Cotransfection experiments in HCC cells of the miR-23b with pGL4.71 Renilla luciferase reporter gene constructs, containing the putative uPA and c-met 3¢-UTR target sites, and with the p
Trang 1downmodulation and a decreased migration of human
hepatocellular carcinoma cells
Alessandro Salvi1, Cristiano Sabelli1, Silvia Moncini2, Marco Venturin2, Bruna Arici1, Paola Riva2, Nazario Portolani3, Stefano M Giulini3, Giuseppina De Petro1and Sergio Barlati1
1 Division of Biology and Genetics, Department of Biomedical Sciences and Biotechnology, IDET Centre of Excellence, University of Brescia, Italy
2 Department of Biology and Genetics, Medical Faculty, University of Milan, Italy
3 Department of Medical and Surgical Sciences, University of Brescia, Italy
MicroRNAs (miRs) are small (21–25 nucleotide)
non-protein-coding RNAs implicated in negative gene
expression regulation [1,2] More than 500 human
miRs have been identified (http://microrna.sanger
ac.uk, version 13.0, updated March 2009), and over
1000 miRs are predicted to exist in the vertebrate and human genome [3,4] The biogenesis of miRs involves
a complex protein system They are generally tran-scribed by RNA polymerase II or III into pri-miR transcripts that are processed by the RNase III enzyme
Keywords
c-met; hepatocellular carcinoma cells;
microRNA-23b; urokinase
Correspondence
G De Petro, Department of Biomedical
Sciences and Biotechnology, Division of
Biology and Genetics, University of Brescia,
Viale Europa n 11, 25123 Brescia, Italy
Fax: +39 30 3701157
Tel: +39 30 3717 264 241
E-mail: depetro@med.unibs.it
(Received 19 December 2008, revised 18
March 2009, accepted 19 March 2009)
doi:10.1111/j.1742-4658.2009.07014.x
Urokinase-type plasminogen activator (uPA) and c-met play a major role
in cancer invasion and metastasis Evidence has suggested that uPA and c-met overexpression may be coordinated in human hepatocellular carci-noma (HCC) In the present study, to understand whether the expression
of these genes might be coregulated by specific microRNAs (miRs) in human cells, we predicted that Homo sapiens microRNA-23b could recog-nize two sites in the 3¢-UTR of uPA and four sites in the c-met 3¢-UTR by the algorithm pictar The miR-23b expression analysis in human tumor and normal cells revealed an inverse trend with uPA and c-met expression, indicating that uPA and c-met negative regulation might depend on miR-23b expression Transfection of miR-23b molecules in HCC cells (SKHep1C3) led to inhibition of protein expression of the target genes and caused a decrease in cell migration and proliferation capabilities Further-more, anti-miR-23b transfection in human normal AB2 dermal fibroblasts upregulated the expression of endogenous uPA and c-met Cotransfection experiments in HCC cells of the miR-23b with pGL4.71 Renilla luciferase reporter gene constructs, containing the putative uPA and c-met 3¢-UTR target sites, and with the pGL3 firefly luciferase-expressing vector showed a decrease in the relative luciferase activity This would indicate that miR-23b can recognize target sites in the 3¢-UTR of uPA and of c-met mRNAs and translationally repress the expression of uPA and c-met in HCC cells The evidence obtained shows that overexpression of miR-23b leads to uPA and c-met downregulation and to decreased migration and proliferation abilities of HCC cells
Abbreviations
GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HGF, hepatocyte growth factor; hsa-miR-23b, Homo sapiens microRNA-23b; miR, microRNA; NM, nitrocellulose membrane; PT, peritumoral; uPA, urokinase-type plasminogen activator.
