Introduction
Hepatocellular carcinoma (HCC) is the sixth most common malignancy and fourth leading cause of cancer-associated death worldwide [
1]. Liver transplantation, surgical resection, and local radiofrequency ablation are the main curative treatments for early staging HCC, however, the early recurrence and metastasis of HCC still make the post-operative survival unsatisfactory [
2]; furthermore, the vast majority of newly diagnosed patients are always with intra- or extra-hepatic metastasis. Therefore, it is urgent to illuminating the underlying mechanisms of HCC metastasis.
It has been widely accepted that epithelial-mesenchymal transition (EMT) devoted to tumor metastasis and recurrence [
3‐
5]. Epithelial cancer cells lose epithelial characteristics and gain the mesenchymal properties during EMT. EMT-related transcriptional factors (TFs), such as Snail/Slug, ZEB1/2, TWIST1, and signaling pathways, for instance, TGF-β1/SMAD, Wnt, VEGF, IGF, and Notch are crucial triggers and regulators for EMT [
6]. Cancer stem cells (CSCs), a subgroup cancer cells with self-renewal and proliferative properties, were recently thought to be the seeds of tumor metastasis and recurrence [
7]. By undergoing EMT, cancer cells could acquire the “stemness”, which establishes a close relationship between EMT, CSCs, and metastasis [
8].
Liver CSCs have been reported to be enriched by several surface markers, including CD13, CD133, CD24, EpCAM, CD44 and CD90 [
9‐
12]. However, the exact mechanism that liver CSCs maintain self-renewal characteristics has been rarely reported. Interestingly, pathways, such as TGF-β1 pathway, Wnt/β-catenin pathway, Notch pathway and Hedgehog pathway, and EMT-related TFs are increasingly shown to regulate the CSCs characteristics [
13‐
15]. TGF-β1 signaling plays a dual role during the progression of HCC, it prevents the progression of HCC in the early stage, while promoting carcinogenesis in the late stage [
16]. In HCC, TGF-β1 induced a partial EMT to maintain stemness characteristics, during which liver cancer cells acquire increased mobility and invasiveness [
17]. However, the exact mechanism that TGF-β1 signaling regulates EMT and stemness needs further investigation.
Accumulating evidences indicated that long non-coding RNAs (lncRNAs) play a significant role in regulating EMT process in cancer cells [
18]. Linc00261, also known as DEANR1, has been found dysregulated in numerous cancers, such as lung cancer [
19], gastric cancer [
20], endometrial cancer [
21], and HCC [
22]. Its downregulation could be associated with transcriptional inhibition by neighbor gene FOXA2, DNMT1-derived CpG islands methylation, and EZH2 catalyzed trimethylation of H3K27at lys27 (H3K27Me3) [
23]. It inhibits cellular proliferation by promoting apoptosis, DNA damage, or G2/M cell cycle arrest, restrains cellular mobility and invasion by restricting the activation of Notch signaling [
24] or accelerating the degradation of Slug [
25]. Interestingly, as an endoderm differentiation specific lincRNA, linc00261 also specifically expressed in adult endoderm-derived tissues and liver shares the highest level; besides, our previous study and others revealed an inhibitory effect of linc00261 on EMT process and metastasis in HCC and gastric cancer. However, whether linc00261 deficiency modulated EMT induced acquisition of stemness, is still undefined.
In this study, we investigated the influence of linc00261 on regulating EMT and cancer stem cell-liked characteristics in HCC, and the exact role of linc00261 in modulating SMAD3, the key factor of TGF-β1 signaling. These findings may provide new strategies for the prevention and therapy for HCC metastasis.
Materials and methods
Cell lines
Liver cancer cell lines SMMC-7721 and Huh7 were bought from the Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, China. HepG2 and Sk-hep-1 were purchased from American Type Culture Collection (ATCC; VA, USA), and MHCC-LM3 was obtained from Liver Cancer Research Institute, Zhongshan Hospital, Fudan University, Shanghai, China as a gift. Cell lines were cultured in DMEM (Gibco) with 10% fetal bovine serum (FBS; Gibco) at 37 °C in a humidified incubator with 5% CO2. Cells was treated with TGF-β1 (5 ng/ml) to induce the EMT.
