MicroRNAs are small non-coding RNA molecules that regulate gene expression post-transcriptionally. They are involved in many biological processes. The document discusses microRNA biogenesis, classification, nomenclature, genomic organization, and roles in ovarian function and follicular development. It specifically examines how microRNA-424 and microRNA-503 are highly expressed in dominant follicles and predicted to target SMAD7 and ACVR2A, genes important for follicular growth. Experiments were designed to validate these targets using luciferase assays and overexpression/inhibition of the microRNAs in bovine granulosa cells.

1. MicroRNAs: endogenous regulators of gene
expression
Hari Om Pandey
hariomvet@gmail.com
ICAR-IVRI, IZATNAGAR, UTTAR PRADESH, INDIA

2. miRNAs as post-transcriptional regulator of
genes
Involved in a range of
biological functions
Regulate genes by
targeting at 3´
untranslated region
Regulates around 30 %
of all protein coding
genes
miRNAs
miRNAs: MicroRNAs
nts: Nucleotides
1Ambros 2004; 2Bartel 2004; 3Lim et al. 2003; 4Rajewsky 2006
 Small, 20-22 nts, single stranded, non coding and endogenous1,2
1, 2
2
3, 4

3. RNAs
mRNA
piRNA
tRNA
snRNA
snoRNA
siRNA
microRNA
rRNA
Classes of RNAs

4.  miRNAs are encoded by the host
genome, whereas siRNAs in most
cases originate from outer source
 siRNA is most commonly a
response to foreign RNA (usually
viral) and is often 100%
complementary to the target
 miRNA regulates post-
transcriptional gene expression and
is often not 100% complementary to
the target
siRNA vs miRNA

5. The first described microRNA, lin-4 was characterised as a
translational repressor of developmental timing from
Caenorhabditis elegans that was involved in silencing of
another gene, lin-14, at appropriate time in the development of
the worm (Lee et al. 1993 and Wightman et al. 1993)
The transcript of this gene was highly unusual as it was non-
coding, and produced extremely small transcripts (22nt) from
hairpin structured RNA precursors
Second microRNA, let-7 was also cloned from C. elegans
(Reinhart et al, 2000)
MicroRNAs

6. Very little is known about functional role of miRNAs
 Total 38589 mature miRNAs are known in 271 species
Species Mature miRNA Precursor
miRNA
Human (Homo sapiens) 2654 1917
Bovine (Bos taurus) 1025 1064
Goat (Capra hircus) 436 267
Sheep (Ovis aries) 153 106
Pig (Sus scrofa) 457 408
 There is a major challenge to decipher the functional role of
miRNAs in bio-physiology
http://www.mirbase.org/ Pandey et al. 2018

7. Nomenclature of miRNA
 The nomenclature guidelines require that novel miRNA
genes should be experimentally verified by cloning or
with evidence of expression and processing
hsa-mir-20a
 The “hsa” tells us it is a human miRNA
 The “20″ tells us that was discovered early — it’s only
the 20th family that was named
 “20b” tells us that it is related to another miRNA that we
can guess is probably called hsa-mir-20a

8. Nomenclature of miRNA
 The mature sequences are designated ‘miR’ in the
database, whereas the precursor hairpins are labelled
‘mir’
 Paralogous sequences - mature miRNAs differ at only
one or two positions are given lettered suffixes—for
example, mmu-miR-10a and mmu-miR-10b

9. Nomenclature of miRNA
 hsa-mir-17; it has two mature products, named hsa-miR-
17 and hsa-miR-17*
 In this case miR-17 arises from the 5′ arm of the mir-17
hairpin, and miR-17* arises from the 3′ arm
 The ‘*’ tells us that miR-17* is considered a ‘minor’
product
 Such mature sequences are currently named of the form
miR-17-5p (5′ arm) and miR-17-3p (3′ arm)

