Current Pharmaceutical Biotechnology, 2009, 10, 569-578
569
Trafficking of Mature miRNA-122 into the Nucleus of Live Liver Cells
Zeno Földes-Papp1,2,*, Karsten König3,4, Hauke Studier4, Reiner Bückle5, H. Georg Breunig4,
Aisada Uchugonova3 and Gerhard M. Kostner1
1
Institute of Molecular Biology and Biochemistry, Medical University of Graz, Harrachgasse 21 , A-8010 Graz, Austria;
Visting Professorship at ISS, 1602 Newton Drive, Urbana-Champaign, IL 61822, USA; 3Faculty of Mechatronics and
Physics, Universität des Saarlandes, D-66123 Saarbrücken, Germany; 4JenLab GmbH, Campus A 1.2, D-66123 Saarbrücken, Germany; 5JenLab GmbH, Schillerstrasse 1, D-07745 Jena, Germany
2
Abstract: The binding of superquencher molecular beacon (SQMB) probes to human single-stranded cellular miRNA122 targets was detected in various single live cells with femtosecond laser microscopy. For delivery of the SQMBprobes, 3D-nanoprocessing of single cells with sub-15 femtosecond 85 MHz near-infrared laser pulses was applied. Transient nanopores were formed by focusing the laser beam for some milliseconds on the membrane of a single cell in order
to import of SQMB-probes into the cells.
In single cells of the human liver cell lines Huh-7D12 and IHH that expressed miRNA-122, we measured target binding in
the cytoplasm by two-photon fluorescence imaging. We found increased fluorescence with time in a nonlinear manner up
to the point where steady state saturation was reached. We also studied the intracellular distribution of target SQMB and
provide for the first time strong experimental evidence that cytoplasmic miRNA travels into the cell nucleus. To interpret
nonlinear binding, a number of individual miRNA-122 positive cells (Huh-7D12 and IHH) and negative control cells,
human VA13 fibroblasts and Caco-2 cells were analyzed. Our experimental data are consistent with the cytoplasmic assembly of nuclear miRNA and provide further mechanistic insight in the regulatory function of miRNAs in cellular physiology. An open issue in the regulation of gene expression by miRNA is whether miRNA can activate gene expression in
addition to the well-known inhibitory effect. A first step for such a regulatory role could be the travelling of miRNA-RISC
into the nucleus.
Keywords: Single live cells, cell trafficking, micro-RNA, RISC, nuclear micro-RNA-122, cytoplasmic assembly of nuclear
miRNA-122, cell biology theory of miRNA role and function, activation of gene expression, in vivo two-photon imaging.
INTRODUCTION
A hot topic in life and medical sciences, RNA interference addresses the question how short pieces of microRNA
(miRNA) regulate the expression of genes [1]. Unlike siRNAs and shRNAs, miRNAs are naturally encoded by cellular
DNA. The expression pattern of miRNA is comparable to
that of many other genes where some members are used for
housekeeping functions and others for defined roles in very
specific processes of organ function [2]. Micro-RNAs comprise a diverse and important branch of the medical and biological sciences. This diversity can be accounted for by the
fact that there exist many different forms of mi-RNAs ranging from precursors to multiprotein complexes as depicted in
Scheme 1 [3-7]. The 17-24 nucleotides, single-stranded (ss)
mature miRNAs are derived from longer, primary transcripts
termed pri-miRNAs. The pri-miRNAs, which can be more
than 1000 nucleotides in length, contain an RNA hairpin in
which one of the two strands includes the mature miRNA.
