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MicroRNA-520g induces epithelial–mesenchymal transition and promotes metastasis of hepatocellular carcinoma by targeting SMAD7
FEBS Letters 589 (2015) 102–109
journal homepage: www.FEBSLetters.org
MicroRNA-520g induces epithelial–mesenchymal transition and promotes metastasis of hepatocellular carcinoma by targeting SMAD7 Heping Kan a, Wenbin Guo b,1, Yuqi Huang a,⇑, Dingli Liu c a b c
Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, No. 1838 Guangzhou Road North, Guangzhou 510515, China Department of Urology, The Third Affiliated Hospital, Southern Medical University, No. 183 Zhongshan Road West, Guangzhou 510000, China Department of Infectious Disease, Nanfang Hospital, Southern Medical University, No. 1838 Guangzhou Road North, Guangzhou 510515, China
a r t i c l e
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Article history: Received 19 October 2014 Revised 18 November 2014 Accepted 19 November 2014 Available online 29 November 2014 Edited by Tamas Dalmay Keywords: MicroRNA-520g Hepatocellular carcinoma Epithelial–mesenchymal transition SMAD family member 7 Cancer metastasis
a b s t r a c t Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Aberrant expression of miRNAs contributes to HCC development. Here, we observed elevated miR-520g expression in tumor samples from HCC patients with relapse and metastasis, and this high miR-520g expression was correlated with poor survival. Through gain- and loss-of-function studies, miR-520g was demonstrated to facilitate HCC cell migration, invasion and epithelial–mesenchymal transition (EMT). SMAD7 was identified as a direct target of miR-520g. Accordingly, we conclude that high miR-520g expression promotes HCC cell mobility and EMT by targeting SMAD7, and this is correlated with reduced survival in HCC patients. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction MicroRNAs (miRNAs) are an abundant group of endogenous non-coding single strand RNAs of 22 nucleotides [1]. They regulate gene expression at post-transcriptional level by translational repression or degradation of target mRNA. In this manner, they participate in various biological process including cell development [2,3], cell cycle [4], and stem cell renewal [5]. Aberrant expression of miRNAs plays a key role in cancer development and progression through modulating oncogenic and tumor suppressor pathways. Using microarray or real-time polymerase chain reaction (PCR), miRNA expressing profiles which can differentiate cancer and normal tissues have been identified [6]. Moreover, different types of cancers have varied expression pattern of miRNAs.
Abbreviations: HCC, hepatocellular carcinoma; EMT, epithelial–mesenchymal transition; SMAD7, SMAD family member 7; ER, estrogen receptor; PR, progesterone receptor ⇑ Corresponding author. Fax: +86 02062787180. E-mail addresses: [email protected] (H. Kan), [email protected] (W. Guo), [email protected] (Y. Huang), [email protected] (D. Liu). 1 Co-first author.
Due to its high stability in serum, miRNAs are becoming promising diagnostic and prognostic biomarkers for patients with malignancy [7,8]. MiR-520g has been proposed as a novel tumor-related miRNA and has been found to be significantly deregulated in tumors. Up-regulated miR-520g in estrogen receptor (ER) and progesterone receptor (PR)-negative tumors indicated that it was a maker for poorer prognosis breast cancer and an attractive target for therapeutic intervention [9]. Furthermore, miR-520g has been considered as an oncogene in glioma, medulloblastoma and other neuroectodermal tumors [10,11]. Recently, the deregulated miRNAs in hepatitis B virus-associated hepatocellular carcinoma (HCC) were identified by Wang et al. [12,13]. They found that the expression of miR-520g was upregulated in HCC tissues as compared with that in adjacent non-tumor tissues. Notably, Zhou et al. reported that the expression of miR-520g was significantly higher in a highly invasive HCC cell line HCCLM3 than that in a low metastatic HCC cell line MHCC97L [14]. Thus, these promoted us to investigate the clinical significance of miR-520g in HCC and its role in cell migration and invasion. In this study, we demonstrate that up-regulation of miR-520g is observed in the aggressive and metastatic phenotype of HCC. Its
http://dx.doi.org/10.1016/j.febslet.2014.11.031 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
H. Kan et al. / FEBS Letters 589 (2015) 102–109
high-expression is associated with poor prognostic features and short survival of HCC patients. In vitro studies demonstrated that miR-520g facilitated HCC cell migration, invasion and epithelialmesenchymal transition (EMT). Furthermore, SMAD family member 7 (SMAD7) was identified as a direct target of miR-520g. Mechanistically, our results demonstrate that miR-520g potentiates the invasive behavior of HCC cells and contributes to tumor metastasis by inhibiting SMAD7 and promoting EMT. 2. Materials and methods 2.1. Clinical samples 130 HCC samples were collected from patients including 98 males and 32 females, who underwent the resection of their primary HCC in the Department of Hepatobiliary Surgery at the Nanfang Hospital of Southern Medical University during January 2006 to December 2008. The clinicopathological data are shown in Table 1. All samples were used after obtaining informed consent. Patients did not receive preoperative chemotherapy or embolization. In these cases, 34 patients had intrahepatic spreading, venous infiltration or tumor invasion into bile ducts, and 66 patients had tumor recurrence.
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2.3. Cell lines and transfection The human immortalized normal hepatocyte cell line, LO2, and six HCC cell lines, HepG2, MHCC97L, Hep3B, Huh7, MHCC97H and HCCLM3 (the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China), were cultured in complete Dulbecco’s modified Eagle medium (DMEM, Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) with 100 units/ml penicillin and 100 lg/ml streptomycin (Sigma, St-Louis, MO, USA) in a humidified containing of 5% CO2 incubator at 37 °C. MiRNA vectors, including miR-520g expression vector (HmiR0334-MR04), the control vector for miR-520g (CmiR0001MR04), miR-520g inhibitor (HmiR-AN0603-AM04) and the negative control for the miR-520g inhibitor (CmiR-AN0001-AM04), were purchased from Genecopoeia (Guangzhou, China). Plasmids carrying wt SMAD7 (pCMV5-SMAD7) were obtained from Addgene (Cambridge, MA, USA). The targeted sequences for SMAD7 siRNA duplex (50 -AA GCU CAA UUC GGA CAA CAA G-30 ) or a non-specific duplex oligonucleotide as a negative control were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Cells were transfected with the vectors mentioned above using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA).
2.2. Real time quantitative reverse transcription-PCR (qRT-PCR) 2.4. Boyden chamber and Transwell assays The PCR amplification for the quantification of the miR-520g and U6 was performed using TaqMan miRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) and TaqMan Human MiRNA Assay Kit (Applied Biosystems, Foster City, CA, USA). The relative expression of miR-520g was shown as fold difference relative to U6. Primer pairs against Homo sapiens gene SMAD7 (HP208950) and GAPDH (HP205798) were purchased from OriGene (Beijing, China). The PCR amplification for the quantification of the SMAD7 and GAPDH mRNAs was performed using an ABI PRISM 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) and a SYBRÒ Premix Ex Taq™ ii (Perfect Real Time) Kit (Takara Bio, Shiga, Japan), as previous reported [15].