10. miRBase
 The miRBase database is a searchable database of published
miRNA sequences and annotation
 All sequence and annotation data are available for download
 The miRBase Registry provides miRNA gene hunters with
unique names for novel miRNA genes prior to publication of
results
 To date, Release 22 of the database contains 38589 entries
representing hairpin precursor miRNAs, expressing 48885
mature miRNA products, in 271 species
(http://www.mirbase.org/)

11. miRBase

12. Genomic context of miRNAs
 The majority of miRNAs are located in intergenic regions,
while few corresponds to intronic regions
 Approximately half of all known miRNAs are found in
close proximity to other miRNAs, clustered miRNAs,
and are expressed as poly-cistronic primary transcripts
 Whereas some miRNAs can be transcribed from their
own promoter as mono-cistronic primary transcripts

13. miRNA biogenesis
Winter et al. 2009

14. miRNAs in ovarian function
1Bernstein et al. 2003; 2 Zhang et al. 2014; 3Hossain et al. 2009
Dicer1 knockout
mice
Embryonic lethal1
 Dicer is one of key enzyme used in miRNA biogenesis
 miRNAs are implicated in primordial follicle formation2
 Expression profilling and next generation sequencing have
identified the spatiotemporal expression of miRNAs in ovarian
tissue3
Function of miRNAs in Animals

15. Roe of miRNA in follicular growth and development
 The initiation of primordial follicle development is essential to
achieve successful reproduction in mammalian females
 miR-145 by targeting TGFBR2 regulated the initiation of
primordial follicle development and maintains primordial follicle
quiescence, whereas down-regulation of miR-145 using an
antagomir (AT) decreased primordial follicles and increased
number of the growing follicles by increasing TGFBR2
expression and activation of SMAD signalling (Yang et al. 2013)
 In another study miR-376a was found to be negatively correlated
with PCNA mRNA expression by directly binding to the 3’UTR of
PCNA in fetal and neonatal mouse

16. miRNA in steroidogenesis
 miR-17-92 cluster increased the progesterone level in the spent
media of obtained from bovine granulosa cell culture (Andreas et al.
2016)
 Overexpression of miR-378 decreased ovarian estradiol production
by post-transcriptionally regulating aromatase whereas
overexpression of the aromatase 3'-UTR enhanced aromatase
expression (Xu et al. 2011)
 miR-133b was involved in follicle-stimulating hormone (FSH)-
induced estrogen production by inhibiting the Foxl2-mediated
transcriptional repression of STAR and CYP19A1 to promote
estradiol production (Dai et al. 2013)
 These miRNAs could be potential miRNA to further study the
disorders of granulosa cells in relation to steroidogenic capacity

17.  The roles of miRNAs in oocyte maturation, identification
and characterization of miRNA populations were done in
pools of bovine germinal vesicle (GV) oocytes,
metaphase II (MII) oocytes, and presumptive zygotes
(PZ); pri-miR-155 was present in MII oocytes, indicating
transcription during maturation; however, levels of pri-let-
7d decreased during maturation (Gilchrist et al. 2016)
 Changes in miRNAs expression were observed in the
cumulus cells matured with or without the oocyte
cytoplasm (Abd El Naby, W S et al. 2013)
MicroRNAs in regulation of cumulus-oocyte
communication and oocyte maturation

18.  Identification of important protein involved in miRNA
biogenesis in villous of trophoblast such as Drosha,
Exportin 5 Argonaute 2 (Ago2), DP103, and miR-378a-5p,
miR-376c and miR-141 implicated in promoting
proliferation, migration and invasion of trophoblast cells
(Tefaye et al. 2016)
 Placenta specific miRNAs, the C19MC, miR-371-3 and the
C14MC clusters are found to be pregnancy-associated
miRNAs during placental development and
pregnancy establishment

19.  Expression profiling was done in bovine endometrium affected
by subclinical or clinical endometritis
 Several miRNAs including let-7 family members were
dysregulated in animals affected by subclinical endometritis,
while 35 miRNAs including let-7e, let-7f, miR1265 and miR-608
were altered in animals with clinical endometritis
 Both clinical and subclinical endometritis were found to show
dysregulation of the let-7 family members which are known to
regulate genes involved in various developmental processes and
immune mechanisms by regulating the expression of cytokines
such as TLR4, IL6, IL13, IL10 associated with host - immune
functions (Salilew-Wondim et al. 2016)
MicroRNAs in uterine infection

