In-silico prediction on effect of Cymbopogon flexuosus plant miRNA

on human target with Cross-kingdom approach

Dhruvam Shukla
BINF732
Prof. Dr. Saleet Jafri, Bioinformatics and computational biology, George Mason University
Content
• Objectives
• Introduction
• Methodology
• Result
• Conclusion
• Future Enhancement
• Acknowledgements
OBJECTIVES

• Identification of novel miRNAs in Cymbopogon

Flexuosus plant

• Find out the interaction of identified novel genes on a

human target.

• Cross-kingdom Analysis of Cymbopogon flexuosus

miRNA on human targets.
 Introduction
• miRNA (Micro RNA).

• MicroRNAs (miRNAs) are small non-coding RNAs 18–

24 nucleotides long that control gene expression by
promoting degradation or repressing translation of target
mRNAs.
• miRNA regulates the expression of hundreds of target
mRNAs, miRNAs can be seen as master-coordinators,
efficiently regulating fundamental cellular processes such
as proliferation, apoptosis, and development.[9]
• miRNAs are involved in a broad range of developmental
and physiological processes their deregulation appears to
play a fundamental role in the onset, progression, and
dissemination of many cancers as well as in many other
human diseases.[10]
Cross-Kingdom Approach
 Cross-Kingdom Approach

• It has been well studied that plant-derived miRNAs may pass through the
gastrointestinal (GI) tract releasing biomolecules like amino acids, fatty acids,
and miRNAs after digestion which get absorbed by human cells.
• The epithelial cell lining of the intestine may absorb these exogenous plant
miRNAs packaged into microvesicles (MVs) along with RNA-induced silencing
complex (RISC) components subsequently reaching the destination through the
circulatory system i.e., the recipient cells by escaping other barriers of the
digestive system and RNase digestion.
• miRNAs get released and participate in the regulation of the target gene.
 METHODOLOGY
Raw data of Transcriptome data Known mature miRNA sequence of
from SRR2970609 virdiplant family from [miRBase]
Assembly

Unigene Reference miRNA ssequences

blastn

Sequence with protein DB (nr) alignment

Candidate pre miRNA

Secondary structure prediction using MFOLD

Hairpin candidate pre-miRNA

Novel miRNA

Target prediction using [psRNATarget] with Human target

Functional annotation
• Retrieval of Data

The Transcriptome data of Cymbopogon Flexuosus was downloaded from

NCBI SRA (Sequence Read Archive) accession SRR2970609
• Quality Control

Sequence quality before trimming Sequence quality after trimming

• Quality check of this data was generated by fastqc tool and

contamination of adapter and everything was observed. A further
trimmomatic tool was used for further trimming of contamination
and making sequence data compatible for assembly.
Transcript Assembly
• The Transcriptome data of Cymbopogon flexuosus were obtained from the NCBI
SRA. The raw data was in SRA format which was converted to FASTQ format
using SRA Toolkit. Then high-quality reads were assembled Denovo using Trinity
assembler. Unigenes were used to predict against known plant (Virdiplantea)
miRNA for the homology.

• Reference miRNA form miRBase database
• miRBASE v22 was used which contains
38589 entries representing hairpin
precursor miRNA, expressing 48885 mature
miRNA products, in 271 species. We use as
a reference 10414 mature virdiplantea
miRNAs.
• The known plant (virdiplantea) miRNA
sequence was used as a query sequence for
homology search by BLASTn. Query
sequence against the assembled unigenes
as a reference database. The blast was
carried out at E-value 1000 and
mismatched less than four. For candidate
miRNA, the length of mature miRNA
sequences should be between 18-24nt.
Secondary Structure Prediction
• The non-protein unigenes are used for the secondary
structure prediction. The secondary structure
prediction of the precursor miRNA sequence is
predicted by using the mfold web server and RNAfold
software with the default parameter. We adjust the
MFE (minimal folding free energy), AMFE (adjusted
minimal folding free energy), and MFEI (minimal
folding free energy index).
• Some different feature was considered in the
prediction of secondary structure:
• 1) loop is not broken in miRNA sequences
• 2)mature RNA should be in one arm
• 3)MFE (minimal folding free energy) must be higher
than negative energy (>-18)
• 4)AMFE (adjusted minimal folding free energy), MFEI
(minimal folding free energy index) must range
between 0.65 to 0.85.
• Target prediction

The identify novel miRNA from the mfold which is used to target
prediction in the psRNATraget tool. It is planted Small RNA Target Analysis
Server. Cymbopogon flexuosus miRNA data were predicted against homo
sapiens with the default parameter.
• Functional annotation