Age (y) Sex HBV Serum AFP level (ng/ml) Tumor size (cm) No. of tumor nodules Cirrhosis Venous infiltration EdmondsonSteiner grading TNM tumor stage
<50 P50 Male Female Absent Present <400 P400 <5 P5 1 P2 Absent Present Absent Present I + II III + IV I + II III + IV
Total No. of patients, n = 130
No. of patients miR520ghigh
miR520glow
54 76 98 32 42 88 48 82 49 81 102 28 54 76 96 34 98 32 97 33
29 36 53 12 20 45 19 46 21 44 47 18 25 40 41 24 43 22 40 25
25 40 45 20 22 43 29 36 28 37 55 10 29 36 55 10 55 10 57 8
2.5. Western blot
0.477
The following primary antibodies were used in the immunoblotting assays: SMAD7 (H-79, sc-11392, Santa Cruz, CA, USA), E-cadherin (24E10, #3195, Cell Signaling Technology, Danvers, MA, USA), vimentin (R28, #3932, Cell Signaling Technology, Danvers, MA, USA) and GAPDH (G8140; US Biological, Swampscott, MA, USA). Horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibodies (Bio-Rad, Hercules, CA, USA) were used at a 1:1000–1:5000 dilution and detected using a Western Blotting Luminol Reagent (sc-2048; Santa Cruz, CA, USA), as described in our previous study [15].
0.103
2.6. Immunohistochemical staining
Table 1 Correlation between the clinicopathologic characteristics and miR-520g expression in HCC. Characteristics
A Boyden chamber assay (NeuroProbe, Gaithersburg, MD, USA) was used to analyze HCC cell migration as previously described [16]. Transwell assays were done in 6 well plates with Transwell inserts equipped with 8-lm pores (Nalge Nunc International Corp, Naperville, IL, USA) coated with matrigel at 1:6 dilution (Becton Dickinson Labware, Bedford, MA, USA) as previously described [17].
P
0.708
0.477
Immunohistochemistry was performed on paraformaldehydefixed paraffin sections. SMAD7 (H-79, sc-11392, Santa Cruz, CA, USA) antibody was used in immunohistochemistry with streptavidin peroxidase conjugated (SP-IHC) method. Immunohistochemistry was performed as previous reported [15]. The percentage of positive tumor cells was graded as per the following criteria: 0, less than 10%; 1, 10–30%; 2, 31–50%; 3, more than 50%.
0.005*
2.7. Luciferase reporter assay
0.069 0.205 0.088
0.015* *
0.001
HCC, hepatocellular carcinoma; HBV, hepatitis B virus; AFP, alpha-fetoprotein; TNM, tumor-node-metastasis. * Statistically significant.
The 30 -untranslated region (UTR) sequence of SMAD7 predicted to interact with miR-520g or a mutated sequence within the predicted target sites was synthesized and inserted into the XbaI and FseI sites of the pGL3 control vector (Promega, Madison, WI, USA). These constructs were named as wt SMAD7-30 UTR or mt SMAD7-30 UTR, respectively. For the reporter assay, HepG2 cells
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were seeded into 24-well plates and transfected with the above constructs and miR-520g expression vector, miR-520g inhibitor, control vector or negative control. After 48 h, the cells were harvested and Renilla luciferase activity was measured using the dual-luciferase reporter assay system (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Results were obtained from three independent experiments performed in duplicate. 2.8. Statistical analysis Results are expressed as mean ± S.E.M. Significance was established, with the SPSS statistical package for Windows Version 13 (SPSS, Chicago, IL, USA) and GraphPad Prism 5 software (GraphPad Software, Inc, San Diego, CA, USA), using a Pearson chi-squared test, a Spearman’s rank correlation coefficient, a two-tailed Student’s t test, a Kaplan–Meier plot, a log-rank test or an ANOVA when appropriate. Difference were considered significant when P < 0.05. 3. Results 3.1. Up-regulation of miR-520g is associated with metastasis and recurrence of HCC We tested the expression of miR-520g by qRT-PCR and normalized against an endogenous control (U6 RNA) in 40 pairs of tumor tissues and matched adjacent non-tumor tissues from HCC patients who received liver resection. The expression level of miR-520g in HCC tissues was significantly higher than that in matched adjacent non-tumor liver tissues (P < 0.05, Fig. 1A). HCC cases that showed intrahepatic spreading, venous infiltration or tumor invasion into bile ducts were considered as aggressive HCC tissues. As compared with non-aggressive HCC tissues, miR-520g levels were prominently up-regulated in aggressive HCC tissues (P < 0.05, Fig. 1B). Furthermore, miR-520g levels were obviously increased in tumor tissues arising from patients with
intrahepatic tumor recurrence or extrahepatic metastasis as compared with those in tumor tissues arising from patients without tumor recurrence (P < 0.05, Fig. 1C). Thus, up-regulation of miR520g level was correlated with metastasis and recurrence of HCC. Next, we analyzed miR-520g expression in a non-transformed hepatic cell line (LO2) and a panel of HCC cell lines (HepG2, MHCC97L, Huh7, Hep3B, MHCC97H and HCCLM3). The miR-520g expression was significantly up-regulated in all HCC cell lines as compared with that in LO2 (P < 0.05, Fig. 1D). Moreover, miR520g expression in the highly metastatic HCC cell lines, MHCC97H and HCCLM3, were obviously higher than those in the low metastatic HCC cell lines including HepG2, MHCC97L, Huh7 and Hep3B (P < 0.05, Fig. 1D). These data indicates that elevated miR-520g expression confers increased metastatic potential of HCC cells. 3.2. Clinical significance of miR-520g expression in HCC cases 130 samples of HCC specimens were subjected to qRT-PCR for miR-520g expression. The expression of miR-520g was considered as either low (n = 65) or high (n = 65) according to the cutoff value, which was defined as the median of the cohort. As shown in Table 1, miR-520g was expressed at prominently higher levels in HCC patients with venous infiltration (P = 0.005), high Edmondson-Steiner grading (P = 0.015) and advanced TNM tumor stage (P = 0.001). Furthermore, 130 HCC patients with clinical survival information (with a median follow-up time of 37.5 months) were analyzed by Kaplan Meier estimation. Tumors with high expression of miR-520g indeed associated with worse overall survival and disease-free survival of HCC patients (P < 0.001, respectively, Fig. 2). These data indicate that miR-520g may act as a potent biomarker for predicting prognosis of HCC patients. 3.3. MiR-520g is correlated with EMT phenotype and invasion ability in HCC cells We transduced HCC cell line, HCCLM3, with miR-520g inhibitor (anti-miR-520g) or negative control (NC). As measured by
Fig. 1. The expression levels of miR-520g in HCC tissues and cells. Comparing differences in the expression levels of miR-520g between (A) HCC and matched non-tumor tissues, (B) aggressive and non-aggressive tumor tissues, (C) HCC tissues arising from recurrent and non-recurrent groups, and (D) HCC cell lines with different metastatic potentials and the immortalized hepatic cell line LO2. ⁄P < 0.05.
