The gene encoding the protein phosphatase 6 catalytic sub-unit PPP6C , a negative cell cycle regulator, was identified as a direct target gene of miR-373 by use of a fluorescent reporter a
Trang 1functions as an oncogene in hepatocellular carcinoma
Nannan Wu*, Xuyuan Liu*, Xuemei Xu*, Xingxing Fan, Min Liu, Xin Li, Qiping Zhong and
Hua Tang
Tianjin Life Science Research Center and Basic Medical School, Tianjin Medical University, China
Introduction
Hepatocellular carcinoma (HCC) accounts for 80–90%
of liver cancers, and is one of the most prevalent
carci-nomas worldwide [1] Liver cancer is a complex genetic
disease in which the expression of many specific genes,
known as oncogenes or tumor suppressor genes, is
abnormally changed Previous studies have revealed
several genes related to human HCC For example, the
cyclin G1 gene is upregulated in HCC [2], and the
phosphatase and tensin homolog (PTEN) gene is
downregulated in HCC [3] Although focusing on
known genes has yielded much new information, previ-ously unknown noncoding RNAs, such as microRNAs (miRNAs), may also provide insights into the biology
of HCC MicroRNAs are a group of noncoding single-stranded RNAs, 22 nucleotides in length, that have emerged as an important class of short endogenous RNAs that post-transcriptionally regulate gene expres-sion by base-paring with their target mRNA [4] Sev-eral lines of evidence have shown that the six to eight nucleotides at the 5¢-end of miRNAs (positions 1–8)
Keywords
cell cycle; hepatocellular carcinoma; miRNA;
miRNA-373; protein phosphatase 6 catalytic
subunit (PPP6C)
Correspondence
H Tang, Tianjin Life Science Research
Center and Basic Medical School, Tianjin
Medical University, Tianjin 300070, China
Fax: +86 22 23542503
Tel: +86 22 23542503
E-mail: htang2002@yahoo.com
*These authors contributed equally to this
work
(Received 9 January 2011, revised 17 March
2011, accepted 5 April 2011)
doi:10.1111/j.1742-4658.2011.08120.x
MicroRNAs are a class of small noncoding RNAs that function as key reg-ulators of gene expression at the post-transcriptional level Recently, micr-oRNA-373 (miR-373) has been found to function as an oncogene in testicular germ cell tumors In our study, we found that miR-373 is upregu-lated in human hepatocellular carcinoma (HCC) tissues as compared with adjacent normal tissues, and promotes the proliferation of the HCC cell lines HepG2 and QGY-7703 by regulating the transition between G1 -phase-and S-phase The gene encoding the protein phosphatase 6 catalytic sub-unit (PPP6C ), a negative cell cycle regulator, was identified as a direct target gene of miR-373 by use of a fluorescent reporter assay The mRNA and protein levels of PPP6C were both inversely correlated with the
miR-373 expression level Overexpression of PPP6C abolished the regulation of cell cycle and cell growth exercised by miR-373 in HepG2 cells These results indicate that miR-373 plays an important role in the pathogenesis
of HCC, and may be a new biomarker in HCC Our results demonstrate that miR-373 can regulate cell cycle progression by targeting PPP6C tran-scripts and promotes the growth activity of HCC cells in vitro The down-regulation of PPP6C by miR-373 may explain why the expression of miR-373 can promote HCC cell proliferation
Abbreviations
ASO, antisense oligonucleotide; EGFP, enhanced green fluorescence protein; FACS, fluorescence-activated cell sorting; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; HCC, hepatocellular carcinoma; miRNA, microRNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PI, proliferation index; PPP6C, protein phosphatase 6 catalytic subunit; SD, standard deviation; shRNA, small hairpin RNA; siRNA, small interfering RNA.