Protein Analysis Through Evolutionary Relationships (PANTHER) is a

• By using the GENEALACART tool of gene cart
collection of protein families that are subdivided into subfamilies. webserver I performed functional annotation for
PANTHER is a multifaceted data resource. Panther has been understanding the nature and property of heat human
mapped to existing InterPro entries. Panther software allows the gene and also find pathway and disease association of
user to categorize new protein sequences to analyze genes list
from the large-scale genomics trial.
hit gene.
• Oncogene recognition

• Reported oncogenes as well as tumour suppresser genes were downloaded

and cross checked again our psRNATarget hit result with help of venny web
tool for bioinformatics and van diagram generated among all 179 hit gene 11
oncogene were present no reported tumour suppresser gene was present.
Results

• After working on the

folding pattern of 6550
precursors I was able to
figure out 51 mature
miRNAs in Cymbopogon
Felxuosus and cross them
with human oncogenes
These 6 MIRs are
responsible as a target to
oncogenes.
• Heat gene involved in human disease
Sr. No. Gene Heat miRNA Gene description Protine class Tissue Cell Pathology Subcellular RNA
Chromosome Cytoband Location Antibody Location tissue
category
1 SEMA4G cfl_miR397a_5p Semaphorin 4G Plasma proteins, 10 q24.31 Intracellular,Membrane,Secreted HPA077752 pancreatic and Vesicles Lipid Tissue
Predicted liver cancers droplets enhanced
intracellular
proteins, Predicted
membrane proteins,
Predicted secreted
proteins,
2 ROR2 cfl_miR6034 Receptor tyrosine Disease related 9 q22.31 Not available HPA021868 Thyroid Cancer NA Mixed
kinase like genes, Enzymes, , Renal Cancer
orphan receptor Potential drug .
2 targets, Predicted
membrane proteins.
3 GFRA1 cfl_miR1436 GDNF family Predicted 10 q25.3 Localized to the Golgi apparatus HPA043829 Breast, Colon, Nucleoplasm, Mixed
receptor alpha 1 membrane proteins (approved) Prostate, Golgi Appratus
Ovarian,
Endometrial,
Liver, Stomach
and Thyroid
cancers
4 KLF12 cfl_miR5148c Kruppel like Predicted 13 q22.1 Localized to the Nucleoplasm HPA064146 Pending Nucleoplasm, Mixed
factor 12 intracellular (Approved) Cancer tissue Cytosol
proteins,Transcriptio analysis.
n factors
5 HNF4A cfl_miR16bO Hepatocyte Disease related 20 q13.12 Localized to the Nucleoplasm HPA004712 Renal, Gastric, Nucleoplasm Group
nuclear factor 4 genes, Nuclear (supported) Pancreatic and enrich
alpha receptors, Predicted Liver cancers
intracellular
proteins,
Transcription factors

6 NANOG cfl_miR2863b Nanog homeobox Predicted 12 p13.31 Not available CAB019380 Most Tissue
intracellular Testicular enhanced
proteins, Cancers
Transcription factors
 • Inhibition graph from psRNA Target results
250

200 193

150

100

50
17

0
Cleavage Translation

Table of Prediction of miRNA target genes of Cymbopogon flexuosus Linn total 51

miRNA and miRNA* has many targets showing the score, target aligned region,
target site, and inhibition type of each gene. This graph concludes that the
prediction of miRNA targets the gene involved in inhibition. A total of 193 genes
are involved in cleavage and 17 genes are involved in the translation process. These
198 genes are breakdown the target gene involved in some diseases.
Functional annotation results
• Cellular Component
1%
Cellular component
1%
synapse (GO:0045202)
12%
cell junction (GO:0030054)

membrane (GO:0016020)
38% 11%

protein-containing complex (GO:0032991)

organelle (GO:0043226)

extracellular region (GO:0005576)

30%
cell (GO:0005623)
7%
• Biological Process
Biological process

0% 1% cellular component organization or biogenesis

3% (GO:0071840)
cellular process (GO:0009987)

biological phase (GO:0044848)

23% 30%
localization (GO:0051179)

reproduction (GO:0000003)

biological regulation (GO:0065007)

response to stimulus (GO:0050896)

2%
developmental process (GO:0032502)
0%
6%
multicellular organismal process (GO:0032501)

3% 10% biological adhesion (GO:0022610)

3%
metabolic process (GO:0008152)
0%
19% cell proliferation (GO:0008283)
• Molecular Function
Molecular functon