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Fig. 2. Prognostic significance of miR-520g in HCC cases. According to the level of miR-520g expression, Kaplan-Meier 5-year overall and disease-free survival curves of HCC patients showed that high expression of miR-520g was correlated with poor prognosis. The median expression value obtained for miR-520g of the 130 HCC samples detected by qRT-PCR was chosen as the cutoff value.
Fig. 3. MiR-520g regulates EMT phenotype and HCC cell mobility. (A) Cell migration and invasion as measured by Boyden chamber and Transwell assays were inhibited by down-regulation of miR-520g in HCCLM3 cells as compared with control cells. n = 3 repeats with similar results; ⁄P < 0.05. (B) miR-520g overexpressing HepG2 cells conferred a higher number of migrated and invaded cells as compared with control cells. n = 3 repeats with similar results; ⁄P < 0.05. (C) Representative Western blot analysis of Ecadherin and vimentin in HCCLM3 cells with miR-520g inhibitor transfection and HepG2 cells with miR-520g transfection was shown. n = 6; ⁄P < 0.05.
qRT-PCR, the expression of miR-520g was significantly downregulated by miR-520g inhibitor in HCCLM3 cells (P < 0.05, data not shown). Boyden chamber assays were performed to test the effect of altering miR-520g levels on HCC cell migration. We found that down-regulation of miR-520g led to a significant reduction of cell migration in HCCLM3 cells (P < 0.05, Fig. 3A). Furthermore, as determined by Transwell assays, the number of invaded HCCLM3
cells was significantly reduced after reduction of miR-520g (P < 0.05, Fig. 3A). Next, miR-520g overexpressing HepG2 cells was established and confirmed by qRT-PCR (P < 0.05, data not shown). As expected, up-regulation of miR-520g obviously promoted HCC cell migration and invasion (P < 0.05, respectively, Fig. 3B). Furthermore, suppression of EMT in HCCLM3 cells with down-regulation of miR-520g was associated with elevated
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expression of E-cadherin and reduced expression of vimentin (Fig. 3C). Otherwise, up-regulation of miR-520g-induced EMT was associated with reduced expression of E-cadherin and elevated expression of vimentin in HepG2 cells. These data indicate that miR-520g is associated with EMT phenotype and cell mobility in HCC cell lines. 3.4. SMAD7 is identified as a functional target of miR-520g and is down-regulated in HCC specimens To disclose the molecular mechanisms by which miR-520g induces EMT in HCC, predicted target genes of miR-520g were retrieved and analyzed using publicly available databases (TargetScan 6.2 and MiRanDa). Among these, EMT-associated genes were selected for further investigating. Significantly, SMAD7, which is known to inhibit EMT and tumor metastasis by blocking TGFb signaling [18,19], was predicted as one of the targets of miR-520g. HepG2 cells that were transfected with miR-520g and control vectors were subjected to qRT-PCR and Western blot for SMAD7 expression. Both SMAD7 mRNA and protein levels were significantly reduced by up-regulation of miR-520g in HepG2 cells (P < 0.05, respectively, Fig. 4A and B). To further demonstrate that SMAD7 is directly targeted by miR-520g in HCC cells, we investigated whether the miR-520g directly interacted with the 30 -UTR of SMAD7 mRNA using a dual-luciferase reporter assay. As expected, miR-520g significantly inhibited the luciferase activity of SMAD7 containing a wild-type (wt) 30 -UTR but did not suppress activity of SMAD7 with a mutant (mt) 30 -UTR (P < 0.05, Fig. 4C and D). When anti-miR-520g was transfected, an increase in luciferase activity of wt SMAD7 30 -UTR was observed. However, with the mt SMAD7 30 -UTR constructs, there was no relative increase in activity (P < 0.05, Fig. 4C and D). Thus, our data strongly suggest that SMAD7 is a target of miR-520g in HCC. Expression of SMAD7 was further detected by qRT-PCR and immunohistochemistry in the previous cohort of 40 pairs of HCC and normal tumor-adjacent tissues. Both SMAD7 mRNA and protein levels were demonstrated to be significantly lower in HCC tissues as compared with those in non-cancerous tissues (P < 0.05,
respectively, Fig. 5A and B). 130 samples of HCC tissues were subjected to immunostaining for SMAD7 expression. SMAD7 immunoreactivity was considered as either negative (score 0) or positive (scores 1-3). In these cases, SMAD7 expression was detected in 46.2% (30/65) of the HCC samples with low expression of miR520g, whereas only 20.0% (13/65) of the HCC specimens with high expression of miR-520g showed a positive SMAD7 signal (P < 0.05, Fig. 5C). Furthermore, Spearman correlation analysis indicated that miR-520g was inversely correlated with SMAD7 expression in HCC tissues (r = 0.7362, P < 0.001). 3.5. MiR-520g promotes EMT and HCC cell mobility by targeting SMAD7 To further confirm the role of SMAD7 in miR-520g-mediated EMT and cell mobility in HCC cells, we restored the expression of SMAD7 in miR-520g overexpressing HepG2 cells by transfecting pCMV5-SMAD7 plasmids. As confirmed by immunoblotting, restoration of SMAD7 suppressed EMT in miR-520g overexpressing HepG2 cells and was correlated with up-regulation of epithelial biomarker, Ecadherin, and reduced expression of mesenchymal biomarker, vimentin (P < 0.05, respectively, Fig. 6A). Furthermore, the migration and invasion abilities of miR-520g overexpressing HepG2 cells was partially abolished after pCMV5-SMAD7 transfection (P < 0.05, respectively, Fig. 6B), which was consistent with above results. Similarly, SMAD7 knockdown promoted the migration and invasion of miR-520g downregulating HCCLM3 cells (P < 0.05, respectively, Fig. 6C). All the data indicate that miR520g promotes EMT and cell mobility by targeting SMAD7 in HCC cells. 4. Discussion Increasing evidences have demonstrated that the deregulation and dysfunction of miRNAs play a critical role in the initiation and progression of HCC [20]. However, miRNAs that are involved in the recurrence and metastasis of HCC remain poorly investigated. In this study, we initially detected the expression level of
Fig. 4. SMAD7 is identified as a functional target of miR-520g in HCC. (A) qRT-PCR and (B) Western blot analysis of SMAD7 expression in HepG2 cells with miR-520g or control vectors transfection. n = 6; ⁄P < 0.05. (C) miR-520g and its putative binding sequence in the 30 -UTR of SMAD7. The mutant miR-520g binding site was generated in the complementary site for the seed region of miR-520g (wt, wild type; mt, mutant type). (D) MiR-520g significantly suppressed the luciferase activity that carried wt but not mt 30 -UTR of SMAD7. Anti-miR-520g led to a noticeable increase in luciferase activity of wt 30 -UTR of SMAD7. n = 3 repeats with similar results; ⁄P < 0.05.
