Background
Hepatocellular carcinoma (HCC), the most common primary malignant liver cancer, is a global public health problem that accounts for approximately 500,000 deaths annually [
1]. The high recurrence and low 5-year survival rate of HCC are mainly due to the intrahepatic and extrahepatic metastases [
2], and the rate of recurrence is 86.5% for intrahepatic metastasis and 13.5% for extrahepatic metastasis [
3]. The epithelial–mesenchymal transition (EMT) plays a pivotal role in local invasion and distant metastasis during HCC progression [
4]. However, the mechanism underlying the EMT of HCC is largely unknown.
Current studies have shown that microRNAs (miRNAs) could act as activators or inhibitors of tumor metastasis by targeting multiple signaling pathways involved in metastasis [
5]. Moreover, miRNAs have been implicated in the process of EMT through the modulation of EMT-related genes [
6]. Our previous results suggested that miR-10b was overexpressed in HCC and promoted HCC cell migration and invasion through the HOXD10/RhoC/uPAR/MMPs pathway [
7]. MMPs help cancer cells spread by breaking down the extracellular matrix (ECM) and other barriers which play an important role in the EMT progress [
8]. So, we speculate that miR-10b may involve in EMT development.
Krüppel-like factor 11 (KLF11) belongs to the family of Sp1/Krüppel-like transcription factors and has been initially characterized as a TGF-β inducible early gene in the EMT progress [
9]. KLF11 promotes the EMT through binding to the Smad7 promoter and suppressing the transcription of Smad7, which interrupts the Smad7-driven negative feedback loop [
10]. Additionally, KLF11 can directly upregulate the Smad2/3 expression to promote EMT development [
11]. In this study, we determined whether miR-10b was involved in KLF11 regulation and whether it participated in HCC EMT progress.
In this study, we first found that miR-10b could promote EMT in HCC cells. Then, we identified the regulation mechanism of miR-10b in EMT through the KLF4/KLF11/Smads pathway. Thus, our data suggested important roles for miR-10b in HCC EMT and implicated miR-10b as a potential target for HCC therapies.
Methods
Cell lines and culture conditions
The two HCC cell lines were used in this study: MHCC-97H (HCC cells with high metastatic potential) and MHCC-97L (HCC cells with low metastatic potential) [
12]. All cell lines were purchased from Shanghai Institute for Biological Sciences (Shanghai, China). All cell lines were routinely cultured in RPMI-1640 medium (Hyclone Laboratories, Logan, UT) supplemented with 10% fetal calf serum (Gibco BRL, Rockville, MD, USA) at 37 °C in a humidified atmosphere of 5% CO
2.
Immunofluorescence
Cells were seeded in 4-well 35-mm dishes (Greiner Bio-One North America Inc., Monroe, NC, USA) at a density of 1000 cells/well and grown for 48 h in culture medium. Then cells were fixed in 4% paraformaldehyde for 20 min and permeabilized in phosphate-buffered saline (PBS) supplemented with 0.5% Triton X-100. After blocking, cells were incubated with the indicated antibodies for 2 h. Cells were washed in PBS, incubated with their corresponding FITC-labeled or TRITC-labeled secondary antibodies (Pierce, Rockford, IL, USA) for 1 h at room temperature and stained with DAPI (Vector Labs, Burlingame, CA, USA). Finally, the cells were mounted using glycerol and observed using a Nikon A1 laser scanning confocal microscope (Japan).
Western blot
Cell samples were lysed with RIPA buffer (Beyotime, China). Equal amounts (10 μg) of total protein were loaded, and then subsequently immunoblotted with the primary antibodies, including anti E-cadherin (BD Biosciences, Franklin Lakes, USA), Vimentin (Invitrogen, Carlsbad, CA, USA), KLF4, KLF11, Smad7, Smad3 and tubulin (Santa Cruz, CA, USA). Proteins were detected using the Amersham enhanced chemiluminescence system (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions.
Real-time RT-PCR
Real-time RT-PCR was performed as described previously [
13]. Expression data were uniformly normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control, and the relative expression levels were evaluated using the
ΔΔCt method [
14,
15]. Primers were used as described previously [
16,
17].
Vector construction, siRNA, and luciferase reporter assay
The miR-10b expression plasmid was constructed by our laboratory previously [
7]. Cultured cells were transfected with miR-10b expression vector, antisense miR-10b (anti-miR-10b), scramble miRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. The sequence were described as before [
7,
18].
The 3′UTR segment of KLF4 and KLF11 were subcloned into the pmirGLO vector (Promega, Madison, WI, USA), respectively [
11,
19]. The coding sequences of KLF4 was amplified and cloned into pcDNA3.1 (Invitrogen) [
20]. The promoter of the KLF11 gene (about − 2000 upstream to the Exon1, containing the KLF4 binding sites CACCC) was amplified by PCR and inserted into the pGL3-basic vector (Promega). The mutant constructs were generated using a QuickChange mutagenesis kit (Stratagene, La Jolla, CA, USA). All constructs were further confirmed by sequencing. siRNAs targeting KLF4, KLF11 and negative control siRNA were purchased from Ambion. Cell transfection and dual luciferase reporter assay were performed as described previously.
