Mir-208 promotes cell proliferation by repressing SOX6 expression in human esophageal squamous cell carcinoma
- Heping Li†1, 2,
- Dayong Zheng†3,
- Bing Zhang†1,
- Liangshuai Liu1,
- Junwei Ou4,
- Wei Chen1,
- Shiqiu Xiong5,
- Yong Gu4Email author and
- Jianyong Yang1Email author
© Li et al.; licensee BioMed Central Ltd. 2014
Received: 5 January 2014
Accepted: 1 July 2014
Published: 15 July 2014
Esophageal squamous cell carcinoma (ESCC) is the major histological type of esophageal cancer in developing countries. The prognosis and survival rate of ESCC are very poor. Recently, microRNAs (miRNAs) have emerged as important regulators of cancer cell biological processes. To better understanding the molecular mechanisms by which they regulate the behavior of cancer cells is needed.
The expression of miR-208 was examined in ESCC cell lines and tumor tissues by real-time PCR. Proliferation capability of ESCC cells upon regulation of miR-208 expression was detected by MTT assay, colony formation assay, anchorage-independent growth ability assay and flow cytometry analysis. The target of miR-208 was determined by western blotting analysis, luciferase reporter assay and real-time PCR.
miR-208 was upregulated in ESCC cell lines and tissues. Overexpression of miR-208 in ESCC cells increased cell proliferation, tumorigenicity and cell cycle progression, whereas inhibition of miR-208 reduced cells proliferation, tumorigenicity and cell cycle progression. Additionally, SOX6 was identified as a direct target of miR-208. Ectopic expression of miR-208 led to downregulation of SOX6 protein, which resulted in the downregulation of p21, upregulation of cyclin D1 and phosphorylation of Rb.
These results suggest that miR-208 represents a potential onco-miR and participates in ESCC carcinogenesis by suppressing SOX6 expression.
KeywordsmiR-208 SOX6 Esophageal squamous cell carcinoma Proliferation
Esophageal cancer, one of the most common malignant tumors, is the eighth most common cancer and the sixth most common causes of cancer mortality in the world . According to its pathological characteristics, it has two main subtypes, ESCC and esophageal adenocarcinoma (EAC) [1, 2]. ESCC is the major histological type of esophageal cancer in developing countries . Although advances have been made in the treatment of ESCC, including surgery, chemotherapy, radiation or a combination of these options, the prognosis of ESCC patients remains very poor, which the overall 5-year survival rate of patient after surgery is only about 14-22% [3, 4]. Some oncogenic and tumor suppressive factors have been reported to be associated with ESCC progression; however, few of them were specific and conclusive [5, 6]. Further information on the biological behavior involved in esophageal cancer initiation and progression, especially in ESCC, is important for the development of effective diagnostic methods and therapeutic strategies.
MiRNAs, a class of small non-coding RNAs of 20–22 nucleotides, are involved in multiple biological processes, such as cell differentiation, proliferation, oncogenesis, angiogenesis and cell invasion [7–9]. MiRNAs play essential roles during human cancer progression by targeting the 3′ untranslated region (3′-UTR) of mRNAs in a sequence-specific manner for translational repression or degradation [10, 11]. In view of the close relationship between miRNAs and the biological progression of multiple cancers, miRNAs are presently considered as potential novel targets for anti-cancer therapies [9–12].
SOX6, a member of the D sub family of the sex determining region Y (SRY)-box-related transcription factors, contains a conserved high-mobility-group (HMG) DNA-binding domain and plays important roles in biological progression [13, 14]. SOX6 has been reported to play a tumor-suppressive function in certain tumors. It is frequently downregulated and significantly associated with better prognosis in primary ESCC and hepatocellular carcinoma [15, 16].