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Fig. 5. An inverse correlation between miR-520g and SMAD7 expression is observed in HCC. Comparing differences in the expression levels of SMAD7 (A) mRNA and (B) protein between HCC and matched non-tumor tissues. ⁄P < 0.05. (C) Representative immunostaining showed negative expression of SMAD7 in miR-520 high-expressing HCC tissue and positive expression of SMAD7 in miR-520 low-expressing tumor. A significant inverse correlation between miR-520g and SMAD7 expression was observed in HCC tissues. Scale bar: 100 lm; ⁄P < 0.05.
miR-520g in 40 samples of HCC and matched normal tumoradjacent tissues. Our data indicated that the expression of miR520g in HCC tissues was obviously higher than that in non-tumor tissues. Moreover, miR-520g was expressed at prominent higher levels in aggressive tumor tissues as compared with that in nonaggressive tumor tissues. Importantly, our results showed that high expression of miR-520g conferred a significant higher recurrence rate for HCC patients. Clinical analysis found that high expression of miR-520g was significantly correlated with venous infiltration, high Edmondson-Steiner grading and advanced TNM tumor stage in HCC. Kaplan–Meier’s plots demonstrated that high expression of miR-520g was significantly associated with poor survival of HCC patients. Furthermore, HCC cell lines, especially high metastatic cell lines (MHCC97H and HCCLM3), showed obviously higher levels of miR-520g as compared with the non-transformed hepatic cell line LO2. Altogether, these results suggest that miR520g is critical for prognosis determination in HCC patients. Next, we further investigated the role of miR-520g in HCC cells. Boyden chamber and Transwell assays were performed to test HCC cell migration and invasion abilities. We found that downregulation of miR-520g inhibited cell migration and invasion in HCCLM3 cells. And up-regulation of miR-520g increased the number of migrated and invaded HepG2 cells. These data suggest that miR-520g indeed promotes HCC cell migration and invasion. EMT, a dynamic and reversible cellular process, is characterized by loss of cell polarity and intracellular junctions and acquirement
of mesenchymal features, resulting in increased HCC cell migration and invasion [21]. Thus, we determined the expression of epithelial marker, E-cadherin, and mesenchymal marker, vimentin in HCC cells with altering expression of miR520g. Interestingly, we demonstrated that down-regulation of miR-520g inhibited EMT and was associated with elevated expression of E-cadherin and reduced expression of vimentin in HCCLM3 cells. Meanwhile, up-regulation of miR-520g promoted EMT and was associated with reduced expression of E-cadherin and elevated expression of vimentin. Taken together, miR-520g may promote HCC cell migration and invasion by inducing EMT. Predicted target genes of miR-520g were retrieved and analyzed using publicly available databases (TargetScan and Miranda). The putative binding sequences of miR-520g were found in the 30 -UTR of SMAD7. SMAD7 is a member of the SMAD family of proteins, which belong to the transforming growth factor (TGF)-b super family of ligands [18]. SMAD7 is involved in cell signaling and is a TGFb receptor I (TGFbRI) antagonist that blocks TGFb1 [22,23]. Smith et at found that miR-106b-25 cluster activated TGF-b signaling pathway through down-regulation of SMAD7 to induce EMT and tumor-initiating cell characteristics in human breast cancer [19]. Notably, recent study reported that miR-216a/ 217 induced EMT by activating the TGF-b signaling pathway by targeting SMAD7 in HCC [18]. We investigated the regulatory effect of miR-520g on SMAD7 in HCC cells. Our data indicated that up-regulation of miR-520g reduced the expression levels of SMAD7
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Fig. 6. MiR-520g induces HCC cell migration, invasion and EMT by targeting SMAD7. (A) Western blot analysis of the expression of E-cadherin and vimentin in pCMV5SMAD7 transfected miR-520g overexpressing HepG2 cells. n = 6; ⁄P < 0.05. (B) Migration and invasion abilities of the stable transfected cells following restoration of SMAD7 in miR-520g overexpressing HepG2 cells were analyzed by Boyden chamber and Transwell assays. n = 3 repeats with similar results; ⁄P < 0.05. (C) SMAD7 knockdown by a specific siRNA promoted the migration and invasion of miR-520g downregulating HCCLM3 cells. n = 3 repeats with similar results; ⁄P < 0.05.
mRNA and protein in HepG2 cells. Herein, we validated SMAD7 as a direct functional target of miR-520g in HCC. Furthermore, expression of SMAD7 in HCC tissues was obviously lower as compared with that in matched normal non-tumor tissues, which was consistent with previous studies [18]. Otherwise, a significant inverse correlation between miR-520g and SMAD7 expression was observed in HCC tissues. Importantly, the regulatory effects of altered expression of miR-520g on EMT and cell mobility were partially abolished by SMAD7 in HCC cells. Accordingly, we suggest that miR-520g promotes EMT and cell mobility by targeting SMDA7 in HCC. In the previous paper, miR-520c expression reduced cell invasiveness and had a tumour suppressive role in breast cancer, and that this was attributed to both the NF-jB and TGF-b/Smad2/3 pathways [24]. MiR-520c has the same seed region as miR-520g and yet high expression appears to result in reduced invasiveness in breast cancer. Interestingly, Toffanin et al. reported that the expression of miR-520c increased proliferation, migration, and invasion of HCC cells in vitro [25]. Therefore, the functional significance of miRNAs in cancer development and progression seem to be cancer-type specific. In conclusion, we find that miR-520g is up-regulated in HCC and its high expression is related to poor prognostic features. Moreover, high expression of miR-520g is a prognostic marker for
predicting poor survival of HCC patients. In vitro studies demonstrate that miR-520g induces HCC cell migration, invasion and EMT. Mechanistically, we suggest that miR-520g promotes EMT and HCC cell mobility by suppressing SMAD7. Taken together, we consider that miR-520g may potentially act as a clinical biomarker, and may also be a therapeutic target, in HCC. Acknowledgments This study was supported by grants from the Natural Science Foundation of Guangdong Province [Nos. S2012020010942 and 8151051501000029]. References [1] Garzon, R., Calin, G.A. and Croce, C.M. (2009) MicroRNAs in cancer. Annu. Rev. Med. 60, 167–179. [2] Rosa, A. and Brivanlou, A.H. (2009) MicroRNAs in early vertebrate development. Cell Cycle 8, 3513–3520. [3] Harfe, B.D. (2005) MicroRNAs in vertebrate development. Curr. Opin. Genet. Dev. 15, 410–415. [4] Vasudevan, S., Tong, Y. and Steitz, J.A. (2007) Switching from repression to activation: microRNAs can up-regulate translation. Science 318, 1931–1934. [5] Croce, C.M. and Calin, G.A. (2005) MiRNAs, cancer, and stem cell division. Cell 122, 6–7.