Trang 2Drosha to release the precursor product, pre-miR,
which consists of about 70 nucleotides Exportin 5
transports these molecules into the cytoplasm, where
they are processed by the RNase III enzyme Dicer into
a transient 22 nucleotide linear duplex Only one
strand of the duplex, called the mature miR, is loaded
into the miR-associated multiprotein RNA-induced
silencing complex Most miRs bind to the 3¢-UTR of
the target mRNAs with imperfect complementarity,
and they direct translational repression and⁄ or mRNA
target degradation [5] It has been predicted that miRs
target up to 30% of protein-coding genes in humans
[6] Accumulating evidence indicates that miRs are
involved in development, differentiation, apoptosis,
proliferation, and several diseases, including cancer [7]
During recent years, there have been several
micro-array studies that have allowed the identification of
new miRs and the determination of global expression
profiles under certain biological conditions [8,9] Array
platforms have also been useful for the definition of
differential miR signatures between neoplastic tissues
and healthy ones, in the case of cancer [10–12]
Although the number of discovered miRs has rapidly
increased, data on possible mRNA targets for most
miRs remain elusive Experimental validation of miRs
predicted by bioinformatic analysis could be
funda-mental for the definition of a biological function of a
given miR [13,14] Therefore, the main purpose of this
study was to validate the miRs predicted by pictar
(http://www.pictar.bio.nyu.edu) [15], targeting
uro-kinase-type plasminogen activator (uPA) and c-met
genes
uPA is a serine protease that, when bound to its
receptor, plays an important role in proteolytic
cas-cades, and governs the extracellular matrix turnover
and the migration and⁄ or proliferation of several types
of tumor cells [16] The tyrosine protein kinase
recep-tor c-met, when bound to its ligand [hepatocyte growth
factor (HGF)], is responsible for several cellular
responses, such as invasive growth in development and
cancer, tissue regeneration, angiogenesis, proliferation,
and migration; c-met may also be activated in an
HGF-independent manner [17] uPA and c-met are
involved in human cancer, through the development
and progression of several types of malignant tumor
An essential role of uPA and c-met in the migration
and proliferation of human hepatocellular carcinoma
(HCC) cells has been assessed by functional studies
carried out using RNA interference technology [18,19]
The evidence suggests that the two systems, uPA–uPA
receptor and HGF–c-met, might cooperate in the
acquisition of malignant phenotypes of cancer cells,
but little is known about the regulatory mechanisms
for expression of these genes [20,21] uPA can be regu-lated at the transcriptional and post-transcriptional levels (i.e promoter methylation⁄ demethylation, and distinct adenylate⁄ uridylate AU-rich element-binding proteins, which can increase⁄ decrease the mRNA sta-bility) [22,23]; c-met expression can be negatively mod-ulated at the transcriptional level by the transcription repressor Daxx [24] For uPA and c-met in human HCC, one of the major cancers worldwide, with a poor prognosis [25], we have shown that the mRNA expression levels are unfavorable prognostic factors for HCC patients, and that uPA mRNA overexpression is strictly associated with c-met mRNA levels [26,27] Thus, in this study, we have verified the hypothesis of miR-mediated gene regulation of uPA and c-met We identified by bioinformatics, and subsequently vali-dated, Homo sapiens miR-23b (hsa-miR-23b) as a putative miR mediating both uPA and c-met down-regulation Overexpression of miR-23b led to uPA and c-met silencing, and to decreased migration ability
of SKHep1C3 HCC cells In addition, inhibition
of endogenous miR-23b by anti-miR-23b molecules led
to upregulated uPA and c-met expression in human normal AB2 fibroblasts
Results
Bioinformatic prediction of miRs targeting uPA and c-met 3¢-UTRs
Bioinformatic prediction, determined using the algo-rithm pictar (also verified by targetscan), showed putative target sites for six and 13 miRs in the uPA and c-met 3¢-UTR sequences, respectively (Table 1) Among the predicted miRs, we focused on hsa-miR-23b, as it recognizes binding sites in both uPA and c-met transcripts (two and four binding sites for uPA and c-met respectively) Furthermore, hsa-miR-23a may possibly target the uPA and c-met 3¢-UTRs, but the free energy of the binding sites (two and four for uPA and c-met, respectively) is generally higher than that for hsa-miR-23b hsa-miR-23b is an intronic miR located in intron 12 of the host gene C9orf3 on chro-mosome 9, and the predicted binding sites in the uPA and c-met 3¢-UTRs are conserved, to varying degrees, across species (Fig 1A,B)
Expression of mature miR-23b and uPA and c-met target genes in human tumor and normal cells
To evaluate the possible effects of miR-23b on the two targets, uPA and c-met, we first verified their
Trang 3expression in human normal and tumor cell lines As
shown in Fig 2A,B, real-time RT-PCR and northern
blot data revealed that miR-23b was highly expressed
in AB2 human dermal fibroblasts, whereas it was
detectable at lower and variable levels in five human
HCC-derived cell lines Regarding uPA expression,
detected by real-time-PCR, western blotting, and
zymography, the undifferentiated HCC-derived
SKHep1C3, SKHep1C3.69.2 and HA22T⁄ VGH cells
had a considerable level of uPA mRNA as well as
high uPA protein and enzymatic activity (Fig 2A,C)
These three cell lines also displayed a higher amount
of c-met protein (Fig 2C) In contrast, lower levels of
uPA and c-met proteins and mRNAs were detectable
in AB2 normal cells and in the differentiated HCC
cells, HepG2 and HuH6 cells (Fig 2A,C) These data
Table 1 PICTAR miR prediction.
