Novel small RNAs expression libraries uncover
hsa-mir30c and hsa-mir30b as key factors
in anoikis resistance
Miguel A. Moreno-Mateos*, Verónica Barragán*, Belén Torres and José Antonio Pintor-Toro
Avda. Americo Vespucio s/n. Edif. CABIMER Parque Cientifico y Tecnolgico Cartuja 93 CP 41092 - Sevilla (SPAIN) Telephone: +34954467838. E-mail: miguelangel.moreno@cabimer.es
*These authors contribute equally to this work.
-m
ir1
9
l
Le
nt
tro
-c
on
Le
nt
-m
ir1
5
tro
Le
nt
-c
on
Le
nt
en
g
l
ht
l
pC
M
Luc
BS mir21(19,15)
pLUC-BS
120
100
80
60
40
20
0
Lentcontrol
Lent-19
Lentcontrol
Luciferase mRNA levels
Luciferase Activity
Probe
Y
Ctr
miR-19
Luc
Plasmids
21 antisense
Probe
Y
Ctr
miR-19
21 sense
Probe:
pCMV-mir21
ll
l
Plasmids
pCMV-control
fu
Lent-21
nt
ro
Lent-control
V21
pLENT-DUAL
pCMV-21 full
lenght
VCo
pCMVcontrol
-m
ir2
1
Lent-21
pC
M
Lent-control
l
20nt
tro
30nt
120
100
80
60
40
20
0
Le
nt
mature miRNAs
Luc
-c
on
40nt
AAAA
180
160
140
120
100
80
60
40
20
0
Le
nt
AAAG
120
100
80
60
40
20
0
Lent-15
120
3’UTR PDCD4
100
80
60
140
40
120
Luciferase activity
H1
E)
D)
Luciferase mRNA levels
U6
C)
Luciferase Activity
B)
Probe
Y
Ctr
miR-21
Ctr
miR-21 FL
A)
Probe
Y
Ctr
miR-21
Ctr
miR-21 FL
INTRODUCTION
During the past ten years, microRNAs (miRNAs) and other small non-coding RNAs (ncRNAs) have been deeply studied in order to elucidate their biological functions. At present, libraries to overexpress
miRNAs are generated by synthesis and cloning of individual miRNAs.
In this work, a patented methodology has been developed to generate a lentiviral expression library containing the whole population of small RNAs present into the cell including matures miRNAs. Using this
library, a functional screening has been completed and several miRNAs involved in anoikis (cell death by loss of attachment) resistance identified. Among these, hsa-mir30c and hsa-mir30b were selected for
further analysis because their expression strongly reduced cell death. Acquisition of anoikis resistance by these miRNAs was achieved through the inhibition of apoptotic pathways by downregulation of
Caspase-3.
20
0
Lent-control
Plasmids
Lent-19
Lent-control
Lent-15
Plasmids
Probe: 19 sense 19 antisense
100
80
60
40
20
0
Lent-control
Lent-21
pCMV control
pCMV 21 Full
Fig 1. Mature miRNAs expressed by dual convergent promoters are functionally similar to full length expressed miRNAs. A) Scheme showing the plasmid used to
Lenght
express mature miRNAs and other small RNAs (at the top) and the plasmid carrying the luciferase ORF followed by a binding site (BS) for miRNA 21, mir19 or mir15
Plasmids
(at the bottom). B) RNAse protection assay of mature hsa-mir21 and hsa- mir19 overexpressed transiently by pLENT-DUAL plasmid. Hsa-mir21 full length (FL) was expressed from
a pCMV-hsa-mir21 plasmid (Ctr:control; Y: Yeast RNA). C) Luciferase assay. Transient transfections were carried out using pLENT-DUAL (hsa-mir21, mir19 or mir15), pLuc-BS (mir21, mir19 or mir15) and pCMV-Renilla (as
normalization control) at 100:10:1 proportions in HEK-293T cells. Hsa-mir21 Full length (FL) was expressed from a pCMV-hsa-mir21 plasmid. All transfections were performed in triplicate, and error bars represent standard error
from three independent experiments. D) Quantification of Luciferase mRNA levels by Real time PCR in the conditions described in section C. Renilla mRNA was used as normalization control. E) Western blot analysis of
Luciferase protein level and quantification in the conditions described in section C. At the bottom, Luciferase assay in a similar experiment described in section C. Transfections were carried out using pLENT-DUAL-hsa-mir21
and pCMV-hsa-mir21 full length and pLUC followed by 3’UTR of pdcd4 gene carrying a binding site of mir21.