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MicroRNA-520g induces epithelial–mesenchymal transition and promotes metastasis of hepatocellular carcinoma by targeting SMAD7 Heping Kan a, Wenbin Guo b,1, Yuqi Huang a,⇑, Dingli Liu c a b c
Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, No. 1838 Guangzhou Road North, Guangzhou 510515, China Department of Urology, The Third Affiliated Hospital, Southern Medical University, No. 183 Zhongshan Road West, Guangzhou 510000, China Department of Infectious Disease, Nanfang Hospital, Southern Medical University, No. 1838 Guangzhou Road North, Guangzhou 510515, China
a r t i c l e
i n f o
Article history: Received 19 October 2014 Revised 18 November 2014 Accepted 19 November 2014 Available online 29 November 2014 Edited by Tamas Dalmay Keywords: MicroRNA-520g Hepatocellular carcinoma Epithelial–mesenchymal transition SMAD family member 7 Cancer metastasis
a b s t r a c t Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Aberrant expression of miRNAs contributes to HCC development. Here, we observed elevated miR-520g expression in tumor samples from HCC patients with relapse and metastasis, and this high miR-520g expression was correlated with poor survival. Through gain- and loss-of-function studies, miR-520g was demonstrated to facilitate HCC cell migration, invasion and epithelial–mesenchymal transition (EMT). SMAD7 was identified as a direct target of miR-520g. Accordingly, we conclude that high miR-520g expression promotes HCC cell mobility and EMT by targeting SMAD7, and this is correlated with reduced survival in HCC patients. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction MicroRNAs (miRNAs) are an abundant group of endogenous non-coding single strand RNAs of 22 nucleotides [1]. They regulate gene expression at post-transcriptional level by translational repression or degradation of target mRNA. In this manner, they participate in various biological process including cell development [2,3], cell cycle [4], and stem cell renewal [5]. Aberrant expression of miRNAs plays a key role in cancer development and progression through modulating oncogenic and tumor suppressor pathways. Using microarray or real-time polymerase chain reaction (PCR), miRNA expressing profiles which can differentiate cancer and normal tissues have been identified [6]. Moreover, different types of cancers have varied expression pattern of miRNAs.
Abbreviations: HCC, hepatocellular carcinoma; EMT, epithelial–mesenchymal transition; SMAD7, SMAD family member 7; ER, estrogen receptor; PR, progesterone receptor ⇑ Corresponding author. Fax: +86 02062787180. E-mail addresses: [email protected] (H. Kan), [email protected] (W. Guo), [email protected] (Y. Huang), [email protected] (D. Liu). 1 Co-first author.
Due to its high stability in serum, miRNAs are becoming promising diagnostic and prognostic biomarkers for patients with malignancy [7,8]. MiR-520g has been proposed as a novel tumor-related miRNA and has been found to be significantly deregulated in tumors. Up-regulated miR-520g in estrogen receptor (ER) and progesterone receptor (PR)-negative tumors indicated that it was a maker for poorer prognosis breast cancer and an attractive target for therapeutic intervention [9]. Furthermore, miR-520g has been considered as an oncogene in glioma, medulloblastoma and other neuroectodermal tumors [10,11]. Recently, the deregulated miRNAs in hepatitis B virus-associated hepatocellular carcinoma (HCC) were identified by Wang et al. [12,13]. They found that the expression of miR-520g was upregulated in HCC tissues as compared with that in adjacent non-tumor tissues. Notably, Zhou et al. reported that the expression of miR-520g was significantly higher in a highly invasive HCC cell line HCCLM3 than that in a low metastatic HCC cell line MHCC97L [14]. Thus, these promoted us to investigate the clinical significance of miR-520g in HCC and its role in cell migration and invasion. In this study, we demonstrate that up-regulation of miR-520g is observed in the aggressive and metastatic phenotype of HCC. Its
http://dx.doi.org/10.1016/j.febslet.2014.11.031 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
H. Kan et al. / FEBS Letters 589 (2015) 102–109
high-expression is associated with poor prognostic features and short survival of HCC patients. In vitro studies demonstrated that miR-520g facilitated HCC cell migration, invasion and epithelialmesenchymal transition (EMT). Furthermore, SMAD family member 7 (SMAD7) was identified as a direct target of miR-520g. Mechanistically, our results demonstrate that miR-520g potentiates the invasive behavior of HCC cells and contributes to tumor metastasis by inhibiting SMAD7 and promoting EMT. 2. Materials and methods 2.1. Clinical samples 130 HCC samples were collected from patients including 98 males and 32 females, who underwent the resection of their primary HCC in the Department of Hepatobiliary Surgery at the Nanfang Hospital of Southern Medical University during January 2006 to December 2008. The clinicopathological data are shown in Table 1. All samples were used after obtaining informed consent. Patients did not receive preoperative chemotherapy or embolization. In these cases, 34 patients had intrahepatic spreading, venous infiltration or tumor invasion into bile ducts, and 66 patients had tumor recurrence.
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2.3. Cell lines and transfection The human immortalized normal hepatocyte cell line, LO2, and six HCC cell lines, HepG2, MHCC97L, Hep3B, Huh7, MHCC97H and HCCLM3 (the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China), were cultured in complete Dulbecco’s modified Eagle medium (DMEM, Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) with 100 units/ml penicillin and 100 lg/ml streptomycin (Sigma, St-Louis, MO, USA) in a humidified containing of 5% CO2 incubator at 37 °C. MiRNA vectors, including miR-520g expression vector (HmiR0334-MR04), the control vector for miR-520g (CmiR0001MR04), miR-520g inhibitor (HmiR-AN0603-AM04) and the negative control for the miR-520g inhibitor (CmiR-AN0001-AM04), were purchased from Genecopoeia (Guangzhou, China). Plasmids carrying wt SMAD7 (pCMV5-SMAD7) were obtained from Addgene (Cambridge, MA, USA). The targeted sequences for SMAD7 siRNA duplex (50 -AA GCU CAA UUC GGA CAA CAA G-30 ) or a non-specific duplex oligonucleotide as a negative control were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Cells were transfected with the vectors mentioned above using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA).