No binding sites Free energies (kcalÆmol)1) uPA miR
c-met miR
)21.2, )22.8
*
, the opposite strand to the mature miRNA.
Fig 1 The uPA and c-met 3¢-UTRs, respectively, harbor two and
four putative binding sites for miR-23b (A) The location of sites 1
and 2 in the uPA 3¢-UTR, and complementarity between miR-23b
and the putative uPA 3¢-UTR target sites The conserved bases of
the putative miR-23b target sequence are also shown (B) The
loca-tion of sites 1, 2, 3 and 4 in the c-met 3¢-UTR, and complementarity
between miR-23b and the putative c-met 3¢-UTR target sites
Con-served bases of the putative miR-23b target sequence present in
the c-met 3¢-UTR are also shown has, Homo sapiens; ptr, P
trog-lodytes (chimpanzee); mmu, M musculus (mouse); rno, R
norvegi-cus (rat); cfa, C familiaris (dog).
Trang 4indicate an inverse trend between the expression levels
of miR-23b and of uPA and c-met AB2 control cells,
which did not express uPA and c-met, displayed a
high level of miR-23b, whereas the most aggressive
HCC cells, which produced high levels of uPA and
c-met, had a lower expression level of miR-23b The
differentiated HuH6 and HepG2 cells showed a
differ-ent profile of uPA and c-met expression, as well as of
miR-23b The HuH6 cells, with undetectable levels
of uPA and c-met, expressed a certain amount of
miR-23b, less than that expressed by normal AB2
cells The HepG2 cells with a c-met mRNA amount
comparable to that displayed by SKHep1C3 cells
expressed miR-23b at levels as low as those of
SKHep1C3 cells
miR-23b decreased uPA and c-met protein expression in SKHep1C3 cells
To assess the effects of miR-23b on uPA and c-met expression, we transiently transfected miR-23b in SKHep1C3 cells to evaluate target gene expression Western blot and zymographic analysis of the condi-tioned media of transfected cells revealed a significant reduction of uPA expression and its enzymatic activity The data shown in Fig 3 clearly show, respectively, 62% and 71% inhibition of uPA protein expression 48 and 72 h after transfection of 100 nm miR-23b (Fig 3A) Maximum inhibition of uPA enzymatic activity (by 51%) was obtained at 72 h after transfec-tion (Fig 3B)
Fig 2 Expression determination of mature miR-23b, uPA and c-met in human normal and HCC-derived cells (A) Real-time RT-PCR detec-tion of miR-23b, uPA and c-met mRNAs The amounts of miR-23b, uPA and c-met mRNAs were evaluated as described in Experimental procedures (B) Northern blot detection of miR-23b and U6 RNA (as control) (C) Western blot and zymographic analysis of conditioned media from normal and tumor cells for the detection of uPA protein and its enzymatic activity, and western blot for c-met detection in cell extracts from the same cell lines Lane 1: AB2 Lane 2: SKHep1C3 Lane 3: SKHep1C3.69.2 Lane 4: HuH6 Lane 5: HepG2 Lane 6: Ha22T ⁄ VGH RQ, relative quantification.