A)
Small RNA isolation from
MDA 231metastasic cell line
5’p
Overexpreesion of
full lenght hsa-mir30b
OH 3’
T4 RNA ligase (no ATP)
3,5
3
2,5
2
1,5
1
0,5
0
Control
30b
ddC 3’
30c
Cell Line
T4 RNA ligase + ATP
pSSFV
ddC 3’
mir30c expression
Independent clones selection
Culture in attached conditions
RT-PCR
20
Overexpreesion of
full lenght hsa-mir30c
Fold change
5’HO
Anoikis screening
(16 days in agar)
OH 3’
ddC 3’
5’p
App
mir30b expression
pSSFV
SubG1/SubG1 control (%)
B)
Fold change
A)
BbvII
120
100
80
60
40
20
0
Control
15
Control
mir30b
-PH
10
mir30c
+PH
5
0
Control
U6
30c
Cell Line
C CA T CAG GTA
H1
30b
Sequencing surviving clones
B)
pLENT-DUAL
sRNA candidates
C)
Ctr 30b 30c
CASP3
BIM
Act
1
Ctr 30b 30c
Pro-Casp3
+PH
SubG1 (%)
mir125b
mir92a
mir30c
mir30b
1,2
-PH
0,8
0,6
0,4
0,2
Fig 2. A functional screening using a small RNAs expression library reveals hsamir30b and hsa-mir30c as important repressors of anoikis. A) Scheme of the small
RNA cloning system to develop expression libraries based on pLENT-DUAL. B)
Immortalized and sensitive to anoikis RPE1 cells were infected with a lentiviral library
containing a small RNAs collection from MDA231 cell line. RPE1 infected cells were
cultured for 16 days in agar dishes at low confluence avoiding any contact. Surviving
clones were transferred to adhesive culture dishes to allow expand anoikis resistant cells.
DNA from these clones was isolated and sequenced. C) Table showing small RNA
sequences obtained from pLENT-DUAL of anoikis resistant clones. D) RPE1 cells were
infected with lentivirus containing mature miRNA candidates or empty vector (control).
Three days after infection, cells were deprived of serum for 24 hours and then were
cultured in polyhema (PH) plates and metyl cellulose (2%) serum free medium for another
24 hours. Apoptosis was determined by FACS
A)
Active Casp3
3’UTR Casp3
(1570 pb)
Mut2
1222-1228
Poorly
conserved
mir30b
Cell Line
mir30c
SubG1 (%)
100
80
Control
mir-30b
mir-30c
Control +
Casp3
mir30b +
Casp3
mir30c +
Casp3
+PH
20
15
10
0
Control
30b
-Trail
30c
Control
30b
30c
+Trail
40
Ctr 30b 30c
20
0
3'UTR
MUT1
-PH
Control
25
5
3'UTR WT
Control +
Casp3
Conclusions
30
60
50
45
40
35
30
25
20
15
10
5
0
Control
Control
35
Luciferase activity
Luc
30b 30c
Fig 3. Full-length hsa-mir30b and hsa-mir30c downregulate anoikis and Caspase-3 expression. A) Levels of mature miRNAs
expressed from a lentiviral plasmid carrying full length hsa-mir30b and full length hsa-mir30c-1 (empty vector as control). On the
right, percentage of apoptosis in the infected cells. B) Western blot analysis of putative targets proteins of hsa-mir30b and hsa-mir30c
(by TargetScan analysis) and related to anoikis. Active Caspase-3 was analysed in cells treated as described in section 2D. On the
right, both protein and mRNA levels (measured by densitometry real time PCR respectively) of pro-Caspase-3 from three
independent experiments. Actin was used as normalization control. C) Caspase-3 complementation. RPE1 cells were coinfected with
two lentiviruses expressing miRNAs and cDNA of Caspase-3. Western blot analysis of Pro-Caspase-3, active Caspase-3 and Actin is
shown. At the bottom, apoptosis determined by FACS of Caspase-3 infected cells growing in normal conditions or as described in
section 2D.