2.2. Real time quantitative reverse transcription-PCR (qRT-PCR) 2.4. Boyden chamber and Transwell assays The PCR amplification for the quantification of the miR-520g and U6 was performed using TaqMan miRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) and TaqMan Human MiRNA Assay Kit (Applied Biosystems, Foster City, CA, USA). The relative expression of miR-520g was shown as fold difference relative to U6. Primer pairs against Homo sapiens gene SMAD7 (HP208950) and GAPDH (HP205798) were purchased from OriGene (Beijing, China). The PCR amplification for the quantification of the SMAD7 and GAPDH mRNAs was performed using an ABI PRISM 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) and a SYBRÒ Premix Ex Taq™ ii (Perfect Real Time) Kit (Takara Bio, Shiga, Japan), as previous reported [15].
Age (y) Sex HBV Serum AFP level (ng/ml) Tumor size (cm) No. of tumor nodules Cirrhosis Venous infiltration EdmondsonSteiner grading TNM tumor stage
<50 P50 Male Female Absent Present <400 P400 <5 P5 1 P2 Absent Present Absent Present I + II III + IV I + II III + IV
Total No. of patients, n = 130
No. of patients miR520ghigh
miR520glow
54 76 98 32 42 88 48 82 49 81 102 28 54 76 96 34 98 32 97 33
29 36 53 12 20 45 19 46 21 44 47 18 25 40 41 24 43 22 40 25
25 40 45 20 22 43 29 36 28 37 55 10 29 36 55 10 55 10 57 8
2.5. Western blot
0.477
The following primary antibodies were used in the immunoblotting assays: SMAD7 (H-79, sc-11392, Santa Cruz, CA, USA), E-cadherin (24E10, #3195, Cell Signaling Technology, Danvers, MA, USA), vimentin (R28, #3932, Cell Signaling Technology, Danvers, MA, USA) and GAPDH (G8140; US Biological, Swampscott, MA, USA). Horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibodies (Bio-Rad, Hercules, CA, USA) were used at a 1:1000–1:5000 dilution and detected using a Western Blotting Luminol Reagent (sc-2048; Santa Cruz, CA, USA), as described in our previous study [15].
0.103
2.6. Immunohistochemical staining
Table 1 Correlation between the clinicopathologic characteristics and miR-520g expression in HCC. Characteristics
A Boyden chamber assay (NeuroProbe, Gaithersburg, MD, USA) was used to analyze HCC cell migration as previously described [16]. Transwell assays were done in 6 well plates with Transwell inserts equipped with 8-lm pores (Nalge Nunc International Corp, Naperville, IL, USA) coated with matrigel at 1:6 dilution (Becton Dickinson Labware, Bedford, MA, USA) as previously described [17].
P
0.708
0.477
Immunohistochemistry was performed on paraformaldehydefixed paraffin sections. SMAD7 (H-79, sc-11392, Santa Cruz, CA, USA) antibody was used in immunohistochemistry with streptavidin peroxidase conjugated (SP-IHC) method. Immunohistochemistry was performed as previous reported [15]. The percentage of positive tumor cells was graded as per the following criteria: 0, less than 10%; 1, 10–30%; 2, 31–50%; 3, more than 50%.
0.005*
2.7. Luciferase reporter assay
0.069 0.205 0.088
0.015* *
0.001
HCC, hepatocellular carcinoma; HBV, hepatitis B virus; AFP, alpha-fetoprotein; TNM, tumor-node-metastasis. * Statistically significant.
The 30 -untranslated region (UTR) sequence of SMAD7 predicted to interact with miR-520g or a mutated sequence within the predicted target sites was synthesized and inserted into the XbaI and FseI sites of the pGL3 control vector (Promega, Madison, WI, USA). These constructs were named as wt SMAD7-30 UTR or mt SMAD7-30 UTR, respectively. For the reporter assay, HepG2 cells
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were seeded into 24-well plates and transfected with the above constructs and miR-520g expression vector, miR-520g inhibitor, control vector or negative control. After 48 h, the cells were harvested and Renilla luciferase activity was measured using the dual-luciferase reporter assay system (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Results were obtained from three independent experiments performed in duplicate. 2.8. Statistical analysis Results are expressed as mean ± S.E.M. Significance was established, with the SPSS statistical package for Windows Version 13 (SPSS, Chicago, IL, USA) and GraphPad Prism 5 software (GraphPad Software, Inc, San Diego, CA, USA), using a Pearson chi-squared test, a Spearman’s rank correlation coefficient, a two-tailed Student’s t test, a Kaplan–Meier plot, a log-rank test or an ANOVA when appropriate. Difference were considered significant when P < 0.05. 3. Results 3.1. Up-regulation of miR-520g is associated with metastasis and recurrence of HCC We tested the expression of miR-520g by qRT-PCR and normalized against an endogenous control (U6 RNA) in 40 pairs of tumor tissues and matched adjacent non-tumor tissues from HCC patients who received liver resection. The expression level of miR-520g in HCC tissues was significantly higher than that in matched adjacent non-tumor liver tissues (P < 0.05, Fig. 1A). HCC cases that showed intrahepatic spreading, venous infiltration or tumor invasion into bile ducts were considered as aggressive HCC tissues. As compared with non-aggressive HCC tissues, miR-520g levels were prominently up-regulated in aggressive HCC tissues (P < 0.05, Fig. 1B). Furthermore, miR-520g levels were obviously increased in tumor tissues arising from patients with
intrahepatic tumor recurrence or extrahepatic metastasis as compared with those in tumor tissues arising from patients without tumor recurrence (P < 0.05, Fig. 1C). Thus, up-regulation of miR520g level was correlated with metastasis and recurrence of HCC. Next, we analyzed miR-520g expression in a non-transformed hepatic cell line (LO2) and a panel of HCC cell lines (HepG2, MHCC97L, Huh7, Hep3B, MHCC97H and HCCLM3). The miR-520g expression was significantly up-regulated in all HCC cell lines as compared with that in LO2 (P < 0.05, Fig. 1D). Moreover, miR520g expression in the highly metastatic HCC cell lines, MHCC97H and HCCLM3, were obviously higher than those in the low metastatic HCC cell lines including HepG2, MHCC97L, Huh7 and Hep3B (P < 0.05, Fig. 1D). These data indicates that elevated miR-520g expression confers increased metastatic potential of HCC cells. 3.2. Clinical significance of miR-520g expression in HCC cases 130 samples of HCC specimens were subjected to qRT-PCR for miR-520g expression. The expression of miR-520g was considered as either low (n = 65) or high (n = 65) according to the cutoff value, which was defined as the median of the cohort. As shown in Table 1, miR-520g was expressed at prominently higher levels in HCC patients with venous infiltration (P = 0.005), high Edmondson-Steiner grading (P = 0.015) and advanced TNM tumor stage (P = 0.001). Furthermore, 130 HCC patients with clinical survival information (with a median follow-up time of 37.5 months) were analyzed by Kaplan Meier estimation. Tumors with high expression of miR-520g indeed associated with worse overall survival and disease-free survival of HCC patients (P < 0.001, respectively, Fig. 2). These data indicate that miR-520g may act as a potent biomarker for predicting prognosis of HCC patients. 3.3. MiR-520g is correlated with EMT phenotype and invasion ability in HCC cells We transduced HCC cell line, HCCLM3, with miR-520g inhibitor (anti-miR-520g) or negative control (NC). As measured by
Fig. 1. The expression levels of miR-520g in HCC tissues and cells. Comparing differences in the expression levels of miR-520g between (A) HCC and matched non-tumor tissues, (B) aggressive and non-aggressive tumor tissues, (C) HCC tissues arising from recurrent and non-recurrent groups, and (D) HCC cell lines with different metastatic potentials and the immortalized hepatic cell line LO2. ⁄P < 0.05.