Trang 5Forty-eight hours after transfection, c-met protein
expression was inhibited by 48% at 100 nm miR-23b, as
detected by a semiquantitative western blotting analysis
Seventy-two hours after transfection, the 170 kDa c-met
precursor form was inhibited by 89% (Fig 3D),
indicat-ing that the translatability of c-met mRNA was strongly
affected The increased amount of the 145 kDa form
(produced by proteolytic processing of the precursor)
may be due to c-met protein turnover
To investigate whether miR-23b targeted uPA and
c-met mRNAs for degradation, a semiquantitative
RT-PCR evaluation was carried out on RNA isolated
from control and transfected cells The data showed
comparable steady-state mRNA levels of uPA
and c-met (Fig 4A,B), providing evidence that
miR-23b in SKHep1C3 cells can lead to decreased
uPA and c-met protein expression without affecting
the amounts of their mRNA Furthermore, regarding
miR expression, transfected cells displayed higher
amounts of miR-23b (Fig 4C, lanes 2 and 3) both at
48 and at 72 h after transfection, with a decline at
72 h (Fig 4C, lanes 5 and 6) As the SKHep1C3 cells
produce very low amounts of miR-23b, the high level detected in transfected cells may be due to the transfected molecules
Anti-miR-23b transfection in normal AB2 cells leads to upregulated uPA and c-met protein expression
To investigate whether the silencing of miR-23b might lead to upregulation of uPA and c-met, AB2 fibro-blasts (at a high expression level of miR-23b) were transfected with 100 nm antisense RNA oligonucleo-tides complementary to miR-23b Forty-eight and 72 h after transfection, the conditioned media, cell lysates and total RNA were examined by zymography, wes-tern blot and RT-PCR to analyze uPA and c-met expression The uPA protein expression levels increased 2.25 ± 0.84-fold at 48 h and 6.68 ± 1.11-fold at 72 h (Fig 5A), and the corresponding enzymatic activity increased 3.1 ± 0.33-fold and 9.55 ± 2.12-fold, as compared with the control, at 48 and 72 h (Fig 5B, lanes 3 and 6, and lanes 2 and 5,
Fig 3 miR-23b inhibits uPA and c-met protein expression in SKHep1C3 cells (A) Western blot analysis of uPA in the conditioned media of control cells (lanes 1 and 4) and miR-23b-transfected cells, at 48 h and 72 h after transfection Lane 2 : 50 n M miR-23b, 48 h Lane
3 : 100 n M miR-23b, 48 h Lane 5 : 50 n M miR-23b, 72 h Lane 6 : 100 n M miR-23b, 72 h (B) Zymographic detection of the corresponding uPA enzymatic activity (C) Western blot detection of GAPDH in control and miR-23b-transfected cells (D) Western blot detection of c-met and GAPDH in cell extracts from control and miR-23b-transfected cells The protein amount of the housekeeping gene GAPDH was compa-rable in all samples tested.
Trang 6respectively) The c-met protein expression level
increased 2.3 ± 0.51-fold at 72 h as compared with
controls (cells plus DOTAP) (Fig 5C, lanes 5 and 6)
Next, we assessed the mRNA expression levels of the
target proteins and miR-23b expression As shown in
Fig 6, for uPA the mRNA expression in anti-miR-23b-transfected cells increased 2.56 ± 0.28-fold and 6.18 ± 1.11-fold, respectively, at 48 and 72 h after transfection as compared with control cells (cells plus DOTAP) (Fig 6A, lanes 3 and 6, P < 0.05) The expression of c-met mRNA in anti-miR-23b-transfected cells increased 1.72 ± 0.27-fold and 2.84 ± 0.64-fold
at 48 and 72 h (Fig 6B, lanes 3 and 6, P < 0.05) As shown in Fig 6C, the detectable miR-23b amount decreased in transfected cells by 91% and 79%, respec-tively, at 48 and 72 h All together, these results showed that transfection of synthetic anti-miR-23b oligo-ribonucleotides leads to uPA and c-met upregulation, both at the mRNA level and at the protein level, and that the annealing of transfected anti-miR with endogenous miR decreases the level of detectable miR expression in transfected cells
miR-23b interacts with the uPA 3¢-UTR
To verify the putative direct interaction between miR-23b and the uPA 3¢-UTR, the regions of the 3¢-UTR