The hairpin, which typically comprises 60-120 nucleotides,
*Address correspondence to this author at the Visiting professorships at ISS
and at the CCFT, Department of Molecular Biology and Immunology,
Health Science Center, University of North Texas, USA;
E-mail: [email protected]
1389-2010/09 $55.00+.00
is cleaved from the pri-miRNA in the nucleus by the doublestrand-specific ribonuclease, Drosha [8]. The resulting precursor pre-miRNA is transported to the cytoplasm via a process that involves Exportin-5 in a Ran GTP-dependent
manner [8]. The pre-miRNA is further cleaved by Dicer to
generate a short, partially double-stranded (ds) RNA in
which one strand is the mature miRNA. The mature miRNA
is taken up by a protein complex that is similar, if not identical, to the RNA Induced Silencing Complex (RISC). The
action of interfering dsRNA in mammals usually involves
two enzymatic steps [9]. First, Dicer, a RNase type-III enzyme, cleaves dsRNA to 17-24-mer siRNA fragments. Then,
RNA-induced silencing complex (RISC) unwinds the RNA
duplex, pairs one strand with a complementary region in a
target mRNA, and initiates cleavage at a site 10 nucleotides
upstream of the 5’end of the siRNA. This process takes place
in the cytoplasm. In mammals, the Argonaute 2 protein
seems to be the key component of the RISC complex responsible for mRNA cleavage [10]. Another common, although
not universal protein of the RNAi machinery is RNAdependent RNA polymerase (RdRP), which synthesizes
dsRNA from ssRNA templates to initiate or amplify the
RNAi reaction [11]. Short, chemically synthesized siRNAs
in the 19-22 mer range do not require the Dicer step and can
enter the RISC machinery directly. RNAs that regulate gene
© 2009 Bentham Science Publishers Ltd.
570 Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 6
Földes-Papp et al.
Scheme 1. RNA interference (RNAi) in a cell. Summarized and modified from ref. [3]. ?: RNA activation by miRNA could be a possible
mechanism that has not yet been proven yet [3-7].
expression post-transcriptionally, affect mRNA degradation
and translation by base-pairing with the 3’ untranslated regions (3’UTRs) [12]. So far, more than 500 miRNAs have
been identified in the human genome [13].
In this work, we provide unequivocal evidence compatible with the long-postulated hypothesis that cytoplasmic
miRNA travels into the cell nucleus. In an era of genomicbased hypotheses, imaging of miRNA molecules in single
live cells is of critical importance to a better understanding
of pathological processes, e.g., in atherosclerosis and related
cardiovascular diseases (CVDs).
MATERIALS AND METHODS
Human Cell Lines
All cell lines were cultured according to guidelines provided by ATCC on plastic ware (100 mm tissue culture dish)
at 37 oC in a humid atmosphere containing 5 % (v/v) CO2 in
air. Cells were grown in Dulbecco's modified Eagle Medium
(DMEM) or Williams´s Medium (IHH) supplemented with
fetal calf serum (FCS), MEM Amino Acids, L-Glutamine,
penicillin-streptomycin and amphothericin B. Supplements
depend on the cell lines used (for specifications see below).
Because miRNA expression varies from highly specific
to ubiquitous, the human hepatoma cell line Huh-7D12 and
the immortalized human hepatocyte cell line IHH, known to
specifically express human microRNA-122 [2], were used to
validate the intracellular delivery and distribution of target.
The human fibroblast VA13 cell line and the human colorectal adenocarcinoma Caco-2 cell line which do not express the
human microRNA-122 [2] were used as miRNA-122 negative target controls.
Huh-7D12
The human Huh-7D12 cell line derived from human hepatocellular carcinoma cells was purchased from ECACC
(European Collection of Cell Cultures, UK). The cells were
cultured in DMEM (Gibco) with 10% FCS (PAA), 1 %
penicillin/streptomycin (PAA), 0.5% amphotericin B (PAA)
and L-Glutamine (Gibco).
IHH
The human IHH cell line, which was transformed by simian virus 40 (SV40), was obtained from ECACC (European
Collection of Cell Cultures, UK). The cells were cultured in
William´s Medium with 10% FCS (PAA), 1 % penicil-
Trafficking of Mature miRNA-122
lin/streptomycin (PAA), 0.5 % amphotericin B (PAA) and LGlutamine (Gibco).
VA13
The VA13 cell line was transformed by SV40 and purchased from LGC Promochem (UK). The cells were cultured
in DMEM (Gibco) with 5% FCS (PAA), 1 % penicillin/streptomycin (PAA) and 0.5 % amphotericin B (PAA).
Caco-2
The Caco-2 cell line was obtained from LGC Promochem (UK). The cells were cultured in DMEM (Gibco) with
20% FCS (PAA), MEM-Amino Acids (100 X; Gibco), 1%
penicillin/streptomycin (100 X; PAA) and 0.5% amphotericin B (250g/ml; PAA).
