smaller than 16 bp and larger than 40 bp, as well as reads matching other ncRNAs (rRNA, tRNA, snRNA, and snoRNAs), were eliminated. The remaining small RNA sequences were aligned against miRbase-22.1 (http://www.mirbase.org, assessed on 10 June 2021) to identify conserved M. koenigii miRNAs. Subsequently, sequences not showing homology were considered for the prediction of novel miRNAs using bowtie [64] and Mireap_0.22b [65]. However, due to the unavailability of the M. koenigii genome, the well-annotated Citrus sinensis (belonging to the same family of M. koenigii) genome was used as the reference. The novel miRNAs with proper precursor secondary structures and MFEI values of ≥0.70 were only considered for this study. The secondary structures of the precursors were predicted using the UNAFold Web Server The conserved and novel miRNAs with copy numbers more than equal to 5 were considered for target gene prediction using the psRNATarget tool (https://www.zhaolab.org/ psRNATarget, assessed on 10 June 2021). For human target prediction, the miRanda tool was used, in which the miRNA sequences were analyzed along with the human cDNA list in strict mode (strict alignment of seed regions), and miRNA hits having minimum free energy ≥ −25 were assumed to be potential targets. GO annotation of the potential M. koenigii miRNAs targets was performed in the BP category (considering top 30 and secondary metabolism associated terms). Moreover, to find the coregulation of the potential targets, a biological network was generated using the MFE values of the miRNA-target interaction, and the biological network of the miRNAs and their targets was visualized using Cytoscape 3.2 (https://cytoscape.org/release_notes_3_2_0.html, assessed on 10 June 2021). Finally, miRNA targets associated with the secondary metabolite biosynthetic pathways were analyzed precisely.

Extraction of Small RNA and Experimental Validation of M. koenigii miRNAs by qPCR
To validate the identified conserved and novel miRNA candidates, small RNAs were isolated from a frozen leaf sample of M. koenigii using the mirVana™ miRNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions. The quality and quantity of the small RNA samples were measured with a NanoDrop One UV-Vis microvolume spectrophotometer (Thermo Scientific™, Wilmington, DE, USA). Following the quality check, small RNAs were polyadenylated and reverse transcribed using the Mir-X miRNA First-Strand Synthesis kit (Clontech, Mountain View, CA, USA), and finally, the qPCR was performed using the TB Green ® Advantage ® qPCR Premix (Takara Bio USA, Inc., San José, CA, USA) in a StepOne™ Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). The reactions were performed in a 48-well optical plate using the following conditions: an initial polymerase activation step for 10 s at 95 • C, followed by 45 cycles of 5 s at 95 • C for denaturation, and 20 s at 55 • C for annealing and extension. The amplification cycle was followed by a melting curve analysis ranging from 56-95 • C, with temperature increases in steps of 0.5 • C every 10 s. The reactions were performed with two biological replicates and three technical replicates for each sample, and the relative expression of the miRNAs was quantified by the 2 −∆∆Ct method utilizing U6 as endogenous control. Additionally, the expression level of miR156a-5p was set as control (taken as 1), and all other miRNA expression was quantified relative to it.

Conclusions
In this study, a total of 142 conserved and 7 novel miRNAs from M. koenigii were identified using high throughput sequencing technology. Among the identified miRNAs, seven conserved and two novel miRNAs were found to target enzymes significantly involved in the terpenoid backbone biosynthesis pathway, while six conserved miRNAs were found to target enzymes of the flavonoid biosynthesis pathway. Moreover, the human target prediction analysis revealed that M. koenigii miRNAs potentially targeted genes implicated in human health. Finally, eight miRNAs were experimentally validated using qPCR. Notably, this is the first report of microRNAs from the medicinal plant M. koenigii and their association with secondary metabolite biosynthesis. This study might strengthen the miRNA-mediated transgenic research for the overproduction of medicinally as well as commercially valuable plant secondary metabolites. Funding: The authors would like to thank to the Regional Department of Bioengineering, South-Central Region of Tecnologico de Monterrey, for their support regarding the equipment and required infrastructure for this project.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
Publicly available datasets were analyzed in this study. This data can be found here: https://www.ncbi.nlm.nih.gov/sra/SRR16796893, assessed on 10 June 2021.