120
Mut1
0
Act
B)
1187-1193
Conserved
Ctr
mRNA Casp3
mir27a
TCACAAGCTAGGGTCTCAGGGA
GCGGAACTTAGACACTGTGAA
Ctr
Active Casp3
p53
mir23b
mir125b
mir27
Ctr 30b 30c
mir23a
TCAACATCAGTCTGATAAGCTT
TGGAAATCCCTGGCAATGTGAT
TTGGAAATCCC TGGCAATGTGAT
ACAGGCCGGGACAAGTGCAATA
GCTGAGAGTGTAGGATGTTTACA
30b 30c
Pro-Casp3
mir21
mir21
mir23a
mir23a
mir92a
mir30c
Ctr
CASP3
Act
Fold change
F11
TCAACATCAGTCTGATAAGCTA
TTACTAGACTGTGAGCTCCT CGA
AGTGGTAATCCCTGGCAATGTGAT
TCAGCATCAGTCTGATAAGCTA
AGCTGAGTGTAGGATGTTTACG
80
70
60
50
40
30
20
10
0
control
B1
B2
B3
D4
F3
F7
mir21
mir151
mir23b
mir21
mir30b
Act
control
Clon small RNA sequence
A8
Ctr
D
)
SubG1(%)
C
)
+PH
-PH
3'UTR
MUT2
Fig 4. Hsa-mir30b and hsa-mir30c downregulate Casp3 through its 3’ UTR.
A) The 3’UTR of Caspase-3 mRNA was cloned downstream of Luciferase open reading frame in the
pLUC-BS plasmid. The 3’UTR of Caspase-3 contains two putative miRNA30b/30c regulatory elements
(MRE); one of them is highly conserved and the other one poorly conserved (by TargetScan analysis). B)
HEK-293T cells were transiently contransfected with plasmids overexpressing miRNAs (hsa-mir30b, hsamir30c or control), pLUC-3’UTR casp3 (3’UTR WT) and pCMV-Renilla at 100:10:1 proportions. The same
experiment was carried out using two mutant versions of pLUC-3’UTR casp3, where six nucleotides were
changed both in the conserved (Mut 1) and poorly conserved (Mut 2) sites.
Fig 5. Hsa-mir30b and
Pro-Casp3
hsa-mir30c reduce
cell death induced
Act
by TRAIL in MCF10A
Cells. Cells were infected with lentivirus
containing full-length hsa-mir30b and hsamir30c. Four days after infection, cells were
cultured in presence of Trail (500 ng/ml) for
seven hours. Apoptosis was determined by
FACS. At the bottom, western blot analysis of
Pro-Caspase-3 in these infected cells. Actin
was used as control.
- An expression library of the complete population of the
cellular small RNAs has been performed.
- Mature miRNAs expressed by this system and full length
expressed miRNAs prove a similar functionality
- Using this library, hsa-mir30b/c have been identified as
important negative modulators of anoikis.
- Caspase-3 is downregulated by hsa-mir30b/c through
translational repression. Hsa-mir30b targets 3’UTR
conserved MRE and hsa-mir30c targets both conserved
and non-conserved MRE.
- Complementation experiments suggest that hsa-mir30b/c
decrease anoikis mainly targeting Caspase-3.
- Other forms of Casp3-dependent cell death are also
downregulated by hsa-mir30b/c.
*This work is supported by grants from the Spanish MEC and the
DGUI of the Junta de Andalucía.