RNA isolation and quantitative real-time PCR (qRT-PCR)
Total RNA was isolated using Trizol reagent RNAisoPlus (Takara, Dalian, China) and reversely transcribed into cDNA using Primescript RT Master Mix (Takara), after which, expression of target gene was evaluated by qRT-PCR using SYBR Green Mix (Takara) according to the manufacturer′s instructions. The primes used were listed as follow: linc00261: 5′-GTCAGAAGGAAAGGCCGTGA-3′ (forward), 5′-TGAGCCGAGATGAACAGGTG-3′ (reverse); Nanog: 5′-TGAACCTCAGCTACAAACAG-3′ (forward), 5′-TGGTGGTAGGAAGAGTAAAG-3′ (reverse); SOX2: 5′-ACGCTCATGAAGAAGGATAAGT-3′ (forward), 5′-GAGCTGGTCATGGAGTTGTAC-3′ (reverse); OCT4: 5′-AGGTGGTCCGAGTGTGGTTC-3′ (forward), 5′-GAGGAGTACAGTGCAGTGAAGTG-3′ (reverse); Slug: 5′-CTGTGACAAGGAATATGTGAGC-3′ (forward), 5′-CTAATGTGTCCTTGAAGCAACC-3′ (reverse); Snail: 5′-CTTCCAGCAGCCCTACGAC-3′ (forward), 5′-CGGTGGGGTTGAGGATCT-3′ (reverse); ZEB1: 5′-AGCAGTGAAAGAGAAGGGAATGC-3′ (forward), 5′-GGTCCTCCTCAGGTGCCTCAG-3′ (reverse); 18SrRNA, 5′-GTAACCCGTTGAACCCCATT-3′ (forward), 5′-CCATCCAATCGGTAGTAGCG-3′ (reverse). 18S rRNA was used as internal control, and 2−ΔΔCT method was applied to analyze expression of target genes.
Small interfering RNA (siRNA) transfection and the construction of linc00261 overexpression cell lines
Liver cancer cell lines were seeded in the 6-well plates. Then the cells were washed three times with PBS and transfected with siRNA using lipofectamine 3000 (Invitrogen), and incubated for 48 h. The siRNA sequences for linc00261 were as follow: si-linc00261-1:5′-GAAAGCTGTAGCCATTCAA-3′, si-linc00261-2:5′-GCAATTAATTCAGGACACT-3′. The linc00261 overexpression lentivirus was constructed and bought from Genechem (Shanghai, China), and the construction of SMMC-7721-linc00261 overexpression model has been introduced in our previous research [
23].
Western blotting
RIPA lysis buffer (Beyotime, Shanghai, China) containing protein inhibitor, Phenylmethanesulfonyl fluoride (Beyotime), and the BCA kit (Beyotime) was used to determine the protein concentration after collecting the supernatant. The lysed proteins were separated on an SDS-PAGE gel and transferred to a PVDF membrane for immunoblotting analysis. The membranes were immersed in TBST solution containing 5% nonfat milk at room temperature for half an hour, incubated with the primary antibodies (Additional file
1: Table S2) overnight at 4 °C, and incubated with horse radish peroxidase-conjugated goat anti-rabbit secondary IgG antibody at room temperature for 1 h. Finally, the expression of proteins were detected using ECL substrate kit (Fdbio Science, Hangzhou, China) and FluorChem E system (ProteinSimple, CA, USA).
Transwell migration and invasion assays
Cells were suspended in medium without FBS and a total of 1 × 105cells were then added to the upper chambers, which were pre-coated with (for invasion assay) or without (for migration assay) Matrigel (BD Biosciences). Medium supplemented with 20% FBS was added to the lower chamber. Cells were cultured at 37 °C for another 48 h, after which, the migrated/invaded cells were fixed with 4% paraformaldehyde and stained using 0.5% crystal violet (Boster Biological Technology, Wuhan, China) at room temperature for 30 min. After washing with PBS, the chambers were air-dried and observed under an inverted light microscope (Olympus, Tokyo, Japan).