Subculturing
Cell lines in exponential growth phase were washed,
trypsinized and re-suspended in complete culture media. All
operations were performed in a biosafety cabinet class II.
For cell splitting, we used cells grown in a confluent
monolayer of a 100 mm tissue culture dish (~ 107 cells) of
each cell line. The cells were washed twice with 10 ml
1xPBS (10xPBS contained 14.4 mg Na2HPO4·2H2O
(Roth)/ml, 2 mg KH2PO4 (Roth)/ml, 80 mg NaCl (Roth)/ml,
2 mg KCl (Roth)/ml diluted with ddH2O to 1xPBS and autoclaved, pH 7.2) and the medium was aspirated after each
washing step. Then, 2 ml of 0.05 % trypsin solution (0.5 mg
Trypsin (Sigma)/ml, 0.02 % of a 2 % EDTA-Solution
(Roth), diluted in 1xPBS, sterilized by filtration and stored at
– 20oC) was added and the dish was let in the incubator for
2-4 min. The trypsinization was stopped by adding 3 ml culture media. If the cells were detached, we gently repipetted
to avoid aggregates. We then transferred the trypsinized cell
suspension into a 30 ml centrifugation tube and centrifuged
at 1250 rpm for 5 min to pellet the cells. We aspirated the
supernatant and resuspended the pellet by gently repipetting
in 4 ml of culture medium. The cell suspension was splitted
to 3 x 60 mm dishes and to a MiniCeM® / MiniCeM® grid
(JenLab, Germany), both equipped with glass cover slips (Ø
40 mm) and medium was added. After at least 24 hours in
the incubator during which a partial monolayer forms, the
cells were attached to the glass surfaces.
Experimental Probe Design
The oligonucleotide sequences corresponding to 206
miRNAs were obtained from Ambion Diagnostics Inc.,
USA (Applied Biosystems). The sequences have previously
been used in gene profiling studies with human tissue or human cell samples [2, 14]. Database matches between miRNAs and target messages in the cholesterol and fatty-acid
metabolism, atherosclerosis as well as angiogenesis are
given here as examples:
miR-122, (5’) 15 – u.gga.gug.uga.caa.ugg.ugu.uug – 36
(3’), database ID hsa-miR-122 [13]. We used the probe sequence 5’-CAA.ACA.CCA.TTG.TCA.CAC.TCC.A-3’, (3’a. cct. cac. act. gtt. acc. aca. aac -5’). The negative control
was a six-mismatch miR-122 probe of sequence 5’A.CAT.ACT.CCT.TTC.TCA.GAG.TCC.A-3’
Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 6
571
Because we jumped from an in vitro laboratory technique
to measurements in live cells, several additional hurdles of
oligonucleotide probes were taken into account as shown in
Table 1. We applied Looked Nucleic Acid oligonucleotides
(LNAs) for the superquencher molecular beacon (SQMB)
probes specifically directed against the selected miRNA.
LNAs are ribonucleotides, containing a methylene bridge
that connects the 2’-oxygen of the ribose with the 4’- carbon
and are now commercially available from Proligo (Texas,
USA).
Table 1.
Third-Generation Oligonucleotide Chemistry [31]
Cellular Hurdle
Experimental Solution by LNA
Oligonucleotides
1. Enhanced resistance to various
nucleases
• Resistance towards nucleases,
predictable melting behaviour
2. Cellular uptake
• Compatible with most enzymes
3. Specific target recognition by
Watson-Crick base pairing
• Basepairs with DNA and RNA
4. Good structural mimicry of the
natural RNA
• Exceptionally high thermal
stability
Superquencher Molecular Beacons
Superquencher molecular beacons (SQMB) that included
the energy transfer pair of Alexa Fluor 488 as donor and
BHQ1 as dark acceptor, were before used for direct probing
of genomic DNA by homogenous hybridization near physiological solvent conditions using two-color fluorescence
cross-correlation technique [15].