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Fig. 2. Prognostic significance of miR-520g in HCC cases. According to the level of miR-520g expression, Kaplan-Meier 5-year overall and disease-free survival curves of HCC patients showed that high expression of miR-520g was correlated with poor prognosis. The median expression value obtained for miR-520g of the 130 HCC samples detected by qRT-PCR was chosen as the cutoff value.
Fig. 3. MiR-520g regulates EMT phenotype and HCC cell mobility. (A) Cell migration and invasion as measured by Boyden chamber and Transwell assays were inhibited by down-regulation of miR-520g in HCCLM3 cells as compared with control cells. n = 3 repeats with similar results; ⁄P < 0.05. (B) miR-520g overexpressing HepG2 cells conferred a higher number of migrated and invaded cells as compared with control cells. n = 3 repeats with similar results; ⁄P < 0.05. (C) Representative Western blot analysis of Ecadherin and vimentin in HCCLM3 cells with miR-520g inhibitor transfection and HepG2 cells with miR-520g transfection was shown. n = 6; ⁄P < 0.05.
qRT-PCR, the expression of miR-520g was significantly downregulated by miR-520g inhibitor in HCCLM3 cells (P < 0.05, data not shown). Boyden chamber assays were performed to test the effect of altering miR-520g levels on HCC cell migration. We found that down-regulation of miR-520g led to a significant reduction of cell migration in HCCLM3 cells (P < 0.05, Fig. 3A). Furthermore, as determined by Transwell assays, the number of invaded HCCLM3
cells was significantly reduced after reduction of miR-520g (P < 0.05, Fig. 3A). Next, miR-520g overexpressing HepG2 cells was established and confirmed by qRT-PCR (P < 0.05, data not shown). As expected, up-regulation of miR-520g obviously promoted HCC cell migration and invasion (P < 0.05, respectively, Fig. 3B). Furthermore, suppression of EMT in HCCLM3 cells with down-regulation of miR-520g was associated with elevated
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expression of E-cadherin and reduced expression of vimentin (Fig. 3C). Otherwise, up-regulation of miR-520g-induced EMT was associated with reduced expression of E-cadherin and elevated expression of vimentin in HepG2 cells. These data indicate that miR-520g is associated with EMT phenotype and cell mobility in HCC cell lines. 3.4. SMAD7 is identified as a functional target of miR-520g and is down-regulated in HCC specimens To disclose the molecular mechanisms by which miR-520g induces EMT in HCC, predicted target genes of miR-520g were retrieved and analyzed using publicly available databases (TargetScan 6.2 and MiRanDa). Among these, EMT-associated genes were selected for further investigating. Significantly, SMAD7, which is known to inhibit EMT and tumor metastasis by blocking TGFb signaling [18,19], was predicted as one of the targets of miR-520g. HepG2 cells that were transfected with miR-520g and control vectors were subjected to qRT-PCR and Western blot for SMAD7 expression. Both SMAD7 mRNA and protein levels were significantly reduced by up-regulation of miR-520g in HepG2 cells (P < 0.05, respectively, Fig. 4A and B). To further demonstrate that SMAD7 is directly targeted by miR-520g in HCC cells, we investigated whether the miR-520g directly interacted with the 30 -UTR of SMAD7 mRNA using a dual-luciferase reporter assay. As expected, miR-520g significantly inhibited the luciferase activity of SMAD7 containing a wild-type (wt) 30 -UTR but did not suppress activity of SMAD7 with a mutant (mt) 30 -UTR (P < 0.05, Fig. 4C and D). When anti-miR-520g was transfected, an increase in luciferase activity of wt SMAD7 30 -UTR was observed. However, with the mt SMAD7 30 -UTR constructs, there was no relative increase in activity (P < 0.05, Fig. 4C and D). Thus, our data strongly suggest that SMAD7 is a target of miR-520g in HCC. Expression of SMAD7 was further detected by qRT-PCR and immunohistochemistry in the previous cohort of 40 pairs of HCC and normal tumor-adjacent tissues. Both SMAD7 mRNA and protein levels were demonstrated to be significantly lower in HCC tissues as compared with those in non-cancerous tissues (P < 0.05,
respectively, Fig. 5A and B). 130 samples of HCC tissues were subjected to immunostaining for SMAD7 expression. SMAD7 immunoreactivity was considered as either negative (score 0) or positive (scores 1-3). In these cases, SMAD7 expression was detected in 46.2% (30/65) of the HCC samples with low expression of miR520g, whereas only 20.0% (13/65) of the HCC specimens with high expression of miR-520g showed a positive SMAD7 signal (P < 0.05, Fig. 5C). Furthermore, Spearman correlation analysis indicated that miR-520g was inversely correlated with SMAD7 expression in HCC tissues (r = 0.7362, P < 0.001). 3.5. MiR-520g promotes EMT and HCC cell mobility by targeting SMAD7 To further confirm the role of SMAD7 in miR-520g-mediated EMT and cell mobility in HCC cells, we restored the expression of SMAD7 in miR-520g overexpressing HepG2 cells by transfecting pCMV5-SMAD7 plasmids. As confirmed by immunoblotting, restoration of SMAD7 suppressed EMT in miR-520g overexpressing HepG2 cells and was correlated with up-regulation of epithelial biomarker, Ecadherin, and reduced expression of mesenchymal biomarker, vimentin (P < 0.05, respectively, Fig. 6A). Furthermore, the migration and invasion abilities of miR-520g overexpressing HepG2 cells was partially abolished after pCMV5-SMAD7 transfection (P < 0.05, respectively, Fig. 6B), which was consistent with above results. Similarly, SMAD7 knockdown promoted the migration and invasion of miR-520g downregulating HCCLM3 cells (P < 0.05, respectively, Fig. 6C). All the data indicate that miR520g promotes EMT and cell mobility by targeting SMAD7 in HCC cells. 4. Discussion Increasing evidences have demonstrated that the deregulation and dysfunction of miRNAs play a critical role in the initiation and progression of HCC [20]. However, miRNAs that are involved in the recurrence and metastasis of HCC remain poorly investigated. In this study, we initially detected the expression level of
Fig. 4. SMAD7 is identified as a functional target of miR-520g in HCC. (A) qRT-PCR and (B) Western blot analysis of SMAD7 expression in HepG2 cells with miR-520g or control vectors transfection. n = 6; ⁄P < 0.05. (C) miR-520g and its putative binding sequence in the 30 -UTR of SMAD7. The mutant miR-520g binding site was generated in the complementary site for the seed region of miR-520g (wt, wild type; mt, mutant type). (D) MiR-520g significantly suppressed the luciferase activity that carried wt but not mt 30 -UTR of SMAD7. Anti-miR-520g led to a noticeable increase in luciferase activity of wt 30 -UTR of SMAD7. n = 3 repeats with similar results; ⁄P < 0.05.