Tumor-sphere culture
The tumor-sphere system mainly consisted of serum-free DMEM/F12 supplemented with 10 μl/ml B27 (Gibco)), 20 ng/ml of epidermal growth factor (EGF), 10 ng/ml of basic fibroblast growth factor (bFGF). Five hundred cell were seeded in a non-adherent 6-well plates (Corning) and maintained for 2 weeks. The non-adherent spheroid clusters (diameter ≥ 20 μm) [
26] were observed under an inverted microscope (Olympus).
Immunofluorescence staining
HCC cells seeded on coverslips were washed 3 times with PBS, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.3% Triton X-100 at room temperature for 40 min for nuclear proteins. Then, the cells were blocked with 5% BSA for 30 min and stained with primary antibodies (Additional file
1: Table S2) overnight at 4 °C (Additional file
1: Table S1). After washing with PBS and incubation with Alexa fluor 594-conjugated goat-anti rabbit secondary antibody (Proteintech) at dark room for 1 h, the cells were incubated with 0.1% 4′,6-diamidino-2-phenylindole (DAPI) for 5 min, washed with PBS, and then observed under an inverted fluorescence confocal microscope (Olympus).
Immunohistochemistry (IHC) analysis
After deparaffinization, the tissue sections (3 μm) were immersed in 10m Mcitrate buffer (pH 6.0) and subjected to microwave treatment for 15 min for antigen retrieval. The samples were subsequently immersed in 3% H
2O
2 for 30 min to block endogenous peroxidase, and then incubated with primary antibodies (Additional file
1: Table S2) at 4 °C overnight. The next day, the sections were incubated with horseradish peroxidase-conjugated goat-anti rabbit secondary antibody (ZSGB-BIO, Beijing, China), and developed with peroxidase substrate diaminobenzidine (DAB; ZSGB-BIO). Finally, the expression of proteins was observed and evaluated semi-quantitatively under an upright microscope (Olympus) as we previously reported.
Patients specimens
HCC tissues and corresponding adjacent non-tumorous (NT) tissues were collected from Nanfang Hospital, Southern medical university between November 2010 and November 2016 in Nanfang Hospital. In paired HCC tissues, the relative linc00261 expression were analyzed by qRT-PCR, and IHC staining of E-cadherin, CD44, CD133, SMAD3 and p-SMAD3 was conducted.
In vivo tumorigenicity
The animal experimental procedures were conducted strictly in accordance with the Guide for the Care and Use of Laboratory Animals. Male NSG mice (2-4 weeks) were bought from Biocytogen (BeiJing, China). A total of 5 × 106 SMMC-7721 cells transfected with linc00261 overexpression or Vector lentivirus were subcutaneously implanted in the same mouse at different side. Tumor growth was recorded every 3 days, and the mice were sacrificed at the 18th day after injection.
Statistical analysis
The statistical significance was determined by Student’s test (unpaired) or one-way ANOVA followed by a post hoc test when appropriate. Data were expressed as mean ± SD, and P value of 0.05 or less was considered significant. IBM SPSS 20.0 or GraphPad prism 5 software was used for the statistical analysis.
Discussion
TGF-β1 had been reported to be a key factor associated with tumor EMT and stemness, leading to tumor metastasis [
26,
28]. In this study, we identified that linc00261 was down-regulated after TGF-β1 treatment, and linc00261 attenuated EMT and stem-like traits in liver cancer cells. Mechanistically, linc00261 facilitates SMAD3 degradation through ubiquitin–proteasome pathway and SMAD3 phosphorylation, thereby inhibiting HCC metastasis.
It’s well known that LncRNAs function as tumor suppressors or promoters through regulating EMT and CSCs by targeting multiple signaling pathways, including TGF-β1 pathway [
29]. The function of linc00261 has been investigated in multiple cancers, it suppressed lung and gastric cancer progression and metastasis by attenuated EMT [
25,
30], and function as a tumor suppressor in varies of human cancers by sponging with miRNA or affecting pathways [
31‐
33]. In contrast, Gao et al. found that linc00261 was at high expression in cholangiocarcinoma, and its higher expression predicted a poorer prognosis [
34]. Our preview study had demonstrated that patients with low expression of linc00261 had a poor progression in HCC, and cells after linc00261 knockdown had increased migratory and invasive capabilities [
22]; moreover, our another study revealed that linc00261 suppresses the formation of microvascular invasion, EMT, and metastasis of HCC through transcriptional upregulation of FOXA2 by recruiting SMAD3 to the FOXA2 promotor regions [
23]. LncRNAs can act as cis or trans to regulate genes expression in a precise temporal and spatial manners [
35]. Considering the close relation of linc00261 with SMAD3, and the observed effect of linc00261 on EMT and CSCs traits, we further investigated whether linc00261 was involved in TGF-β1-regulated progression of HCC.