5% 9%

8%
transcription regulator activity (GO:0140110)
molecular transducer activity (GO:0060089)
30%
binding (GO:0005488)
structural molecule activity (GO:0005198)
molecular function regulator (GO:0098772)
catalytic activity (GO:0003824)

39% transporter activity (GO:0005215)

6%
3%

Function annotation of genes in panther database shown in Biological Process (BP),

Molecular Function (MF), and Cellular Component (CC). All 198 hit genes of humans
studied with this and in Biological Process 30% gene involved in a cellular process, in
Molecular Function 39% gene involved in binding and 30% gene involved in catalytic
activity. Last, in cellular components total 30% of gene are responsible for organelle
development.
 Interaction Of Oncogene

This protein-protein interaction only interacts with each other cancer genes in strings databases. A total of 5 genes of 11
oncogenes are associated with each other. Different interactions such as experimentally and curated database interactions also
predicted interactions like gene fusion, gene neighborhood, and gene co-occurrence.
 • Network analysis of Cancer gene

In network analysis, a total of 11 oncogenes represent the interaction network with functionally related
other genes in geneMANIA which shows the network from a different perspective. These interacted genes
function blue color shows involvement in the metabolic pathway which is 2.8% where purple color shows
Co-expression and 95.24% gene are involved in that and green color shows genetic interactions 1.96% gene
are involved in it.
Conclusion
• cross-kingdom regulation seems to be the key to answering the
inevitable question of how these medicinal plants regulate human
transcriptome and hence identification of these novel mechanisms
could greatly enhance our understanding of molecular signaling
between species. Collectively, this analysis not only prompts toward
the role of these plant miRNAs in regulating the human target genes
having a great significance in various diseases but also paves the path
for future studies that might explore the potential of miRNA mediated
cross-kingdom regulation in, the prevention and treatment of various
human diseases including cancer.
Future Scope
• The miRNA mimic technology (miR-Mimic) is an innovative approach
for gene silencing. This approach is to generate nonnatural double-
stranded miRNA-like RNA fragments.
• Once introduced into cells, this RNA fragment, mimicking an
endogenous miRNA, can bind specifically to its target gene and
produce posttranscriptional repression, more specifically translational
inhibition, of the gene.
• During covid pandemic I figured out 44 CFL MIRs are involved in the
antiviral activity and can be used as therapeutics for COVID.
References
1. Kumar S, Dwivedi S, Kukreja AK, Sharma JR, Bagchi GD, editors. Cymbopogon: The Aromatic Grass
Monograph. Lucknow, India: Central Institute of Medicinal and Aromatic Plants; 2000. [Google Scholar]
2. Melo, C. A., & Melo, S. A. (2014). Biogenesis and physiology of microRNAs. In Non-coding RNAs and Cancer (pp. 5-
24). Springer, New York, NY.
3. MacFarlane, L. A., & R Murphy, P. (2010). MicroRNA: biogenesis, function and role in cancer. Current genomics, 11(7),
537-561.
4. Dai, X., & Zhao, P. X. (2011). psRNATarget: a plant small RNA target analysis server. Nucleic acids
research, 39(suppl_2), W155-W159.
5. Griffiths-Jones, S., Grocock, R. J., Van Dongen, S., Bateman, A., & Enright, A. J. (2006). miRBase: microRNA
sequences, targets and gene nomenclature. Nucleic acids research, 34(suppl_1), D140-D144.
6. Thomas, P. D., Campbell, M. J., Kejariwal, A., Mi, H., Karlak, B., Daverman, R., ... & Narechania, A. (2003). PANTHER: a
library of protein families and subfamilies indexed by function. Genome research, 13(9), 2129-2141.
7. Szklarczyk, D., Franceschini, A., Kuhn, M., Simonovic, M., Roth, A., Minguez, P., ... & Jensen, L. J. (2010). The STRING
database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic acids
research, 39(suppl_1), D561-D568.
8. Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic acids
research, 31(13), 3406-3415.
9. Melo, C. A., & Melo, S. A. (2014). Biogenesis and physiology of microRNAs. In Non-coding RNAs and cancer (pp. 5-
24). Springer, New York, NY.
10. MacFarlane, L. A., & R Murphy, P. (2010). MicroRNA: biogenesis, function and role in cancer. Current genomics, 11(7),
537-561.

Acknowledgement
1. This work was financially supported by the Financial Assistance Programme - Department of Science and Technology [Grant
Number GSBTM/MD/JDR/1409/2017-18] and Gujarat Council on Science and Technology
[GUJCOST/Supercomputer/2019-20/1359].
Email: dshukla4@gmu.edu