Trang 2are important for target site recognition, and they have
been designated as the ‘seed’ region Animal miRNAs
target mRNA 3¢-UTRs predominantly by seed
sequence complementarity, and are rarely fully
comple-mentary; therefore, they function through translational
repression rather than cleavage [5] On the basis of
this, miRNAs could control as many as 30% of all
protein-coding genes [6] MicroRNAs play important
roles in developmental timing, and participate in the
regulation of processes such as cell fate determination,
proliferation, differentiation, and cell death [7–10]
Pre-vious studies have identified cancer-specific miRNAs in
many types of cancer, including B-cell chronic
lympho-cytic leukemia [11], colorectal cancer [12,13], lung
can-cer [14], breast cancan-cer [15], and brain cancan-cer [16,17]
A recent study described miR-373 as a tumor
suppres-sor gene in prostate cancer [18]; other studies provided
evidence that miR-373 was upregulated in breast
can-cer, testicular germ cell tumors, and human esophageal
cancer [19–21] However, the regulatory effects of
miR-373 on the tumorigenesis of other cancers remain
to be elucidated
In this study, we found, through real time reverse
transcription PCR (real time RT-PCR), that miR-373
was overexpressed in human HCC tissues as compared
with adjacent normal tissues, and identified the gene
encoding protein phosphatase 6 catalytic subunit
(PPP6C) as a direct target of miR-373 We also
observed that upregulation of miR-373 promoted cell
cycle progression through the G1⁄ S checkpoint in HCC
cells Taken together, our results suggest that miR-373
regulates the proliferation of a human HCC cell line by
negatively regulating PPP6C expression
Results
MicroRNA-373 is upregulated in HCC
To determine the expression of miR-373 in human
HCC tissues and adjacent normal tissues, we used
quantitative real time RT-PCR to detect 26 pairs of
HCC samples It was shown that miR-373 expression
level was generally and significantly higher in cancer
tissues than in adjacent nontumor tissue (Fig 1) Thus,
we speculated that miR-373 might be involved the
pathogenesis of HCC
Alteration of miR-373 affects cell growth of HCC
in vitro
First, we transfected either miR-373 antisense
oligonu-cleotides (ASOs) or an miR-373 expression vector
(pcDNA3⁄ pri-miR-373, pri-373) into HCC cells, and
detected miR-373 levels by real time RT-PCR Expres-sion of miR-373 was increased 4.5-fold in the HepG2 cells transfected with pcDNA3⁄ pri-miR-373 as com-pared with controls; miR-373 ASOs resulted in an
75% reduction of miR-373 levels (Fig 2A) Cell via-bility of HCC cells transfected with miR-373 ASOs or pri-373 was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay; miR-373 ASOs reduced cell viability at 48 or 72 h after transfection, whereas pri-373 increased cell viability (Fig 2B) In parallel, we analyzed colony formation and cellular proliferation to assess the effect of
miR-373 on the proliferative capacity of HCC cells The colony formation rate of HepG2 cells after transfection with miR-373 ASOs was 30% lower than that of HepG2 cells transfected with control oligomers Con-versely, transfection with pri-373 increased colony for-mation by 29% in HepG2 cells (Fig 2C,D) We observed similar results in another HCC cell line, QGY-7703 (Fig 2) These results indicate that
miR-373 can promote the cell proliferation of HCC cells
miR-373 facilitates the G1⁄ S-phase transition in HepG2 cells
To explore whether the promotion of proliferation caused by miR-373 in HCC cells is attributable to an alteration in cell cycle progression, we performed
Fig 1 Differential expression of miR-373 in HCC tissues The expression level of miR-373 in 26 pairs of HCC tissues (cancer) and matched normal tissues (normal) was detected by real-time RT-PCR Box-plot lines represent medians and interquartile ranges
of the normalized threshold values; whiskers and spots indicate 10–90th percentiles and the remaining data points The expression
of miR-373 is normalized to U6 small nuclear RNA (*P < 0.05).
Trang 3fluorescence-activated cell sorting (FACS) analysis.