Interestingly, we observed that linc00261 was significantly down-regulated after treatment with TGF-β1, which is consistent with TGF-β1 induced-suppression of linc00261/Foxa2 in lung cancer cells [
19]. According to our findings, overexpression of linc00261 induced an epithelial-like appearance, inhibited the tumor spheres formation, and also abolished TGF-β1-induced EMT, migration, and invasion in SMMC-7721; moreover, both linc00261 knockdown and overexpression affect the mRNA and protein expressions of the EMT-TFs (Slug and ZEB1) and CSCs-TFs (OCT4 and SOX2), the core downstream targets of TGF-β1 pathway, besides, the activated TGF-β1 signaling after linc00261 knockdown was blocked by TGF-βR inhibitor, SB431542. All these results demonstrated that linc00261 down-regulation is necessary for TGF-β1-induced EMT, and even CSCs traits acquisition. However, the exact mechanism that TGF-β1 suppresses linc00261 expression, and even TGF-β1 could forms a feedback loop with linc00261, still need further investigation.
TGF-β1/Smad signaling has a dual role among the tumor igenicity depending on cellular context and tumor stages [
36,
37]. After TGF-β signaling activation, SMAD2 and SMAD3 acquire phosphorylation and then translocate into nucleus, thereafter combined with SMAD4 to trans activate downstream target genes [
38,
39]. The functions of SMAD3 in HCC were still controversial; some reports showed that it was a tumor suppressor, while others proposed that it was a tumor promoter. A recent study had demonstrated that SMAD3 could promote migration, invasion, and metastasis of HCC cells in vitro and in vivo, binding directly to PTPRε promoters to activate its expression, and then feedback to activate TGF-β1/SMAD3 signaling to promote HCC metastasis [
40]. However, our preview study indicated that linc00261 guides SMAD3 protein to the promoter region of FOXA2 genome to enhance its transcription, thereafter contributes to the prevention of HCC progression [
23]. Herein, we found that linc00261 decreases TGF-β1-induced upregulation of SMAD3 and
p-SMAD3; furthermore, linc00261 promotes the degradation of SMAD3 by ubiquitin–proteasome pathway and inhibits the phosphorylation of SMAD3 in vitro and in vivo. These results indicated that linc00261 inhibits TGF-β/SMAD3 signaling to prevent the progression of HCC, which could be a strong evidence supporting the oncogenic role of SMAD3.
The intracellular regulators of TGF-β1 signaling includes Smad-dependent and -independent pathways, the former is known as receptor-regulated SMADs (SMAD1, 2, 3, 5, and 8), inhibitory SMADs (SMAD 6 and 7), and common mediator SMADs. The inhibitory SMADs antagonize the receptor-regulated SMADs’ activity by interacting with TGF-βR1, and then prevent the R-SMADs from phosphorylation, followed with degradation via the ubiquitin proteasome pathway [
41]. Therefore, linc00261 could interact with inhibitory SMADs, or potentially E3 ubiquitin ligase and phosphorylases to suppress SMAD3 phosphorylation and accelerate ubiquitin proteasome pathway-based degradation. Besides, our previous study demonstrated a lower expression of linc00261 in cytoplasm compared to nucleus in liver cancer cell lines [
23], which supports the current phenomenon that reduced linc00261, especially cytoplasmic linc00261, allows SMAD3 to get phosphorylation (Ser423/425), and escape from ubiquitination and degradation, thereby translocate into the nucleus, and ultimately promote the progression of HCC.
In conclusion, our results demonstrated that TGF-β1-induced deficiency of linc00261 facilitates EMT and stemness via inhibiting SMAD3 in HCC. It is the first study to reveal the inhibitory role of linc00261 on TGF-β1/SMAD3 signaling, providing a novel mechanism underlying TGF-β1-induced EMT and stem-like traits in HCC. Also, our work provides a new potential therapeutic target for the treatment of HCC.
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