of human uPA mRNA containing the two putative hsa-miR-23b-binding sites were cloned into the Renilla luciferase report plasmid construct pGL4.71 In partic-ular, a 150 bp sequence (pGL4.71 uPA-3¢-UTR-1S) containing miR-23b-binding site 1 (1S construct) and a
102 bp sequence (pGL4.71 uPA-3¢-UTR-2S) containing miR-23b-binding site 2 (2S construct) were cloned The respective control constructs were obtained by cloning the same fragments in an antisense orientation (pGL4.71 UTR-1AS and pGL4.71 uPA-3¢-UTR-2AS) The Renilla constructs were cotransfected with a control firefly luciferase reporter plasmid into SKHep1C3 cells After 48 h of transfection with
100 nm miR-23b (Fig 7A), the 1S and 2S constructs inhibited the luciferase relative activity, respectively, by 32% (P < 0.01) and 36% (P < 0.001) (Fig 7A) Dose–response experiments showed that the inhibition
of Renilla luciferase by the 1S construct was 20% and 27%, respectively, at 75 nm and 100 nm miR-23b; for the 2S construct, the maximum inhibition obtained was 29% at 100 nm miR-23b (Fig 7B) There was no inhibition by the corresponding antisense sequences (Fig 7C) The data obtained showed that the two putative binding sites of the uPA mRNA 3¢-UTR were targets for hsa-miR-23b, and thus miR-23b could modulate gene expression directly at the uPA 3¢-UTR
As well as assessment of the possible direct inter-action between miR-23b and the c-met 3¢-UTR, the entire c-met 3¢-UTR (2278 bp) was cloned into Renilla luciferase-expressing plasmids and then cotransfected with the firefly luciferase-expressing plasmids The
Fig 4 (A,B) RT-PCR evaluation of uPA and c-met mRNAs on total
RNA isolated from control cells (lanes 1 and 4) and
miR-23b-trans-fected cells Lane 2 : 50 n M miR-23b, 48 h Lane 3 : 100 n M
miR-23b, 48 h Lane 5 : 50 n M miR-23b, 72 h Lane 6 : 100 n M miR-23b,
72 h miR-23b did not affect the steady-state amounts of uPA and
c-met mRNAs (C) Real-time RT-PCR evaluation of miR-23b in
control and in transfected cells RQ.
Trang 7results showed a greater decrease in luciferase activity
as compared with controls, both in miR-23b-transfected
and untransfected SKHep1C3 cells Inhibition was 73%
without exogenous miR-23b and 65% (P < 0.001) with
miR-23b (Fig 8A) These results would suggest that the
c-met3¢-UTR is an important target for control of the
protein expression level
In Hela cells, the c-met 3¢-UTR was a target that
was responsive to miR-23b (Fig 8B, P < 0.01),
indi-cating that SKHep1C3 rather than Hela cells produce
different molecules (including miRs) that may interact
with the c-met 3¢-UTR
Subsequently, four regions of the 3¢-UTR of c-met
mRNA containing putative hsa-miR-23b-binding sites
cloned into the pGL4.71 Renilla luciferase report
plas-mid, called pGL4.71 c-met-3¢-UTR-1S (175 bp),
pGL4.71 UTR-2S (166 bp), pGL4.71
c-met-3¢-UTR-3S (179 bp), and pGL4.71 c-met-3¢-UTR-4S (157 bp), were also tested Constructs 1S, 2S and 4S inhibited the luciferase relative activity by, respectively, 26%, 20%, and 10% Construct 3S did not affect the luciferase activity (Fig 8B) Furthermore, results obtained with SKHep1C3 cells treated or untreated with miR-23b showed that the 1S, 2S and 4S con-structs were miR-23b sensitive (Fig 8C) Furthermore,
as shown in Fig 8E, the c-met-3¢-UTR-1S construct was not sensitive to the control miR-107
miR-23b reduced the migration and proliferation abilities of SKHep1C3 cells
To assess the biological effects of miR-23b, we exam-ined its effects on SKHep1C3 cell migration and proliferation As shown in Fig 9A, the migration
Fig 5 Anti-miR-23b enhances uPA and c-met protein expression in AB2 cells Western blot analysis (A) of uPA in the conditioned media of control cells (lanes 1,
2, 4, and 5) and anti-miR-23b-transfected cells (lanes 3 and 6) at 48 and 72 h after transfection, and the corresponding uPA enzymatic activity (B) verified by zymography (C) Western blot detection of c-met expression in cell extracts from control cells (lanes 1, 2, 4, and 5) and transfected cells (lanes 3 and 6) at 48 and
72 h after transfection.