SQMB hsa-miRNA-122 Target Probe with LNA
5’-Alexa488-ACCGCG-caa+Aca+Ccat+Tgtc+AcactccaCGCGGT-BHQ1-3’, where +N represents LNA base. The
probe possessed a certain ‘key’ region indicated by small
letters, which fits into the single-stranded human miRNA122 target containing the appropriate ‘lock’ region. If hybridization onto human miRNA-122 occurred, its force was
strong enough to continuously straighten the loop, breaking
the hairpin’s double-stranded structure of the molecular beacon. In contrast, unbound probe molecules remain in the
closed structure at room temperature. Using perfectly
matched spectral overlaps between the fluorophore Alexa488 – quencher BHQ1 combination of the probe, the reduction of the fluorescence signals from the unbound probes,
i.e. the large decrease in fluorescence background signal to
the background signal of double-distilled water (Table 2
given in ref. [15]), greatly enhanced the sensitivity of detection.
SQMB Negative Control Probe with LNA
5’-Alexa488-ACCGCG-cat+Act+Cct+Ttc+Tca+GagtccaCGCGGT-BHQ1-3’, where +N represents LNA base.
SQMB Synthesis and Purification
The SQMB probes were synthesized and purified by
ProOligo Inc. (Texas, USA) according to the design and
specifications given above.
572 Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 6
Femtosecond Laser Scanning Microscopy
Experimental Setup
Femtosecond laser microscopes have become an important tool for non-contact 3D-nanoprocessing of living cells.
The sub-15 femtosecond 85 MHz near-infrared laser scanning microscope FemtOgene™ (JenLab GmbH, Germany) is
a compact device for nanobiotechnology and nanomedicine
(see Fig. 1A). The use of these extremely short pulses enables very efficient excitation of multiphoton effects. The
high efficiency makes it possible to perform intracellular and
intratissue surgery, dissection and optical knock-out of cells
[16-21] as well as targeted transfection by transient opening
of the cellular membrane [22, 23] using very low mean laser
powers. This ensures to avoid collateral damage in the microenvironment due to photodisruptive effects based on the
formation of bubbles and shock waves [24, 25]. Formerly,
using longer pulses mean powers of about 100 mW (~1 nJ)
were required to induce transient holes [26-28]. For a successful self-repair of the membrane damage the size of these
holes should be in the submicron range. With FemtOgene™
used here optical transfection of cells can easily be performed with laser mean powers of even less than 7 mW (<
100 pJ) [29, 30]. For achieving sub-15 femtosecond laser
pulses behind the focussing optics the FemtOgene™ is
equipped with dispersive mirrors for dispersion precompensation (s. Fig. 1B). The system uses the femtosecond
Ti:sapphire oscillator Integral ProTM (Femtolasers Produktions GmbH, 85 MHz, M2 < 1.3, 400 mW mean power output (~5 nJ)). The microscope is equipped with fast galvoscanners for beam scanning and with focussing optics
consisting of a highly-dispersive, large-NA objective (Zeiss
EC Plan-Neofluar 40x/1.3 oil). The mean laser power was
determined behind the objective with a power meter (Ophir
Optronics Ltd., Israel). To be able to control the number of
pulses that irradiate the sample a fast mechanical shutter was
integrated into the beam path. For detecting two-photon fluorescence a photomultiplier (Hamamatsu H 7732) is attached
to the front port of the microscope and on-line video imaging
of the cells is provided by a CCD camera attached to the side
port.
In preparation for the cell experiments the pulse duration
was measured at the focus of the objective using a second
order interferometric scanning autocorrelator (FemtometerTM, Femtolasers Produktions GmbH). A non-linear photodiode (NL-PD) placed directly into the focus of the objective
was used to detect the autocorrelator signal induced by the
Michelson interferometer of the autocorrelator which was
positioned in the beam path between the laser and the microscope (see Fig. 1B). Pulse durations <15 fs were measured in
the focal plane of the objective. Fig. (2) shows the autocorrelation function, the laser output spectrum and the corresponding laser output parameters obtained directly before the
cell measurements.
Experimental Procedure
The different cell lines were each transferred into special
300 l sterile miniaturized cell chambers (MiniCeM, JenLab
GmbH, Germany) consisting of two 170 Hm thick glass windows and a silicon ring as spacer in a plastic housing. For
imaging, each cell sample was taken out of the incubator and
Földes-Papp et al.
immediately placed to the microscope. By carefully illuminating the sample with the halogen lamp and taking transmission images a suitable amount, i.e. a group of a few cells
was selected and put into the field of view of the microscope.