Interestingly, in miR-373 ASO-treated HepG2 cells,
the percentage of cells in G1-phase increased to 54%,
whereas scramble ASO-treated cells had only 40% of
cells in G1-phase The percentage of miR-373
ASO-treated cells in S-phase decreased to 18%, as compared
with 31% in the control group (Fig 3A) The
prolifer-ation index of miR-373 ASO-treated HepG2 cells was
85.2%, as compared with 150% in controls In
contrast, after transfection with pri-373, the percentage
of HepG2 cells in G1-phase was 37%, as compared
with 50% in the scramble pcDNA3-treated cells, and
the percentage of cells in G2-phase was 20%, as
com-pared with 34% in scramble pcDNA3-treated cells
(Fig 3B) These results indicate that miR-373 plays an
important role in the G1⁄ S-phase transition of the cell
cycle
miR-373 targets PPP6C and negatively regulates its expression
MicroRNAs regulate a variety of cellular activities through regulation of the expression of target genes
To determine the mechanism of miR-373-mediated cell cycle dysregulation in HCC cells, we next identified target genes that could be responsible for the effect of miR-373 Taking into consideration that miR-373 was upregulated in HCC tissues (Fig 1), we reasoned that its target genes should be correspondingly downregu-lated Twelve candidate genes were predicated by three bioinformatics software packages (pictar, target-scan, and microcosm) Among these genes, the tumor suppressor gene PPP6C, which was predicted to have
an miR-373-binding site in its 3¢-UTR (Fig 4A), was chosen for further study
Fig 2 Alteration of miR-373 levels affects the growth of HCC cells (A) The miR-373 expression level in HCC cells was effec-tively altered by transfection of an miR-373 ASO vector or a pri-373 vector as detected
by real-time RT-PCR U6 small nuclear RNA was used for normalization (B) HCC cells were transfected with an miR-373 ASO vec-tor or a pri-373 vecvec-tor The MTT assay was used to determine relative cellular prolifera-tion at 48 and 72 h A 570 nm is the absor-bance of MTT measured at 570 nm After transfection with miR-373 or pri-373, the
48-h and 72-48-h data points s48-howed a statistically significant difference (72-h data not shown) (C) HCC cells were transfected with an miR-373 ASO vector or a pri-373 vector (D) Cell growth was measured with colony formation assays and proliferation curve assays The data represent the mean ± SD
of three different experiments (NC, negative control; *P < 0.05, **P < 0.005,
# P < 0.0005).
Trang 4To confirm whether miR-373 could bind to this
predicted region and suppress the expression of
PPP6C protein, we constructed an enhanced green
flu-orescence protein (EGFP) reporter vector (pcDNA3⁄ EGFP-PPP6C-3¢UTR), in which the 3¢-UTR fragment
of PPP6C, including the region encoding the putative binding site, was inserted downstream of the EGFP coding region HepG2 cells were transfected with the reporter vector together with either miR-373 ASOs or pri-373 As shown in Fig 4B, the intensity of EGFP fluorescence was higher in the miR-373-blocked group than in the control groups, whereas ectopic expression
of miR-373 decreased the intensity of EGFP fluores-cence when compared with the control groups In addition, we constructed another EGFP reporter vec-tor containing mutations in the regions encoding the miR-373 binding sites (Fig 4A) Neither blocking of miR-373 with ASOs nor overexpression of miR-373 had any effect on the intensity of EGFP fluorescence from the vector containing mutations in the region encoding the miR-373 binding sites These results show that miR-373 binds directly to the 3¢-UTR of the PPP6C transcript to repress gene expression
To determine whether miR-373 negatively regulates PPP6C expression at the mRNA or protein levels, we assessed endogenous PPP6C expression in HepG2 cells with altered miR-373 expression HepG2 cells were transfected with miR-373 ASOs or pcDNA3⁄
pri-miR-373 to block or overexpress miR-pri-miR-373, respectively, and the expression level of PPP6C mRNA was measured
by real time RT-PCR When miR-373 was blocked, PPP6C mRNA was elevated 3.8-fold as compared with the control group, whereas overexpression of miR-373 resulted in an 80% decrease in PPP6C mRNA
Fig 3 miR-373 can promote the
progres-sion from G 1 -phase to S-phase in HepG2
cells After transfection with an miR-373
ASO vector or a pri-373 vector, HepG2 cells
were detached, rinsed, fixed and stained as
described in Experimental procedures Cell
cycle phase distribution was analyzed by
FACS (A) The fraction of cells in G 1 -phase
was significantly increased in the miR-373
ASO group, and the fraction of cells in
S-phase was significantly decreased in the
miR-373 ASO group (B) In the pri-373
group, the fraction of cells in G1-phase was
significantly decreased The PI increased
noticeably in the pri-373 group The data
represent the mean ± SD of three different
experiments (NC, negative control;
*P < 0.05, **P < 0.005).