Trang 8abilities of miR-23b-transfected cells were significantly affected, by 45% (P < 0.05) and 39% (P < 0.01), respectively, as compared with controls, at 50 nm and
100 nm 23b Regarding cell proliferation, miR-23b decreased the proliferative ability of SKHep1C3 cells by up to 25% (P < 0.05) at 24 h after seeding, as compared with controls (Fig 9B) These results suggest that migration and proliferation of SKHep1C3 cells can be modulated by expression of miR-23b On the other hand, the anti-induced inactivation of miR-23B in AB2 fibroblasts did not modulate the migration ability of these normal cells (data not shown) This suggests that anti-miR-induced uPA and c-met expression is not sufficient to induce motility in these normal cells
Dysregulation of miR-23b expression in human HCC
In order to determine the potential role of miR-23b in human HCC, we assessed the miR-23b expression level
in HCC biopsy specimens and corresponding peritu-moral (PT) tissues As shown in Fig 10, among the samples derived from 17 HCC patients, there are two subsets: one (14⁄ 17, 82%) showing miR-23b down-regulation in HCC specimens as compared with PT tis-sues; and another (3⁄ 13, 18%) showing miR-23b upregulation in HCC specimens (Fig 10A) This indi-cates dysregulated expression of miR-23b in HCC specimens The average expression level of miR-23b
in HCC specimens is about two-fold lower than in PT tissues (P < 0.01) (Fig 10B)
Discussion
MicroRNAs have emerged as important negative regu-lators of gene expression that primarily block transla-tion of target mRNAs Many miR genes and several tissue⁄ organ-specific miRs have been identified, and the miR signature in several human diseases, including cancer, has been assessed, but little is known about target mRNAs As uPA and c-met overexpression in human HCC are strictly associated [27], and uPA regu-lation can also occur at the post-transcriptional level [23], we examined the possibility that a given miR might negatively coregulate uPA and c-met gene expression in human cells Among the putative miRs targeting uPA or c-met (six and 13, respectively; see Table 1), specific computational searches have shown target sites in the uPA and c-met 3¢-UTRs for miR-23a and miR-23b, indicating two and four target sites in the uPA and c-met 3¢-UTRs, respectively Therefore,
Fig 6 Anti-miR-23b enhanced uPA and c-met mRNA expression
in AB2 cells AB2 cells were transfected with the anti-miR
oligonu-cleotides as described in Experimental procedures Forty-eight
hours and 72 h after transfection, total RNA isolations from control
cells (lanes 1, 2, 4, and 5) and transfected cells (lanes 3 and 6) were
examined by RT-PCR to detect uPA mRNA (A) and c-met mRNA
(B), and by real-time PCR to detect miR-23b expression (C) RQ.
Trang 9according to thermodynamic parameters, we decided
to use miR-23b for experimental validation
First, we ascertained the expression of miRN-23b,
uPA and c-met in AB2 human dermal fibroblasts and
in four HCC-derived human cell lines All cell lines
expressed variable levels of miR-23b and the putative
uPA and c-met mRNA targets mR-23b was highly
expressed in AB2 normal cells, whereas uPA and c-met
were physiologically downregulated Lower levels of miR-23b expression were detected in the three undif-ferentiated HCC cell lines, SKHep1C3, SKHep1 C3.69.2, and HA22T⁄ VGH, with high levels of uPA (at both the mRNA and enzymatic activity levels)
We then examined the role of miR-23b in targeting uPAand c-met following transfection of miR-23b mol-ecules into SKHep1C3 cells In the transfected cells, the endogenous uPA and c-met protein were downreg-ulated without affecting the steady-state amount of the corresponding mRNAs Delivery of anti-miR-23b oli-goribonucleotides into AB2 normal cells increased the detectable levels of endogenous uPA and c-met at the mRNA and protein levels These findings show that uPA and c-met downregulation can be induced by ectopic miR-23b expression in SKHep1C3 malignant cells The mechanisms by which the endogenous miR-23b might play a biological role in the physiological negative regulation of uPA and c-met expression in normal AB2 cells (as anti-miR-23b transfection induced uPA and c-met upregulation) remain to be elucidated These findings suggest a regulatory mecha-nism involving the molecules miR-23b, uPA, and c-met, which until now have not been known to interact