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Fig. 5. An inverse correlation between miR-520g and SMAD7 expression is observed in HCC. Comparing differences in the expression levels of SMAD7 (A) mRNA and (B) protein between HCC and matched non-tumor tissues. ⁄P < 0.05. (C) Representative immunostaining showed negative expression of SMAD7 in miR-520 high-expressing HCC tissue and positive expression of SMAD7 in miR-520 low-expressing tumor. A significant inverse correlation between miR-520g and SMAD7 expression was observed in HCC tissues. Scale bar: 100 lm; ⁄P < 0.05.
miR-520g in 40 samples of HCC and matched normal tumoradjacent tissues. Our data indicated that the expression of miR520g in HCC tissues was obviously higher than that in non-tumor tissues. Moreover, miR-520g was expressed at prominent higher levels in aggressive tumor tissues as compared with that in nonaggressive tumor tissues. Importantly, our results showed that high expression of miR-520g conferred a significant higher recurrence rate for HCC patients. Clinical analysis found that high expression of miR-520g was significantly correlated with venous infiltration, high Edmondson-Steiner grading and advanced TNM tumor stage in HCC. Kaplan–Meier’s plots demonstrated that high expression of miR-520g was significantly associated with poor survival of HCC patients. Furthermore, HCC cell lines, especially high metastatic cell lines (MHCC97H and HCCLM3), showed obviously higher levels of miR-520g as compared with the non-transformed hepatic cell line LO2. Altogether, these results suggest that miR520g is critical for prognosis determination in HCC patients. Next, we further investigated the role of miR-520g in HCC cells. Boyden chamber and Transwell assays were performed to test HCC cell migration and invasion abilities. We found that downregulation of miR-520g inhibited cell migration and invasion in HCCLM3 cells. And up-regulation of miR-520g increased the number of migrated and invaded HepG2 cells. These data suggest that miR-520g indeed promotes HCC cell migration and invasion. EMT, a dynamic and reversible cellular process, is characterized by loss of cell polarity and intracellular junctions and acquirement
of mesenchymal features, resulting in increased HCC cell migration and invasion [21]. Thus, we determined the expression of epithelial marker, E-cadherin, and mesenchymal marker, vimentin in HCC cells with altering expression of miR520g. Interestingly, we demonstrated that down-regulation of miR-520g inhibited EMT and was associated with elevated expression of E-cadherin and reduced expression of vimentin in HCCLM3 cells. Meanwhile, up-regulation of miR-520g promoted EMT and was associated with reduced expression of E-cadherin and elevated expression of vimentin. Taken together, miR-520g may promote HCC cell migration and invasion by inducing EMT. Predicted target genes of miR-520g were retrieved and analyzed using publicly available databases (TargetScan and Miranda). The putative binding sequences of miR-520g were found in the 30 -UTR of SMAD7. SMAD7 is a member of the SMAD family of proteins, which belong to the transforming growth factor (TGF)-b super family of ligands [18]. SMAD7 is involved in cell signaling and is a TGFb receptor I (TGFbRI) antagonist that blocks TGFb1 [22,23]. Smith et at found that miR-106b-25 cluster activated TGF-b signaling pathway through down-regulation of SMAD7 to induce EMT and tumor-initiating cell characteristics in human breast cancer [19]. Notably, recent study reported that miR-216a/ 217 induced EMT by activating the TGF-b signaling pathway by targeting SMAD7 in HCC [18]. We investigated the regulatory effect of miR-520g on SMAD7 in HCC cells. Our data indicated that up-regulation of miR-520g reduced the expression levels of SMAD7
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Fig. 6. MiR-520g induces HCC cell migration, invasion and EMT by targeting SMAD7. (A) Western blot analysis of the expression of E-cadherin and vimentin in pCMV5SMAD7 transfected miR-520g overexpressing HepG2 cells. n = 6; ⁄P < 0.05. (B) Migration and invasion abilities of the stable transfected cells following restoration of SMAD7 in miR-520g overexpressing HepG2 cells were analyzed by Boyden chamber and Transwell assays. n = 3 repeats with similar results; ⁄P < 0.05. (C) SMAD7 knockdown by a specific siRNA promoted the migration and invasion of miR-520g downregulating HCCLM3 cells. n = 3 repeats with similar results; ⁄P < 0.05.
mRNA and protein in HepG2 cells. Herein, we validated SMAD7 as a direct functional target of miR-520g in HCC. Furthermore, expression of SMAD7 in HCC tissues was obviously lower as compared with that in matched normal non-tumor tissues, which was consistent with previous studies [18]. Otherwise, a significant inverse correlation between miR-520g and SMAD7 expression was observed in HCC tissues. Importantly, the regulatory effects of altered expression of miR-520g on EMT and cell mobility were partially abolished by SMAD7 in HCC cells. Accordingly, we suggest that miR-520g promotes EMT and cell mobility by targeting SMDA7 in HCC. In the previous paper, miR-520c expression reduced cell invasiveness and had a tumour suppressive role in breast cancer, and that this was attributed to both the NF-jB and TGF-b/Smad2/3 pathways [24]. MiR-520c has the same seed region as miR-520g and yet high expression appears to result in reduced invasiveness in breast cancer. Interestingly, Toffanin et al. reported that the expression of miR-520c increased proliferation, migration, and invasion of HCC cells in vitro [25]. Therefore, the functional significance of miRNAs in cancer development and progression seem to be cancer-type specific. In conclusion, we find that miR-520g is up-regulated in HCC and its high expression is related to poor prognostic features. Moreover, high expression of miR-520g is a prognostic marker for
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