Chromatin immunoprecipitation (ChIP)
ChIP assays were performed using a EZ ChIP assay kit (Millipore Corporation, Billerica, MA, USA). Immunoprecipitation was carried out with anti-KLF4 antibody or rabbit IgG at 4 °C overnight with rotation. The immunoprecipitated DNA was amplified by the promoter-specific primers: forward 5′-ACG CTG AGT ACA GTG GGA GCC AC-3′; reverse 5′-TCC TCG AGC CTG CAT T-3′. The PCR products were analyzed on 1% agarose gel.
Statistical analysis
All statistical analyses were performed using the SPSS statistical software package (SPSS, Chicago, IL, USA). The significance of the data was determined using Student’s t test or one-way ANOVA. All the statistical tests were two-sided, and a P value < 0.05 was considered significant.
Discussion
In our study, we found that overexpression of miR-10b could promote HCC EMT. miR-10b induces upregulation of KLF11 and Smads signaling activity to promote EMT. Specifically, there was no direct regulation of miR-10b in KLF11 expression. Our results showed that miR-10b downregulates KLF4, the inhibitory transcriptional factor of KLF11 to upregulate KLF11 expression. Our study presents the regulation mechanism of miR-10b in EMT through the KLF4/KLF11/Smads pathway for the first time.
HCC is the third leading cause of cancer-related death worldwide. Since clinical symptoms are not easily observed during the early stage, the prognosis is poor at the time of diagnosis, which, in most cases, is during the advanced stage [
21]. Thus, it is of much significance to explore new diagnostic and therapeutic molecular targets for HCC. miRNAs have been demonstrated to have close relationship with HCC. miR-10b locates in the HOX gene cluster on chromosome 2, suggesting that it is closely related to tumor invasion and metastasis. Previous studies showed that miR-10b was overexpressed in a variety of human cancers, such as breast cancer, malignant glioma, nasopharyngeal carcinoma, pancreatic cancer, and HCC [
22].
EMT is a key process driving cancer metastasis and the loss of E-cadherin and increase in vimentin expression are considered to be the most important molecular markers of EMT. Recent studies have revealed that miRNAs act as crucial modulators of EMT through the regulation of E-cadherin and other molecules such as vimentin and ZEB [
23]. Our previous work has demonstrated that miR-10b was overexpressed in HCC and promoted HCC cell migration and invasion [
7]. In this study, we found that transfected with miR-10b expression plasmid exhibited a greater number of mesenchymal cells. The expression of E-cadherin was downregulated, and the mesenchymal marker vimentin was upregulated after transfection of miR-10b compared with that of control. These findings demonstrated that miR-10b promoted EMT in HCC cells.
Next, we explored the underlying mechanisms involved in the regulation of EMT by miR-10b. We found that KLF11 protein and mRNA expression levels increased after transfected with miR-10b. Consequently, the Smad7 expression was decreased. Subsequently, the Smad3 protein and mRNA expression levels were increased. So, our results indicated that the levels of KLF11 were increased by miR-10b, which induced Smads signaling activity to promote EMT. We speculated that miR-10b could bind to the KLF11 3′UTR and regulate its expression. We constructed KLF11-3′UTR luciferase reporter plasmid. Cotransfection of KLF11-3′UTR and miR-10b caused no decrease in the luciferase activity compared with the negative control. The similar result was found in transfection with mutation plasmid. These results indicated that there was no direct regulation of miR-10b in KLF11 expression. Thus, we speculated that there was a regulation mediator factor between miR-10b and KLF11.
Bioinformatic prediction tools indicated that KLF4 is a putative target of miR-10b [
24]. Zhang has identified that miR-10b targeted KLF4 levels in human NPC cells [
25]. Consistently, we found that miR-10b exerts inhibitory effects on KLF4 expression via interaction with the 3′UTR of KLF4. KLF4 is a transcription factor involved in cell cycle regulation, apoptosis, and differentiation. Its expression increases in response to DNA damage, serum deprivation, and contact inhibition [
26]. Specifically, KLF4 has been shown to negatively regulate EMT in cancers. Down-regulation of KLF4 is required for EMT, cell migration, and for the induction of apoptosis [
27]. Together, our results indicate that KLF4 may take part in the EMT progress induced by miR-10b in HCC.
Emerging evidence suggests that the Sp/KLF family member could regulate each other [
28,
29]. We found that the transcriptional activity of KLF11 was reduced by KLF4 overexpression. The ChIP assays revealed that endogenous KLF4 directly bound to KLF11 promoter. Western-blot showed that KLF11 protein expression was downregulated or upregulated after overexpression or siRNA knockdown of KLF4, respectively. Altogether, KLF4 can bind to its special binding motifs on KLF11 promoter to down-regulate their transcription. Furthermore, the induction role of miR-10b in HCC EMT could be blocked by KLF11 siRNA. miR-10b promotes EMT in HCC cells via upregulation of KLF11.
Conclusions
We first found that miR-10b could promote EMT in HCC cells. miR-10b downregulates KLF4, the inhibitory transcriptional factor of KLF11, which induces Smads signaling activity to promote HCC EMT. This newly identified miR-10b KLF4/KLF11/Smads pathway provides a new, potential therapeutic target to treat HCC.
Authors’ contributions
HG and SHZ carried out the molecular genetic studies and drafted the manuscript. HZ and JQ participated in the confocal immunofluorescence study. HG, XWX and HO participated in cell biology experiments. HG performed the statistical analysis. CGL and SEY conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.