20. MiRNA expression in bovine granulosa cells
miRNA enriched Total RNA
Granulosa cells
SF (n=43)
(n= 6)
DF (n=9)
Day 3
Granulosa cells
SF (n=58) DF (n=3)
(n= 7)
Day 7
Granulosa cells
Day 19
SF (n=76) PDF (n=5)
(n= 7)
miRNA expression profiling using NGS

21. Role of miRNA in follicular growth and developement
(Gebremdhn et al. 2015; Reviewed in Tesfaye et al. 2018 Reproduction) 9
 miRNA-424/503 cluster members were highly enriched in bovine
granulosa cells of preovulatory dominant follicle at day 19 of
estrous cycle compared to their subordinate counterpart1
MiRNA-424: bta-microRNA-424-5p
MiRNA-503: bta-microRNA-503-5p
miR-183-96-182
miR-132-212
miR-20a-92a
miR-23a-27a-24
miR-222-221
miR-214-199a
miR-424-503-450a
miR-665-127
miR-452-224-3431
1, 2

22. Experimental strategies
1. MicroRNA target gene validation using luciferase assay
2. Overexpression of miRNAs using miRNA mimics
3. Inhibiton of miRNAs activity using miRNA inhibitors
4. Knockdown of the target genes using siRNAs
miRNA: MicroRNA
siRNA: Small interfering RNA

23. Genomic context of miRNA-424/503 cluster members
miRNA Accession no. Sequence
bta-miR-424-5p MIMAT0013593 CAGCAGCAAUUCAUGUUUUGA
bta-miR-503-5p MIMAT0025557 UAGCAGCGGGAACAGUACUG
https://www.ncbi.nlm.nih.gov/nuccore/563320293
http://www.mirbase.org/cgi-bin/mirna_entry.pl?acc=MI0022315
MiRNA-424: bta-microRNA-424-5p
MiRNA-503: bta-microRNA-503-5p
miR-424 and miR-503 are evolutionary conserved among mammals
miRNA Genomic position
bta-mir-424 chrX:18185439-18185534[-]
bta-mir-503 chrX:18185095-18185177[-]

24. MiRNA target prediction
miRWalk
(http://www.umm.uni-
heidelberg.de/apps/zmf/mirwalk/)
Analyze potential target genes
miRNA-mRNA binding site
analysis
(http://www.targetscan.org/)
In-silico target gene prediction
Target site prediction
PITA
(http://genie.weizmann.ac.il/pubs/mir07/mir07_predicti
on.html)
PITA: Probability of interaction by target accessibility

25. Target gene mRNA-miRNA binding sites
ACVR2A: Activin receptor type II A
SMAD7: Mothers against decapentaplegic homolog family 7
miRNAs
 SMAD7 and ACVR2A were identified as potential putative targets for
miR-424/503 cluster members
Pandey et al. 2018

26. Wild type and mutant plasmid vector construction
Wild type SMAD7 3´ UTR
Mutant SMAD7 3 ´ UTR
Wild type ACVR2A 3´ UTR
Mutant ACVR2A 3´ UTR
Pandey et al. 2018

27. Generation of target
fragment
Cell lysate
Measured luciferase
activity
Wild type SMAD7
Cloning into pmirGLO
vector
Plasmid isolation
Mutant Wild type ACVR2A Mutant
Plasmid co-transfection
with miR-424 mimic
miR-503 mimic or
mimic NC into bGCs
bGCs: bovine granulosa cells; NC: Negative control
Target gene validation using luciferase assay