Fig 4 PPP6C is a direct target of miR-373 (A) The PPP6C 3¢-UTR
carries one potential miR-373-binding site (B) The direct interaction
of miR-373 and PPP6C mRNA was confirmed by a fluorescent
repor-ter assay HepG2 cells were transfected with an EGFP reporrepor-ter
vec-tor together with an miR-373 ASO or a pri-373 vecvec-tor, and the EGFP
intensity was measured The data represent the mean ± SD of three
different experiments (NC, negative control; hsa, Homo sapiens;
*P < 0.05, **P < 0.005, # P < 0.0005) Similar results were obtained
in QGY-7703 cells (data not shown).
Trang 5(Fig 5A) Western blot assay indicated that miR-373
ASOs resulted in a 2.3-fold increase in the PPP6C
pro-tein level, whereas overexpression of miR-373 reduced
the PPP6C protein level by 70% (Fig 5C)
To confirm the results obtained from the cell lines, we
also examined the expression of PPP6C mRNA in 16
pairs of hepatocarcinoma tissue samples Figure 5B
shows that, as compared with adjacent normal tissues,
PPP6C mRNA was consistently downregulated in HCC
tissue samples These results suggest that miR-373
regu-lates endogenous PPP6C expression through mRNA
degradation
Knockdown of PPP6C promotes HCC cell growth
Sequence-specific small interfering RNA (siRNA) can
effectively suppress gene expression We constructed a
plasmid expressing a small hairpin RNA (shRNA)
tar-geting PPP6C (pSilencer⁄ shRNA-PPP6C) Western
blot assay showed that the level of PPP6C expression
was reduced by 90% in HepG2 cells that were
trans-fected with pSilencer⁄ shRNA-PPP6C, as compared
with HepG2 cells transfected with a control plasmid
(Fig 6A) Inhibition of PPP6C expression increased
HCC cell growth as compared with the control group
(Fig 6B–D), which was consistent with the effects of
miR-373 overexpression Next, we examined the effects
of PPP6C knockdown on the cell cycle (Fig 6E)
Knockdown of PPP6C resulted in a significant
decrease in the proportion of cells in G1-phase and an
increase in the proportion of cells in S-phase These
findings indicate that PPP6C decreases the
prolifera-tion of HCC cells by inducing cell cycle arrest at the
G1⁄ S checkpoint, which is consistent with the effect of
miR-373 on HCC cells
Overexpression of PPP6C counteracts the effects
of miR-373 expression on the G1⁄ S-phase
transition
PPP6C induces cell cycle arrest at the G1⁄ S checkpoint
in cancer cells [22] We generated a plasmid (pcDNA3⁄
PPP6C, lacking the 3¢-UTR) to increase the protein
expression of PPP6C (Fig 7A) We transfected HCC
cells with the plasmid, and analyzed cell growth and cell
cycle progression In this experiment, ectopic expression
of PPP6C abrogated the promotion of cell growth
(Fig 7B–D) and the increase in the rate of G1⁄ S-phase
transition (Fig 7E) caused by miR-373 in HepG2 cells
The overall effect of PPP6C overexpression was
compa-rable to that of miR-373 ASO treatment, suggesting
that PPP6C is a key mediator of the miR-373
regula-tion of cell growth and cell cycle progression in HCC
Discussion
MicroRNAs regulate diverse biological processes, including tumorigenesis It has been reported that miR-373 functions as an oncogenic miRNA in testicu-lar germ cell tumors [20] and in human esophageal cancer [21] We wanted to determine whether miR-373 also functions as an oncogenic miRNA in HCC cells