Subsequently, a high resolution two-photon image was recorded as reference of the untreated cells (<3 mW laser mean
power). The membrane of a single cell, the nucleus or both
were then up to three times consecutively single-point illuminated with a train of fs-laser pulses with duration of 0.5 s
(total energy deposited at 10 mW mean power ~0.2 nJ) to
open the cellular membrane transiently or check the effect of
the pulses on the nucleus, respectively. Typically, mean laser
powers in the range of 8-10 mW were employed. The effects
of the single-point illumination on the cells and any external
changes were documented by acquiring multiphoton images
directly after the single-point illumination, after one minute,
after three minutes, after five minutes, after seven minutes
and after ten minutes of darkness. Afterwards, as a comparison a further transmission image using the halogen lamp was
recorded. Finally, also fluorescent images using a second
microscope (IX70, Olympus) with an Hg lamp were recorded. For these fluorescence images a 482/35 nm bandpass filter was used in the excitation-light path and a 536/40
nm band-pass filter in the signal-light path. This procedure
was repeated with several cell clusters located on different
parts of each of the different cell chambers.
RESULTS AND DISCUSSION
We constructed superquencher molecular beacons
(SQMB, see ref. [15]) for a highly selective and sensitive
detection of single-stranded miRNA-122 in living cells by
hybridization without amplification, transcription or immobilization. SQMB-probes operate on the principle of
DNA/RNA base pairing. They are synthetic DNA molecules,
which form a stem-loop or hairpin structure, and provide the
target recognition. The sensitivity of a molecular beacon
probe depends on the brightness of the fluorescent donor, the
efficiency of the fluorescence resonance energy transfer
(FRET) to the quencher (acceptor), and properties of the
quencher, i.e. the quencher was a dark molecule that is not
fluorescent under the experiment conditions used [15, 31].
Because the efficiency of extracellular probe delivery to a
single live cell is still a limiting factor for basic single-cell
research, we circumvented the use of electroshocks in bulk
suspensions, the so-called electroporation, or chemical
means for perforation of cell membranes in bulk suspensions. Instead we used 3D-nanoprocessing of single cells
with near-infrared sub-15 femtosecond laser pulses. Transient nanopores were formed by focusing the laser beam for
some milliseconds on the membrane of a single cell [24, 30].
Through these pores, SQMB probes can enter into the single
cell by diffusion from the surrounding solution. In Fig. (3),
we demonstrated the non-invasive, gentle formation of a
transient nanopore in the cell membrane of a single Huh7D12 cell by low mean power sub-15 femtosecond 85 MHz
near-infrared laser pulses. The applied conditions for optoporation do not induce collateral damage and disturbances of
the self-repairing potency of cells [24, 30, 32-34].
After addition of SQMB hsa-miRNA-122 target probe
with LNA to the cell suspension, we imaged the Huh-7D12
cells by two-photon microscopy as depicted in Fig. (4A) and
Trafficking of Mature miRNA-122
Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 6
573
A)
B)
Fig. (1). Experimental setup.
A: Photograph of the sub-15 femtosecond non-linear laser scanning microscope FemtOgene™.
B: Scheme of the FemtOgene™ system in arrangement used to optimize the dispersion pre-compensation and measure the pulse duration.
performed single-point illuminations of cell membranes and
nuclei. After random optoporations, the extracellular SQMB
hsa-miRNA-122 target probe molecules with LNA diffused
into the cytoplasm before cellular self-repair occurred. Fig.
(4B-C) shows that we could clearly distinguish between the
dark (non-fluorescent) unbound probes in the extracellular
solution and the bright (fluorescent) bound miRNA-122 target probes within the single Huh-7D12 cells. This high
quenching efficiency is a consequence of improved absorption extinction coefficient of the quencher BHQ and increased dipole-dipole interaction between the fluorophore
and quencher molecule in the closed unbound conformation
of the molecular beacon probe [15, 31]. Because we also
optically knocked out the nuclei membranes, we observed a
fluorescence steady-state across the whole cells within 5 min
after optoporations of cell and nuclei membranes (Fig. 4BC). The fluorescence increase was nonlinear with time
within the 5 min corresponding to intracellular target hybridizations. We found many single cells with bright intracellular fluorescence. Upon identifying and localizing the
diffuse fluorescent spots, we counted a total of 50 different
single Huh-7D12 cells out of 50 responding to miRNA-122
specific target probes.
