miRNA as Potential and Emerging

Biomarkers for
Diagnosis Infectious Diseases

Sofia Mubarika Haryana

Presented at the 15th BMBC 2017
Malang, 12 – 14 May 2017
 SDG’s Target
Post-2015 Development Agenda
THE WORLD We Want:
THE WORLD WE WANT
To be a Healthy and Prosperous World
(status: 20 February 2012)
Targets of NCD : CV, Metabolic Syndromes, Cancer , COPD
© United Nations Development Programme
 MicroRNA (MiRs) Emerging Biomarkers

Outline of Presentation
• Non-coding RNAs (ncRNAs) and microRNAs
(MiRs)-background and functions
• MiR expression in tumors and cancer cells
• Exosome - miRs in body fluids and their potential as
prognostic and diagnostic biomarkers
• MiRNA in Infectious Diseases
•MiRs as Biomarkers and drug targets
 Post Genomic Era -
Personalized Medicine

• Biomarkers for Diagnosis, Prognosis and

Target Therapy
• Biomarker – minimal invasive
• Exosomes - fluid biopsies : serum, plasma,
saliva, urine , breast milk , CSF
Challenged : Personalized to
Precision Medicine

• Role of genes
• Phenotypes of
expressions

• Early Diagnosis
• Follow up Treatment
• Prediction of Diseases
• Drug Development
Personalised Med – Patient safety
 The Human Genome Project
Cancer’s Precision Medicine
GWS
NGS
Microarray

TCGA 3 billion bases 30,000 genes

EPIGENOME http://www.genome.gov/
 The New Genetics: The Human Genome
 22,000 genes based on current
Dark Matter is 98% of the algorithms =
mass of the universe 5% of genome
 30% have instructions to make
How is our genome ? proteins
 70% have instructions to regulate
the protein-coding genes

 The other 70% (regulating)

SHINING A LIGHT ON THE GENOME’S
‘DARK MATTER’
NON-CODING RNAs (nc RNAs) :
MICRORNAs & CANCER
• Ribosomal (rRNA)
• Transfer RNA (tRNA)
• Small nucleolar RNA
(snoRNA)
• Micro RNA (miRNA)
 95% Junk DNA:
 Pseudogenes
 Other functions?

© 2007 The American Society for Investigative Pathology

 Non-coding RNA: the RNA formerly known as “junk”
RNA Transcripts

Protein-coding mRNA Non-coding RNA Transcripts

Regulatory RNA Housekeeping RNAs

snoRNAs
miRNA
siRNA
•tRNA
piRNA
•rRNA
Anti-sense RNA •snRNA
•tmRNA
•Rnase P RNA
• Transcription/chromatin structure regulators •vRNAs
•gRNAs
• Translational regulators
•MRP RNA
• Protein function modulators •SRP RNAs
• RNA/Protein localization regulators •Telomerase RNA

NC-RNAs compose majority of transcription in complex genomes

 Nobel Prize in Physiology or Medicine 2006
Andrew Z. Fire
Craig C. Mello

Cho WC. MicroRNAs in cancer - from research to therapy.

Biochim Biophys Acta - Rev Cancer 2010;1805(2):209-217. C. elegans
 miR oncogene ( OncomiRs) and TSG ( T supressosor miRs)
Correlation between Fragile chromosome sites and location of miRNA genes

Copyright © 2004, The National Academy of Sciences Calin G. A. et.al. PNAS 2004;101:2999-3004
Genomic Organization of miRNA Genes

•Intronic miRNAs often in antisense direction, made from own promoter

•Exonic miRNAs - non-coding (or in alternatively spliced exons)

Zhao Y, Srivastava D, TIBS 32:189,2007
 miRNA biogenesis and function.

Copyright © American Heart Association Suárez Y, Sessa W. Circulation Research 2009;104:442-454

 Micro RNA (miRNA)

• miRNAs-1st discovered in 1993 by Victor Ambros, Rosalind Lee

and Rhonda Feinbaum

• Endogenously expressed small Non-coding RNA, function as post-

transcriptional regulators of gene expression

• 19 – 25 nucleotides in length RNA sequences - bind to

complementary sequences in the 3’ UTR

• multiple target mRNAs ~60% of all genes, resulting in silencing.

miRNAs Affect Everything

© Frank Slack frank.slack@yale.edu

In facts :
mRNA and miRNA
Ratio Coding : Non coding in the
Genomic DNA

Ryan et.al, BioEssays 29:288–299 (2007 )

 miRNAs in Tumor and Cancer
miRNA – promising biomarkers
Small molecules
Stable
Target mRNA – post transcritional
One miRNA can target multiple mRNA 
Master of the Regulators
MDG‘s and SDG’s Challenges

• Acute to chronic conditions: MADAT –

NCD ( CV, DM, Cancer, COPD)

• Aging Population – RISKESDAS 2014

• Health Disparities : Double/Triple Burden of

Diseases

• Emerging Diseases , Re-emerging , Neglected :

EBOLA, ZICA, Rabies, Leprosy etc.