In this study, we found that miR-208 is upregulated in ESCC cell lines and tissues. We verified a functional role for miR-208 in ESCC cell proliferation, tumorigenicity and cell cycle regulation. Furthermore, our data indicated that SOX6 mRNA is a target of miR-208 and that SOX6 is essential for the regulation of miR-208 in ESCC cells in vitro. Our results suggest that miR-208 may promote cell proliferation, tumorigenicity and cell cycle progression in ESCC through the SOX6-mediated signaling pathway.
Material and methods
Primary culture of normal esophageal epithelial cells (NEEC) was established from fresh specimens of the adjacent noncancerous esophageal tissue, which is over 5 cm from the cancerous tissue, according to a previous report . The ESCC cell lines, including Kyse140, Kyse30, Kyse510, Kyse520, Eca109, TE-1, Kyse410, Kyse180, EC18, HKESC1 and 108CA, were grown in the Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA), 100 units of penicillin and 100 units of streptomycin at 37°C in a 5% CO2 atmosphere in a humidified incubator.
For the use of the clinical materials for research purposes, prior patient consent and approval from the Institutional Research Ethics Committee were obtained. This study was conducted on 10 pairs of snap-frozen ESCC tumor and matched normal tissues from adjacent regions, which were histopathologically diagnosed at the First Affiliated Hospital of Sun Yat-sen University from 2001 to 2006. The 10 ESCC tissues and the matched adjacent noncancerous esophageal tissues were frozen and stored in liquid nitrogen until further use.
Generation of stably engineered cell lines
The miR-208 expression plasmid was generated by cloning the genomic pre-miR-208 gene, with 300-bp on each flanking side, into retroviral transfer plasmid pMSCV-puro (Clontech Laboratories Inc., Moutain View, CA, USA) to generate plasmid pMSCV-miR-208. The non-targeting control microRNA (negative control mimic), which are designed computationally to have no perfect seed-sequence matches to the transcriptome, was subcloned into retroviral transfer plasmid pMSCV to generate the plasmid pMSCV-NC. pMSCV-miR-208 or pMSCV-NC was cotransfected with the PIK packaging plasmid into 293FT cells using the standard calcium phosphate transfection method . Thirty-six hours after cotransfection, supernatants were collected and incubated with cells to be infected for 24 hours in the presence of polybrene (2.5 μg/ml). After infection, puromycin (1.5 μg/ml) was used to select stably transduced cells over a 10-day period.
RNA extraction, reverse transcription (RT) and real-time PCR
Total cellular RNA was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. cDNAs were amplified and quantified in an ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using dye SYBR Green I (Molecular Probes, Invitrogen). The primers selected were as follows:
p21 forward: 5′-CATGGGTTCTGACGGACAT -3′,
p21 reverse: 5′- AGTCAGTTCCTTGTGGAGCC -3′;
Cyclin D1 forward: 5′-AACTACCTGGACCGCTTCCT -3′,
Cyclin D1 reverse: 5′-CCACTT GAGCTTGTTCACCA-3′.
Expression levels of genes were normalized to that of the housekeeping gene GAPDH as the control (GAPDH forward primer, 5′-GACTCATGACCACAGTCCA TGC-3′; reverse primer, 3′-AGAGGCAGGGATGATGTTCTG-5′), and calculated as 2-[(Ct of p21, CyclinD1) – (Ct of GAPDH)], where Ct represents the threshold cycle for each transcript. The expression of the miRNA was defined based on the threshold cycle (Ct), and relative expression levels were calculated as 2-[(Ct of miR-208) – (Ct of U6)] after normalization with reference to expression of U6 small nuclear RNA.
Western blotting analysis was performed according to standard methods, as previously described . The membranes were probed with polyclonal rabbit antibodies against anti-SOX6 (1:500; Abcam, Cambridge, MA, USA), anti-p21, anti-cyclinD1 and anti-Rb, anti-phosphorylated Rb (1:1,000; Cell Signaling, Danvers, MA, USA). The membranes were stripped and re-probed with an anti-α-Tubulin mouse monoclonal antibody (1:1,000; Sigma, Saint Louis, MO, USA) as a loading control.