There is mounting evidence that small RNAs like
miRNA and piRNA may induce transcriptional activation [38], the actual mechanism is elusive. One theory put forward
is the hypothesis that miRNA assembled in the cytoplasm is
loaded into the nucleus. In order to directly test our hypothesis of the functional role of miRNA in the nucleus, we performed the following experiments with the human hepatocyte cell line IHH. 3D-nanoprocessing of single IHH cells
with respect to intracellular delivery of SQMB hsa-miRNA122 target probe with LNA by optoporation was carried out
as established for the human hepatocellular carcinoma cells
574 Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 6
Földes-Papp et al.
Fig. (2). Laser parameters applied in the study.
Left: Autocorrelation function measured behind the objective directly before cell experiments.
Right: Laser spectrum.
Huh-7D12 without optical knock-out of nuclei membranes.
We first analyzed molecular interactions and trafficking of
miRNA-122 targets in single IHH cells as shown in Fig.
(4D-I). To better demonstrate the new findings, we did not
perform single-point illuminations (optoporations) of cell
membranes in the right and central part of the images. The
flux of SQMB hsa-miRNA-122 target probe molecules with
LNA through the nanopore of the cell membrane of single
human hepatocytes after single-point illuminations caused a
cytoplasmic spread of fluorescence in the 1 min to 2 min
two-color images of Fig. (4E-F). If the adjacent cell nucleus
is coupled by a loading mechanism, the target fluorescence
will also appear in the nucleus and label it. Indeed, after 2
min we observed bright target fluorescence inside the intact
nucleus as depicted in Fig. (4G-I). The nucleus membrane
was found to be intrinsically ‘permeable’ to those
probe/probe-target molecules and thus the nuclear fluorescence increased non-linearly. Because human hepatocyte
nuclei enabled a selective loading of probe/probe-target
molecules, we conclude from the measured data that there is
an intracellular communication between intact live cytoplasm and nucleus with respect to the target distribution. The
bound fluorescent SQMB hsa-miRNA-122 target probe
molecules were initially found in the cytoplasm but elevated
in a time-dependent manner in the intact nucleus of human
single hepatocytes. In total, we identified and localized cytoplasmic and nuclear fluorescence in 40 – 50 counted single
IHH cells.
To verify our obtained results of Fig. (4), we carried out
negative control experiments as depicted in Fig. (5). As
shown in Fig (5A-B), we explored the SQMB negative control probe with LNA and, in Fig. (5C-D), cells known to not
specifically express human microRNA-122 [2]. We measured levels of cellular autofluorescence only but not bright
target fluorescence as in Fig. (4).
Taken together, the cell biological concept of RNA activation by miRNA (see Scheme 1, refs [3-8]) has proved
quite a challenge to us. In this study, we have tried to directly prove the first step in this concept, namely the cytoplasmic assembly of loaded nuclear miRNA-122. Only mature cytoplasmic miRNAs are single-stranded RNA molecules that consist of 17-24 nucleotides. Thus, we exclude the
possibility that our probes detected de-novo synthesized
miRNAs precursors in the nucleus or cytoplasm.
CONCLUSIONS
An open issue in the regulation of gene expression by
miRNA is whether miRNA can do some activation in addition to the well-known inhibitory effect on gene expression.
A first step for such a regulatory role could be the travelling
of miRNA-RISC into the nucleus.
The primary transcripts pri-miRNAs in the cell nucleus
are generated by polymerase II. The pri-miRNA transcripts
are predicted to form specific "hairpin-shaped", doublestranded secondary structures [8], in which one of the two
strands includes the mature miRNA. They are processed by a
microprocessor complex comprising Pasha/DGCR8 that is a
double-stranded RNA binding protein and the double-strandspecific ribonuclease Drosha (see ref. [8]). The microproces
Trafficking of Mature miRNA-122
Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 6
A)
B)
C)
D)
E)
F)
575
Fig. (3). Formation and self-repair of a nanopore in the cell membrane of a single Huh-7D12 cell. The nanopore of a single cell (middle
part of the images) is induced by single-point illumination with sub-15 femtosecond laser pulses for 0.8 s pulse duration and a mean power of
9 mW. Imaging was performed: before (A) and after (B-F) optoporation, the nanopore was closed (F) within ca. 1 s after its formation (B).