To address this question, we first examined miR-373 expression in HCC tissues and matched adjacent non-tumor tissues by real-time RT-PCR, as previously described [23] The results show that the level of miR-373 is uncreased in tumor tissues as compared with the matched normal tissues in 16 pairs of matched specimens It has been reported that the upregulation of miR-373 promotes the growth of the cell lines in many cancers, e.g breast cancer [19], tes-ticular germ cell tumors [20], and human esophageal cancer [21] We determined the effect of miR-373 on the HCC cell lines HepG2 and QGY-7703 cells by gain and loss of function approaches MTT, colony forma-tion and growth curve assays show that miR-373 can increase the growth of those cells, and FACS analysis indicates that miR-373 can promote progression of the
G1⁄ S-phase transition in the cell cycle Thus, we inferred that miR-373 might be a growth-promoting fac-tor in HCC In breast cancer, miR-373 can promote invasion of the cancer cells [19] The function of miR-373
on HCC cells needs to be elucidated in the future The fundamental function of miRNAs is to regulate their targets by direct cleavage of mRNA or by inhibi-tion of translainhibi-tion [18], depending on the degree of complementarity with the 3¢-UTR of their target genes Computational algorithms were used to predict miRNA targets, which are based mainly on base pair-ing of miRNAs and the 3¢-UTRs of genes [6,24–26] Twelve candidate genes were predicted by three bioin-formatics software packages (pictar, targetscan, and microcosm), which may be correlated with the phenotype of the HCC cell lines caused by the alter-ation of miR373 Among them, a tumor suppressor gene, PPP6C, which regulates the G1⁄ S-phase transi-tion [22], was selected for further study It was pre-dicted that the 3¢-UTR region of PPP6C transcript would have an miR-373-binding site Given that miR-373 can target PPP6C mRNA, it may suppress the expression of PPP6C Western blot and real time RT-PCR assays show that miR-373 decreases PPP6C expression at the protein and mRNA levels in the HCC cell lines as compared with the control (Fig 5A,C), and also that PPP6C expression is down-regulated in HCC tissue as compared with normal tis-sues (Fig 5B), in which miR-373 is upregulated
Trang 6(Fig 1) The regulation of genes by miRNA occurs
mainly through direct targeting of the 3¢-UTR region
[5] We confirmed, with an EGFP-PPP6C-3¢UTR
reporter assay, that miR-373 can bind directly to the
PPP6C 3¢-UTR and negatively regulate PPP6C
expres-sion (Fig 4B) A previous study has demonstrated the
direct regulation of CD4, a signal molecule involved in
cell growth and adhesion, by miR-373 [18] Here, we
have enough evidence to confirm the
tumor-suppress-ing role of PPP6C in HCC cells, because knockdown
of PPP6C by siRNA promoted HCC cell proliferation,
whereas ectopic expression of PPP6C effectively
allevi-ated the miR-373-induced promotion of HCC cell
proliferation Together, these findings indicate that
miR-373 might exert its effects in HCC mainly by
tar-geting PPP6C However, Ivanov et al [27] recently
reported that PPP6C can be targeted by miR-31, and
functions as an oncogene in mesothelioma, which is in
contrast to our results obtained in HCC The possible
explanation is that the biological molecules have
differ-ent influences in differdiffer-ent tumor cells For example,
KLF4 was found to be an oncogene in breast cancer
[28], but Guan et al [29] reported KLF4 as a tumor
suppressor in B-cell non-Hodgkin lymphoma and in
classic Hodgkin lymphoma Also, miRNAs have
dif-ferent functions in difdif-ferent tissues; for instance, miR-9
is upregulated in breast cancer cells [30], but
downreg-ulated in human ovarian cancer [31] In addition,
dif-ferent miRNAs can target the same gene; for example,
CCND1 is directly regulated by the miR-16 family
[32], and miR-19a can also regulate the expression of CCND1 [33] Although miR-31 can target the PPP6C transcript, the binding sites in nucleotides 1363–1369