Plasmid, oligonucleotides, siRNA and transfection
The region of the human SOX6 3′-UTR, from 1 to 1100 bp, generated by PCR amplification from DNA of the Eca109 cells, was cloned into vector pGL3 (Promega, Madison, WI, USA). The primers selected were as follows: SOX6-3′UTR-wt-up: 5′- GCCCCGCGGTGGCTCCACAATTACATCAGC -3′, SOX6-3′UTR-wt-dn: 3′- GCCCTGCAGCATAAAATCACTATGTACACAGGA -5′. The miR-208 mimic, miR-208 inhibitor and negative control (NC) were purchased from RiboBio (RiboBio Co. Ltd, Guangzhou, Guangdong, China). For depletion of SOX6, the siRNA was synthesized and purified by RiboBio. The SOX6 siRNA sequences used were: CGGGAAACTGTCCTCCATAAA. Transfection of oligonucleotides and siRNA were performed using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction.
ESCC cells (3.5 × 104) were seeded in triplicate in 24-well plates and allowed to settle for 12 h. One hundred nanograms of pGL3-SOX6-luciferase plasmid was transfected into ESCC cells using the Lipofectamine 2000 reagent (Invitrogen). Medium was replaced after 6 h, and luciferase and renilla signals were measured 48 h after transfection using the Dual Luciferase Reporter Assay Kit (Promega) according to the manufacturer’s protocol. Three independent experiments were performed and the data were presented as the mean ± SD.
3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay
Kyse30 and Kyse410 cells, seeded on 96-well plates, were stained at the indicated time points with 100 μl sterile MTT dye (0.5 mg/ml, Sigma) for 4 h at 37ºC, followed by removal of the culture medium and addition of 150 μl dimethyl sulfoxide (Sigma). The absorbance was measured at 570 nm, with 655 nm as the reference wavelength. All experiments were performed in triplicate.
Colony formation assay
Cells were plated on a 6-well plate (0.5 × 103 cells per well) and cultured for 10 days. The colonies were stained with 1.0% crystal violet for 1 min after fixation with 10% formaldehyde for 5 min. The experiment was performed independently three times for each cell line.
Anchorage-independent growth ability assay
One thousand cells were trypsinized and suspended in 2 ml complete medium plus 0.33% agar (Sigma). The agar-cell mixture was plated on top of a bottom layer comprising 0.66% complete medium agar mixture. After 10 days, colony sizes were measured with an ocular micrometer and colonies greater than 0.1 mm in diameter were counted. The experiment was performed for independently three times for each cell line.
Flow cytometry analysis
All cells in a culture dish were harvested by trypsinization, washed in ice-cold PBS, and fixed in 80% ice-cold ethanol. Before staining, the cells were pelleted in a cooled centrifuge and resuspended in cold PBS. Bovine pancreatic RNAase (Sigma) was added at a final concentration of 2 μg/ml, and cells were incubated at 37°C for 30 min, followed by incubation in 20 μg/ml propidium iodide (Sigma) for 20 min at room temperature. 20,000 cells were analyzed on a flow cytometer (FACSCalibur; BD Biosciences, Bedford, MA, USA).
Student’s t test was used to evaluate the significant difference of two groups of data in all the pertinent experiments. A P value <0.05 (using a two-tailed paired t test) was considered significantly different for two groups of data.
miR-208 expression is elevated in ESCC cell lines and tissues
Ectopic expression of miR-208 enhances proliferation of ESCC cells
Inhibition of miR-208 reduces proliferation of ESCC cells
SOX6 is a direct target of miR-208 in ESCC cells
SOX6 suppression is critical for miR-208-induced cell proliferation in ESCC
In the current study, we demonstrated that miR-208 is upregulated in ESCC cell lines and tissues. Overexpression of miR-208 promotes the proliferation and tumorigenicity of ESCC cells, probably through post-translationally downregulating SOX6 expression by targeting its mRNA 3′- UTR. The negative regulation of SOX6 by miR-208 leads to upregulation of p21, downregulation of cyclinD1, and Rb phosphorylation. We demonstrated that miR-208 might play essential role via the SOX6-mediated pathway during ESCC progression.