28. SMAD7 and ACVR2A are direct targets of miR-424/503 cluster
Mean ± SEM; *p<0.05; **p<0.01
SMAD7
pmirGLO-SMAD7 3´UTR
bta-miR-424-5p mimic
pmirGLO-SMAD7 3´UTR Mut
bta-miR-503-5p mimic
+ + + - - -
- - - + + +
- + - - + -
- - + - - +
SMAD7
Mimic NC + - - + - -
ACVR2A
+ + + - - -
- - - + + +
- + - - + -
+ - - + - -
pmirGLO-ACVR2A 3´UTR
bta-miR-424-5p mimic
pmirGLO-ACVR2A 3´UTR Mut
Mimic NC
ACVR2A
bta-miR-503-5p mimic - - + - - +
Pandey et al. 2018

29. Overexpression of miRNA-424/503 cluster members
enhanced granulosa cell proliferation
Mean ± SEM; **p<0.01; ***p<0.001
PCNA: Proliferating cell nuclear antigen
PCNA
 Granulosa cell proliferation is necessary for follicular growth and
creation of unique micro-environment for oocyte maturation (Maruo
1995)
Cell proliferation assay
PCNA
β -ACTIN
mimic inhibitor
Pandey et al. 2018

30. Functional analysis of miR-424/503 cluster members in
granulosa cells
Bovine granulosa cells were
collected from Ø 3-5 mm
follicles
Cultured in DMEM/F12
supplemented with 10% FBS
Sub-confluent bovine granulosa cells
were transfected
Harvested 48 h post-transfection
DMEM/F12:Dulbecco's modified eagle's medium/nutrient F12 Ham
FBS: Fetal bovine serum
mR-424 mimic mR-503 mimic Inhibitor NC
Mimic NC
miR-424
Inhibitor
miR-503
Inhibitor
Cell proliferation
assay
Gene expression
SMAD7, ACVR2A,
PCNA and STAR
Protein analysis
SMAD7, ACVR2A,
PCNA and STAR
Cell cycle analysis

31. NC siRNA SMAD7 siRNA NC siRNA ACVR2A siRNA
Knockdown of miR-424/503 cluster members target genes
using siRNA
Bovine granulosa cells were
collected from Ø 3-5 mm
follicles
Cultured in DMEM/F12 HAM
supplemented with 10% FBS
Sub-confluent bovine granulosa cells
transfected with
Harvested 48 h post-transfection
Gene expression
SMAD7, ACVR2A,
PCNA and STAR
Protein expression
SMAD7, ACVR2A and
PCNA
Cell proliferation
assay
Cell cell cycle analysis

34. Knockdown of SMAD7 enhanced granulosa cell
proliferation
SMAD7 siRNA
Cell proliferation
assay
Mean ± SEM; *p<0.05; **p<0.01
Pandey et al. 2018

35. Expression pattern of miR-424 in cumulus cell and oocyte
during in vitro oocyte maturation
In vitro maturation
39 °C for 22 h, 5% CO2
miR-424 expression
analysis
COCs: Cumulus-oocyte complexes
GV: Germinal vesicle, MII: Metaphase II
COCs collection from Ø 2-8
mm follicle
Cumulus
cells
Oocytes
Cumulus cell-oocyte
separation
1% hyaluronidase
GV stage
COCs
MII stage COCs
Oocytes
Expanded
cumulus
cells
Pandey et al. 2018

36. miR-424 expression is increased in cumulus cells and oocytes
during in vitro maturation
GV: Germinal vesicle
MII: Metaphase II
Mean ± SEM; ***p<0.001
 The miR-424 expression is increased from GV
to MII stage in both cumulus cells and oocytes
Pandey et al. 2018

37. Future Perspective
 Potential biomolecule, which could be utilized as
therapeutic and diagnostic molecules
 Indeed the misregulation of several miRNAs has already
been linked to the development of several disease
 ‘miRNA replacement therapy,’ which involves introducing
synthetic miRNAs or miRNA mimic into diseased tissues
 This again comes with various other challenges such as drug
delivery to the right organs or tissues and choosing the
appropriate technology to modulate the miRNA expression