of the 3¢-UTR are different from the miR-373-binding sites in nucleotides 1338–1344 of the 3¢-UTR, which
do not overlap Whether miR-373 and miR-31 can simultaneously regulate PPP6C in HCC cells remains
to be elucidated
In conclusion, miR-373 functions as an oncogene and is upregulated in HCC tissues as compared with adjacent normal tissues Suppression of miR-373 repressed cell growth, possibly through inhibition of the cell cycle by targeting PPP6C Thus, the identifica-tion of the oncogene, miR-373, and its target gene, PPP6C, may help us to understand the molecular mechanism of tumorigenesis in HCC and may have potential diagnostic and therapeutic value in the future
Experimental procedures
Clinical specimen and RNA isolation
Twenty-six pairs of clinical specimens, including 26 human HCC tissue samples and 26 matched normal liver tissue samples, were obtained from the Tumor Bank Facility of Tianjin Medical University Cancer Institute and Hospital and the National Foundation of Cancer Research, with patients’ informed consent, which was approved by the eth-ics committee The category of specimens was confirmed by
Fig 5 The expression level of PPP6C is
inversely correlated with the level of
miR-373 (A) When miR-373 was blocked or
overexpressed, the mRNA level of PPP6C
was subsequently elevated or diminished,
respectively, as compared with the control
group (B) Relative expression level of
PPP6C in HCC tissues or matched
noncan-cerous tissues, as measured by real-time
RT-PCR The expression level of PPP6C is
normalized to b-actin (C) When miR-373
was blocked or overexpressed, the protein
level of PPP6C was subsequently elevated
or diminished, respectively, as compared
with the control group (NC, negative control;
*P < 0.05,#P < 0.0005).
Trang 7pathological analysis Large and small RNAs were isolated
from tissue samples with the mirVana miRNA Isolation
Kit (Ambion, Austin, TX, USA), according to the
manu-facturer’s instructions
Cell culture and transfection
Two human HCC cell lines (HepG2 and QGY-7703) were
maintained in MEMa or RPMI-1640 (Gibco, Grand
Island, NY, USA), respectively, and supplemented with
10% fetal bovine serum, 100 IUÆmL)1 penicillin, and
100 lgÆmL)1streptomycin Cells were incubated at 37C in
a humidified chamber supplemented with 5% CO2
Trans-fection was performed with Lipofectamine 2000 Reagent
(Invitrogen, Carlsbad, CA, USA), following the
manufac-turer’s protocol
Construction of expression vectors
To construct an miR-373-expressing vector (pcDNA3⁄ pri-miR-373, pri-miR-373), we first amplified a 476-bp DNA fragment carrying pri-miR-373 from genomic DNA; the amplified fragment was then cloned into the pcDNA3 at the XhoI and HindIII sites For construction of 3¢UTR reporter vectors (pcDNA3 ⁄ EGFP-PPP6C-3¢UTR and pcDNA3 ⁄ EGFP-PPP6C-EGFP-PPP6C-3¢UTR mutant), the 3¢-UTR and mutant 3¢-UTR fragments of PPP6C tran-scripts amplified by RT-PCR were inserted into the vector backbone downstream of the EGFP gene between the BamHI and EcoRI sites of the pcDNA3⁄ EGFP vector, as previously described [34] The pSilencer⁄ shRNA-PPP6C plasmid expressing an siRNA targeting the PPP6C transcript was constructed by annealing single-stranded
Fig 6 Knockdown of PPP6C shows con-cordant effects with miR-373 overexpres-sion in HCC cells (A) Western blot analysis showed that the expression of PPP6C was successfully suppressed by PPP6C siRNA (B–D) PPP6C was knocked down in HCC cells, and cell growth ⁄ viability activity was analyzed with the (B) MTT, (C) colony for-mation and (D) proliferation curve assays (E) Cell cycle phase distribution was ana-lyzed by FACS The data represent the mean ± SD of three different experiments (NC, negative control; *P < 0.05,