576 Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 6
Földes-Papp et al.
A
B
C
D
E
F
G
H
I
Fig. (4). Single human Huh-7D12 cells (A-C) were imaged by two-photon microscopy after addition of 2 1M SQMB hsa-miRNA-122
target probe with LNA. Two-photon images showing fluorescent spots corresponding to autofluorescence (A) and intracellular target binding
of SQMB-probe molecules directed against single-stranded human miRNA-122 targets (B, C). A: Before optoporation; B, C: 1 min and 5
min after optoporation, respectively.
Single human hepatocyte IHH cells (D-I) were imaged by two-photon microscopy after addition of 2 ,M SQMB hsa-miRNA-122 target probe
with LNA. Two-photon images showing fluorescent spots corresponding to autofluorescence (D) and intracellular target binding of SQMBprobe molecules directed against single-stranded human miRNA-122 targets (E-I). D: Before optoporation; E-I: 1 min, 2 min, 5 min, 7 min,
and 10 min after single-point optoporation of single cell membranes, respectively. For better comparison, hepatocytes in the right and central
part of the images were not optoporated.
The abbreviations N and C in the images stand for nucleus and cytoplasm.
Trafficking of Mature miRNA-122
Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 6
A
B
C
D
577
)
Fig. (5). Negative control experiments to Fig. (4).
A, B: Two-photon Imaging of single cells known to specifically express human miRNA-122 [2] (Huh-7D12) before (A) and after (B) optoporation in the prescence of 2 ;M SQMB negative control probe with LNA. No target binding fluorescence was observed.
C, D: Two-photon Imaging of single cells know to not specifically express human microRNA-122 [2] (V13) before (A) and after (B) optoporation in the presence of 2 ;M SQMB hsa-miRNA-122 target probe with LNA. No target binding fluorescence was observed.
The abbreviations N and C in the images stand for nucleus and cytoplasm.
sor complex recognizes the distinct hairpin secondary structure of the pri-miRNA and specifically cleaves at the base of
the stem loop releasing the hairpin, a 60- to 70-nucleotide
double-stranded pre-miRNA intermediate, enabling Exportin
5–mediated export into the cytoplasm. The stem–loop structures predicted for all miRNA precursors are reminiscent of
the partially double-stranded fold-back structures [35]. In the
cytoplasm, the pre-miRNA is further cleaved by Dicer, a
second RNase III endonuclease, to generate a short, partially
double-stranded RNA, in which one strand is the mature
miRNA. The miRNA ‘guides’ a ribonucleoprotein complex,
which is the RNA induced silencing complex RISC. After
enzymatic processing, one of the strands of the doublestranded miRNA is incorporated into RISC and the other
miRNA strand is enzymatically degraded as soon as it is
released. Cytoplasmic RISC recruitment can inhibit mRNA
translation or induce mRNA degradation. The target mRNAs
display sequence complementary of their 3’UTRs of protein
coding genes to the single-stranded, mature miRNA [36].
For the first time, we give unequivocal proof for the load of
cytoplasm-assembled single-stranded miRNA-122 into the
cell nucleus by 3D-nanoprocessing of single human hepatocyte cell membranes with sub-15 femtosecond 85 MHz nearinfrared laser pulses and two-photon high-resolution imag-
578 Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 6
ing. Mechanisms of miRNA action in the nucleus are so far
unknown [37]. Our results provide further evidence for a
potential regulatory function of miRNAs in cellular physiology.
Földes-Papp et al.
[15]
[16]
ACKNOWLEDGEMENTS
We thank Jens Müller, Stefan Blatter (JenLab, GmbH,
Germany) and Dr. Ben Barbieri (ISS, USA), who supported
this study, for their generosity. PD Dr. Dr. Zeno Földes-Papp
acknowledges financial support in part from his FWF Austrian Science Fund Research Project P20454-N13.
We thank Anna Schreilechner (Institute of Molecular
Biology and Biochemistry, Medical University of Graz, Austria) for cell care and excellent technical assistance.
[17]
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