The fact that miR-23b ectopic expression in HCC cells induced endogenous uPA downregulation via translation repression, whereas the endogenous miR-23b in AB2 cells seemed to target uPA mRNA for deg-radation, may be due to the presence of different aden-ylate⁄ uridylate AU-rich element-binding proteins in these tumor cells and in normal cell lines, respectively increasing or decreasing the stability of uPA mRNA and therefore impairing or favoring their degradation
As the uPA mRNA of certain human tumor cell lines
is more stable than that produced by normal cells [20],
we speculate that miR-23b may target the uPA mRNA
A
B
C
Fig 7 miR-23b interacted with uPA 3¢-UTR mRNA The 150 bp sequence pGL4.71 uPA-3¢-UTR-1S containing miR-23b-binding site 1 and the 102 bp sequence pGL4.71 uPA-3¢-UTR-2S containing miR-23b-binding site 2 were transfected in SKHep1C3 cells to assess the luciferase activity The respective antisense constructs were used as controls (pGL4.71 uPA-3¢-UTR-1AS and pGL4.71 uPA-3¢-UTR-2AS) The Renilla constructs were cotransfected with a control firefly luciferase report into SKHep1C3 cells Both site 1 and site 2 inhibited the luciferase relative activity, by 32% (P < 0.01) and 36% (P < 0.001), respectively, 48 h after 100 n M
miR-23b transfection (A) Dose–response experiments showed that the inhibition of Renilla luciferase for the 1S region was 20% and 27%, respectively, at 75 and 100 n M miR-23b; for the 2S region, the maximum inhibition obtained was 29% at 100 n M miR-23b (B).
No inhibition was obtained for the corresponding antisense sequences (C).
Trang 10molecules at the translation and⁄ or degradation level,
depending on the high or low stability of the mRNAs
Regarding the interaction of miR-23b with target
mRNA, the decreased luciferase activity obtained with
uPA 3¢-UTR constructs (1S and 2S) and with c-met
3¢-UTR constructs (1S, 2S, 3S, and 4S) indicated that
miR-23b can interact with the 1S and 2S target sites
within the uPA 3¢-UTR, and with the 1S and 2S target
sites within the c-met 3¢-UTR Furthermore, the
inter-action of the 1S target site was sensitive only to
miR-23b and not to miR-107 Hence, regulation of human
uPA and c-met expression could be directly mediated
by miR-23b In addition, other miRs targeting the c-met 3¢-UTR (i.e miR-34a and miR-199a*) [33,34] and⁄ or unknown regulatory factors may interact with the c-met 3¢-UTR in SKHep1C3 cells, as indicated by the strong inhibition of luciferase activity occurring with or without the presence of cotransfected miR-23b Furthermore, we cannot exclude an indirect influence
of miR-23b on c-met, even if it is very unlikely, at least through uPA Evidence has been provided that c-met activation may be concomitant with the induc-tion of the uPA proteolysis network [20,21], but not vice versa
Fig 8 miR-23b interacts with c-met 3¢-UTR mRNA The Renilla luciferase plasmids containing the entire c-met 3¢-UTR (2278 bp) were tested for luciferase activity in SKHep1C3 cells (A) and in Hela cells (B) The luciferase inhibition obtained in SKHep1C3 cells was 73% in the absence of exogenous miR-23b, and 65% (P < 0.001) in the presence of miR-23b (A) The luciferase inhibition obtained in Hela cells was 22% in the presence of miR-23b (B) The Renilla luciferase plasmids pGL4.71 c-met-3¢-UTR-1S (175 bp), pGL4.71 c-met-3¢-UTR-2S (166 bp), pGL4.71 c-met-3¢-UTR-3S (179 bp), and pGL4.71 c-met-3¢-UTR-4S (157 bp), harboring, respectively, putative miR-23b-binding sites 1, 2, 3, and 4, were analyzed for luciferase activity Sites 1, 2 and 4 inhibited the luciferase relative activity by 26%, 20%, and 10%, respectively Binding site 3 did not affect luciferase activity (C) Furthermore, results obtained with SKHep1C3 cells treated or untreated with miR-23b showed that sites 1, 2 and 4 were miR-23b-sensitive (D) Site 1 was found to be responsive to miR-23b and not to miR-107, used as control (E).