**P < 0.005,#P < 0.0005).
Trang 8hairpin cDNA and inserting it into a pSilencer2.1⁄ neo
vec-tor (Ambion), using BamHI and HindIII sites To construct
the PPP6C expression plasmid, the coding sequence (ORF)
without the 3¢-UTR of human PPP6C was amplified by
RT-PCR and inserted into the EcoRI and XhoI sites of
pcDNA3 All of the primers used are shown in Table 1
Cell proliferation assay
Cells were seeded in 96-well plates at a density of 5000 cells
per well, and then transfected with pri-miR-373 or miR-373
ASOs on the next day The MTT assay was used to deter-mine relative cell viability at 48 and 72 h Ten microliters
of MTT solution was added to 100 lL of culture medium, and incubated for 4 h at 37C; the absorbance at 570 nm (A570) was then measured
For cell proliferation measurements, HCC cells were seeded in 24-well plates at 10 000 cells per well (HepG2 cells) and 3000 cells per well (QGY-7703 cells) after trans-fection with pri-miR-373 or miR-373 ASOs Cell numbers were then counted every day for 6 days Each experiment was performed in triplicate
Fig 7 Effects of PPP6C overexpression in
HCC cells HCC cells were transfected with
pcDNA ⁄ 373 (pri-373) After 8 h, cells were
transfected with a pcDNA3⁄ PPP6C vector
or pcDNA3 empty vector The
pcDNA3⁄ PPP6C vector did not contain the
3¢-UTR of PPP6C, and miR-373 could not
therefore regulate ectopic PPP6C
expres-sion (A) At 48 h after transfection, PPP6C
expression was measured by western blot.
Cell growth⁄ viability was analyzed with the
(B) MTT, (C) colony formation and (D)