Recent studies showed that miRNAs regulated various cellular pathways by affecting the expression of multiple target genes [8, 20]. The expression of miRNAs might contribute to human carcinogenesis and cancer progression, and are considered as potential novel targets for cancer diagnosis and therapy [21–24]. miR-208 has been identified as a myomiR. It is specifically expressed at much higher levels in cardiac tissue and is dysregulated in various cardiovascular diseases [25–27]. Inhibition of miR-208 improved cardiac function and patient survival during heart failure [27, 28]. However, the expression and function of miR-208 in cancers remain unknown. For the first time, we have demonstrated that miR-208 expression is correlated with ESCC progression and might play an important role during ESCC development. It has also been reported that miR-208 downregulates the Ets1 proto-oncogene, which is closely involved in the regulation of cell proliferation, differentiation, metastasis, apoptosis, and angiogenesis, by targeting the Ets1 mRNA 3′-UTR . Nevertheless, the expression level of miR-208 in esophageal cancer or other cancers and its clinical relevance, require further study.
Systematic reporter studies have shown that functional regulation by miRNAs is highly sensitive to base pair mismatches within nucleotides 2–8 of the miRNA, which have been defined as the seed region . The functional importance of seed region complementarity as the major determinant of miRNA targeting has well established , which the microRNAs with mutation in the seed sequences could not genetically match with target genes and fail to inhibit the target genes expression [8, 32]. In the current study, we found that miR-208 negatively regulated SOX6 expression by targeting SOX6 3′ untranslated region (3′-UTR) in the sequence-specific manner. Consistently, miR-208 with mutated seed sequence failed to show an inhibitory effect on the luciferase activity of SOX6. SOX6 is reported to have a tumor-suppressive function in tumors [15, 16]. SOX6 suppressed ESCC cells proliferation and cell motility, and inhibited tumor formation. SOX6 also inhibits cell cycle G1/S phage transition by upregulating p53 and p21WAF1/CIP1, and by downregulating cyclin D1/CDK4, cyclin A and β–catenin [14, 15]. In our study, downregulation of SOX6 by miR-208 led to downregulation of p21, upregulation of cyclin D1, and induced phosphorylation of Rb, resulting in the promotion of cell proliferation and cell cycle progression.
These data suggested that overexpression of miR-208 favored ESCC progression. Most patients with ESCC are diagnosed at an advanced stage with lymph node (LN) metastasis, resulting in a poor prognosis . Therefore, a better understanding of the development of LN metastasis may lead to therapeutic improvements for ESCC patients. SOX6 is closely associated with LN metastasis of ESCC . Whether miR-208 is involved in SOX6-associated LN metastasis needs further study.
In summary, we describe, for the first time, that miR-208 plays an important role in ESCC development and progression. The results reveal that miR-208 is upregulated in ESCC cell lines and tissues. Furthermore, miR-208 overexpression promotes cell proliferation and tumorigenicity in human ESCC cell lines in vitro. Additionally, we identified SOX6 mRNA as a direct and functional target of miR-208. Finally, we revealed that SOX6 suppression is essential for miR-208-induced cell proliferation in ESCC. Based on these results, we propose that miR-208 might be used as a therapeutic agent for ESCC. Further study is required to identify the clinical relevance and utility of miR-208 in ESCC diagnosis and therapy.
This study was supported by the Science and Technology Projects Foundation of Guangdong Province (No.2012B031800501) and Natural Science Foundation of Guangdong Province (No.S2012010008827).
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