• Biodefense - Biosafety and Biosecurity

 TOP 10 MALIGNANCIES IN DHARMAIS NCH 1993 – 2010

Trachea, Bronchus, Lung * HPV & EBV related Ca

* * HBV – related Ca
*
*
* *
*

* *
* Trachea, Bronchus, Lung
*
Men *

*
Courtesy:
Hospital-Based Cancer Registry Women
Dharmais NCC
 HPV and EBV related Ca
Cervical
HPV - EBV - HIV ?? Ca
NPC
Hodgkin Vaginall/ Oropha
Gastric Lympho Squam ryngeal
Ca ma Ca Ca
HPV
EBV
Lympho Anal Prostate
B/ T/ NK
prolif
Lympho
Disease
Ca Ca
ma

Paramita, et al., 2006, J Fachiroch, 2010,

S. Hutajulu, et al 2012)

Laryngeal Ca : A. Surono , 2010 Lowy DR, et al, 2014, Chesson HW, 2014, Shi R, 2015
 TCGA :
miRNA – biomarkers for Cancer
• Non coding RNA ( > 95% of the genome )
• Conserved in the genome
• Stable , secreted as exosome in the circulation
• Regulate protein expression – bind at 3’UTR,
Coding sequence or 5’UTR  inhibit protein
translation or degrade Protein
• miRNA regulate more 30% mRNA
 miRNAs in Serum …..

Circulating microRNAs as stable blood-based

markers for cancer detection
PNAS, 2008 Jul

Characterization of microRNAs in serum:

a novel class of biomarkers for diagnosis of cancer and
other diseases
Cell Res. 2008 Oct
 miRNA is potential biomarkers

Diagnostic
biomarker

Targeted Prognostic
therapy biomarker

Annual 12th miRNA as Biomarkers and Diagnostic, Cambridge, April 2016

Sampel detection for microRNA
Can detected from
body fluids like :
• serum,
• plasma,
• saliva,
• urine,
• Breast milk ,
• Cerebrospinal
fluid

Collin et al. Nature. 2012

Quantification microRNA from body fluids

Exicon ,2014
 Figure 2

Trends in Molecular Medicine 2014 20, 460-469DOI: (10.1016/j.molmed.2014.06.005)

Copyright © 2014 Elsevier Ltd Terms and Conditions
Serum biomarkers with changes Before and After
Chemotherapy in Relapsed NPC Patients
EP: Biomarker: 7,659 Da GC: Biomarker: 7,765 Da
% Diff = 206.8% p = 4.8E-6 % Diff = 194.4% p = 6.1E-6
increased decreased Mean +/- SD increased decreased Mean +/- SD

1.727 0.835
2.382
4 +/- 0.753
+/- 0.781 6 +/- 1.128

3 5

4 1.225
2 +/- 0.734

3
1
2

0
1

-1 0
Before After Before After
Chemotherapy (N=35) Chemotherapy (N=29)

EP, Etoposide and Cisplatinum; GC, Gemcitabine and Cisplatinum.

Cho WC et al. ProteinChip array profiling for identification of disease- and
chemotherapy-associated biomarkers of nasopharyngeal carcinoma. Clin Chem. 2007;53(2):241-50.
miRNA for treatment – hope
Hallmarks of cancer – viral related cancers
Communicable vs Non
Communicable Disease
 miRNA signature – Functional Genomics – its implication

Diagnostic
Prognostic
Target therapy
 Oncology-directed miRNA
replacement therapy.

Bader A G et al. Cancer Res 2010;70:7027-7030

©2010 by American Association for Cancer Research

 Exosome – MVB
• Cell to cell communication
• Donor – Recipient cell
• Miniature of cell – states of cell
 Exosome – Cell to Cell Communication

Donor Recipient
Biomarkers potential for cancer – miRNA

Small mol, stabil, plasma,

Minimal invasive !!! --. Big Hope

Sharon A. Ross, and Cindy D. Davis Adv Nutr 2011;2:472-485 ©2011 by American Society for Nutrition
Exosomes
Exosome Composition
Exosome Composition
Getting traction
 SERUM AND BODY FLUID MiRs AS
BIOMARKERS

• Multiple miRs have been characterized not only in serum

but also tears, urine, breast milk, seminal fluid, saliva,
amniotic fluid, bronchial lavage, cerebrospinal fluid,
pleural fluid, peritoneal fluid and colostrum
(Clin. Chem 56, 1733, 2010)

• A select number of miRs may serve as diagnostic markers

for different tumor types
(Mol. Cancer 9:306, 2010)
 Frontiers in Microbiology
Circulating miRNA
as Potential and Emerging Biomarkers for
Human Infectious Diseases

Verma et al. Front Microbiol, 2016

miRNA regulation in Immune Response
miRNAs IN INNATE IMMUNITY

J. Allergy Clin & Immunology, 2015

 miRNA
Regulate
Innate &
Adaptive
Immune
Response
 miRNA in
Innate
Immunity
Bacterial/fungal
miR 155
miR-146a
Virus :
miR-155