prolif-eration curve assays (E) Cell cycle phase
distribution was analyzed by FACS The data
represent the mean ± SD of three different
experiments (*P < 0.05, **P < 0.005,
# P < 0.0005).
Trang 9Colony formation assay
After transfection, cells were counted and seeded in 12-well
plates (in triplicate) at a density of 500 cells per well
(HepG2 cells) or 100 cells per well (QGY-7703 cells) The
culture medium was replaced every 3 days Colonies were
counted only if they contained more than 50 cells, and the
cells were stained with crystal violet The rate of colony
for-mation was calculated with the following equation: colony
formation rate = (number of colonies⁄ number of seeded
cells)· 100%
Flow cytometry analysis
At 48 h after transfection, the cells were detached from the
plates by trypsin incubation, rinsed with NaCl⁄ Pi, and fixed
in 95% (v⁄ v) ethanol The cells were then rehydrated in
NaCl⁄ Piand incubated with RNase (100 lgÆmL)1) and
pro-pidium iodide (60 lgÆmL)1) (Sigma-Aldrich, MO, USA)
Cells were analyzed with the FACS Calibur System
(Beck-man Coulter, Brea, CA, USA), and the cell cycle phase was
determined by cell quest analysis The proliferation index
(PI) was calculated as follows: PI = (S + G2⁄ M) ⁄ G1 (S,
G2⁄ M and G1 refer to the percentages of cells in S-phase,
G2⁄ M-phase and G1-phase, respectively) [35]
Bioinformatics
The miRNA targets predicted by computer-aided
algo-rithms were obtained with pictar, targetscan, and
microcosm For identification of the genes commonly
dicted by the three different algorithms, the results of
pre-dicted targets were intersected with matchminer
EGFP reporter assay
To confirm the direct interaction between miR-373 and
PPP6C mRNA, HCC cells were simultaneously transfected
with pri-miR-373 or control vector pcDNA3 and the repor-ter vectors in 48-well plates The red fluorescent protein expression vector pDsRed2-N1 (Clontech) was used for normalization The cells were lysed with radioimmunopre-cipitation assay buffer (150 mm NaCl, 50 mm Tris⁄ HCl,
pH 7.2, 1% Triton X-100, 0.1% SDS) 72 h later, and the proteins were harvested The intensities of EGFP and red fluorescent protein fluorescence were detected with an F-4500 Fluorescence Spectrophotometer (Hitachi, Tokyo, Japan)
Real time RT-PCR
The stem–loop real time RT-PCR method was performed
to detect the miRNA level, as previously described [23] Real time RT-PCR was performed with SYBR Premix Ex Taq (TaKaRa, Otsu, Shiga, Japan) on a 7300 Real Time PCR system (ABI, Foster City, CA, USA) The relative expression of miR-373 was defined as follows: quantity of miR-373⁄ quantity of U6 in the same sample The real time RT-PCR results were analyzed and expressed as relative expression of CT (threshold cycle) value, using the 2)DDCT method [36]
To detect the relative levels of PPP6C transcript, real time RT-PCR was performed Briefly, a cDNA library was gener-ated through reverse transcription, using Moloney murine leukemia virus reverse transcriptase (Promega, Madison,
WI, USA) with 5 lg of the large RNA, and used to amplify the PPP6C gene (and the b-actin gene as an endogenous control) by PCR PCR primers were as follows: PPP6C sense and PPP6C antisense as above; b-actin sense, 5¢-CGTGAC-ATTAAGGAGAAGCTG-3¢; and b-actin antisense, 5¢-CT-AGAAGCATTTGCGGTGGAC-3¢ PCR cycles were as follows: 94C for 5 min, followed by 40 cycles at 94 C for
1 min, 56C for 1 min, and 72 C for 1 min Real time RT-PCR was performed as described above, and the relative expression level of PPP6C was defined as follows: quantity
of PPP6C⁄ quantity of b-actin in the same sample
Table 1 The oligonucleotides used in this work.
TCAAATTACTTACAAGTTTTTTGAATTCTCGAGA
TGATCTCTTGAACAAATTACTTACACAAAGAG
Trang 10Western blot
HCC cells were transfected and lysed 48 h later with
radio-immunoprecipitation assay buffer, and proteins were
har-vested All proteins were resolved on a 10% SDS denatured
polyacrylamide gel, and then transferred onto a
nitrocellu-lose membrane Membranes were incubated with an
antibody against PPP6C or an antibody against
glyceralde-hyde-3-phosphate dehydrogenase (GAPDH) overnight at
4C The membranes were washed and incubated with
horseradish peroxidase-conjugated secondary antibody
Protein expression was assessed by enhanced
chemilumines-cence and exposure to chemiluminescent film lab works
image acquisition and analysis software was used to
quan-tify band intensities Antibodies were purchased from
Tian-jin Saier Biotech and Sigma-Aldrich
Statistical analysis
Data are expressed as mean ± standard deviation (SD),
and P < 0.05 is considered to be statistically significant
with the Student–Newman–Keuls test
Acknowledgements
This work was supported by the National Natural
Science Foundation of China (No 30873017; No
31071191) and the Natural Science Foundation of
Tianjin (No 08JCZDJC23300 and No 09JCZDJC
17500)
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