Pathogen TLR :
miR-223
miR-155
miRNA Regulate Immune Response
 miRNAs in Innate Immunity

miRNA Targets mRNA Effect

• miR-9 NF-κB1 Negative regulator of TLR4 signaling
• miR-19 TLR2 Decreases TLR2-med. inflammation
• miR-21 PDCD4, IL-12 p35 Negative regulator of TLR4 signaling
• miR-27b PPAR-γ Enhances response to LPS
• miR-105 TLR2 Decreases TLR2-med. inflammation
• miR-106a IL-10 Decreases IL-10
• miR-125b TNF-α; IRF4 Diminishes inflammation; enhances
macrophage activation
• miR-145 TIRAP Inhibits TLR signaling
• miR-146a TRAF6, IRAK1, IRAK2 Negative regulator of TLR signaling
• miR-155 AID; MyD88; TAB2; Pellino-1;
IKKε; SHIP-1; SOCS1; C/EBP-β Enhances inflammation; negative
feedback regulation
• miR-223 IKKα; Pknox1 Proinflammatory activity
• Let-7i, let-7e TLR4 Downregulate inflamm signaling
 miRNA in Adaptive
Immunity
B Cell development &
differentiation :
miR 181-a1
miR 17-921
miR-155

T cell development &

differentiation :
miR 17-921
miR-155
miR-181a
 Exosome
as Antigen Presenting Cells
miRNA an emerging powerful biomarkers
MiRNAs are a class of gene regulators :
• Regulate at the level of transcription, translation,
post translational of the genome
• miRNA regulates all signaling pathways in healthy/
physiological 
• The small, stable , circulating miRNA  proposed as
molecular specific biomarkers of infectious diseases
• Dysregulated – caused by SNP, Mutations,deletion,
hypermethylation, translocation, amplification

Cho et al., 2011, Zhang et al, 2012, Verma et al., 2016

 miRNA & infectious diseases
1. HIV/AIDS

2. Hepatitis

3. Tuberculosis

4. Helicoibacter Pylorii

5. Malaria

6. Trypanosomiasis
 miRNA in HIV : as Molecular Diagnostic
62 miRNA
Up regulated
• miR- 223; miR-382; miR-
125b , miR 28  target
3’UTR HIV mRNA –I>
decrease HIV replication
High Expression miR-21 and mi- R122
Low expression of miR – 223
Down regulated
• miR-146-5p; miR—150;
miR-16 and miR-191  esp
Found in B, T lymphocytes
indicating HIV +
• miR-1465p, miR-15-
decreased in HIV with ARV
resistance
Discriminate HIV + and HIV -
Thapa et al., 2014
lncRNA & miRNA in HIV infection
miRNA 155 inhibited HIV integration

HIV 
Immunocomromised
through CD4 cell

miR – 155 – inhibit nuclear

integration through
binding to ncRNA comlex

Verma et al, 2016

Post transcriptional regulation of host MIRNA on
miRHIV

Thapa et al, 2014, Verma et al, 2016

Molecular Regulation of MiR on HIV

Verma et al, 2016

miR and Inflammation

Physiol Genomic 2010

MicroRNA and Tissue Injury

Physiol Genomic 2010

 miRNA in Tuberculosis Pathogenesis
• miR-144 highly expressed in pulmonary Tuberculosis
• miR-29c and miR 22 – discriminate between latent and active
TB ( Zhang et al, 2013)

• miR -361- 5p, miR-889, miR- 576-3p up regulated  non

invasive molecular biomarker in pulmonary tuberculosis

• miR – 378, miR- 483-5, miR-22 and miR- 29c, miR-101 and miR-
320 – molecular diagnosis for TB + and TB - ( Qi et al, 2012)

• miR – 378 and miR-101 target MAPA1 signalling

• miR-483-5p, miR-320, miR22 affect AKT3 and BCL9L signalling
Zhang et al. 2013
Verma et al, Frontier in Microbiology, 2016
miRNA as specific Biomarkers for Tuberculosis
Hepatitis C Virus-Induced Upregulation of MicroRNA
miR-146a-5p in Hepatocytes Promotes Viral Infection
and Deregulates Metabolic Pathways Associated with
Liver Disease Pathogenesis
 miR for molecular Diagnosis of HBV
infection
• miR – 122; miR – 34A; miR-16; miR-21: biomarkers
for hepatitis related HCC (Cermelli, 2011)
• miR -122 and miR – 34a : upregulated during HCV
infection ; miR -21 : normal during Infections)
• Hepatic fibrosis – increase miR-21 / activate TGF-
beta ( Behrer et al., 2011)
• miR-149, miR- 638; miR-491 is upregulated in HCV
Infection by inhibiting PI3K Frontier in Microbiology, ( Conrad
and Liman, 2014)

Verma etal., Frontiers in Microbiology, 2016

The development of MiRNA for Therapy
 MiR-122
antagonist for
H CC
 Summary
• miRNA non coding DNA, small molecul , stable,
that give a big promise to better Diagnostic and
Therapy
• miRNA and non coding lnc RNA play in harmony
with coding DNA carried by exosomes – but
when disrupted that make diseases
• ncRNA - miRNA is emerging powerful diagnostic
for infectious diseases/ diseases
• MiRNA for targeted therapy is in progress
Thank You

Exosomes &
miRNA –
Toward
Personalised
Medicine