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ERK pathways
Cancer Letters 337 (2013) 226–236
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
MicroRNA-21 suppresses PTEN and hSulf-1 expression and promotes hepatocellular carcinoma progression through AKT/ERK pathways Longlong Bao a,1, Yan Yan a,1, Can Xu b,1, Weidan Ji a, Shuwen Shen a, Gaoya Xu a, Yong Zeng a, Bin Sun a, Haihua Qian a, Lei Chen a, Mengchao Wu a, Changqing Su a,⇑, Jie Chen b,⇑ a b
Department of Molecular Oncology, Eastern Hepatobiliary Surgical Hospital & Institute, Second Military Medical University, Shanghai 200438, China Changhai Hospital, Second Military Medical University, Shanghai 200168, China
a r t i c l e
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Article history: Received 1 April 2013 Received in revised form 27 April 2013 Accepted 4 May 2013
Keywords: Hepatocellular carcinoma MicroRNA Signaling Epithelial–mesenchymal transition Human sulfatase-1
a b s t r a c t MicroRNAs (miRNAs) have been believed to associate with malignant progression including cancer cell proliferation, apoptosis, differentiation, angiogenesis, invasion and metastasis. However, the functions of miRNAs are intricate, one miRNA can directly or indirectly target multiple genes and function as oncogene or tumor suppressor gene. In this study, we found that miR-21 inhibits PTEN and human sulfatase-1 (hSulf-1) expression in hepatocellular carcinoma (HCC) cells. The hSulf-1 is a heparin-degrading endosulfatase, which can inhibit the heparin binding growth factor-mediated signaling transduction into cells. Therefore, miR-21-mediated suppression of both hSulf-1 and PTEN led to activation of AKT/ERK pathways and epithelial–mesenchymal transition (EMT) in HCC cells, and finally enhance the activity of HCC cell proliferation and movement and promote HCC xenograft tumor growth in mouse models. These findings may provide candidate targets for prevention and treatment of HCC. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cancer is a complex multipronged physiological and pathological process that involved in the activation of oncogenic pathways and/or inactivation of tumor suppressor signals [1]. The proliferation and invasion of tumor cells are considered as a foundation of cancer survival and development. Accumulating evidences demonstrated that the induction of epithelial–mesenchymal transition (EMT) and aberrant expression of microRNAs (miRNAs) are associated with tumorigenesis, tumor progression, metastasis and relapse in cancers, including hepatocellular carcinoma (HCC) [2–4]. EMT describes the process which epithelial cell gradually lose polarity and typical epithelial characteristics, then show characteristics of mesenchymal cells [5]. EMT phenomenon endows epithelial cells with enhanced migratory and invasive properties, and Abbreviations: HCC, hepatocellular carcinoma; EMT, epithelial–mesenchymal transition; AKT, PI3 K-protein kinase B; ERK, extracellular signal regulated kinase; hSulf-1, human sulfatase-1; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; HSPG, heparan sulfate proteoglycans. ⇑ Corresponding authors. Addresses: Department of Molecular Oncology, Eastern Hepatobiliary Surgical Hospital & Institute, Second Military Medical University, Shanghai 200438, China. Tel./fax: +86 21 8187 5351 (C. Su), Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China. Tel./fax: +86 21 8187 3222 (J. Chen). E-mail addresses: [email protected] (C. Su), [email protected] (J. Chen). 1 These authors contributed equally to this work. 0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2013.05.007
increasing data are provided to show the existence of EMT phenotypes in the invasion and migration process of cancer cells [6]. Inducers of EMT include some protein polypeptides, transcription factors, growth factors and miRNAs. As a marker of epithelial cells, E-cadherin regulates homotypic cell–cell adhesion and binds to cytoskeletal components to mediate the morphology, polarity and behavior of epithelial cells [7]. Accompanied by the change of E-cadherin, the expression of mesenchymal markers such as N-cadherin, Vimentin, and alpha-SMA also have corresponding changes [8,9]. miRNAs are a family of small highly conserved endogenous non-coding RNAs which inhibit translation of target genes by base pairing to the 30 -UTR of mRNAs. Recently, a series of miRNAs have been shown to play critical roles in the processes of EMT of cancer cells to promote the progression and metastasis of human malignancies, including human HCC [2,4,10–12], in which miR-21 is an important component involved in the cellular signaling pathways that regulates EMT processes [3,13] and is always up-expressed in many kinds of malignancies. Recent studies showed that several tumor suppressors including PTEN, TPM1, PDCD4, maspin, and TIMP3 were targets of miR-21 [14–18], suggesting that miR-21 is an important oncogenic miRNA which is closely related to tumor growth and metastasis [2]. As a typical target gene of miR-21, PTEN negatively regulates the PI3K-protein kinase B (AKT) pathway [19] and mitogen-activated protein kinase/extracellular signal regulated kinase (ERK) pathway [20], but it always lowly expressed in
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some cancer cells [14], partly because of inactivation by miR-21. Antagonism of miR-21 could re-activate PTEN and inactivate AKT and ERK pathways through PTEN up-regulation and finally reverse EMT in MDA-MB-231 cells [2]. Up-regulation of miR-21 can inhibit PTEN expression and lead to an activation of AKT and ERK pathways, finally enhance HIF-1a and VEGF expression and induce tumor angiogenesis [21]. However, we found that, in PTEN-silenced cancer cells, manipulation of miR-21 expression also results in the changes of AKT and ERK pathway activity, demonstrating that there is another mechanism involved in the regulation of AKT and ERK signaling besides PTEN. In this study, we have revealed that up- or down-regulation of miR-21 expression in HCC cells could change the human sulfatase-1 (hSulf-1) expression and downstream AKT and ERK signaling activity, then regulate cancer cell migration and invasion by inducing cellular EMT. The hSulf-1 is always expressed at lower levels in several tumor types, including ovarian, breast, and hepatocellular cancers, compared with the expression levels in the corresponding normal tissues [22,23]. Over-expression of hSulf-1 could reduce the activity of ERK and AKT signaling and suppress cell growth, migration and invasion [24,25]. The results strongly suggested that miR-21 might regulate hSulf-1-mediated AKT and ERK pathways in HCC, finally influence cancer progression, invasion and metastasis. Anyway, we provided the first evidence that miR-21 promotes cancer progression, invasion and metastasis by hSulf-1-mediated AKT and ERK pathways in HCC, which may provide a candidate target for cancer gene therapy. 2. Materials and methods 2.1. Cell lines and cell culture The human HCC cell lines MHCC-97H and MHCC-97L were obtained from the Liver Cancer Institute of Fudan University (Shanghai, China). SMMC-7721 was purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). The human liver cell line WRL-68 and the fibroblast cell line MRC-5 were purchased from the ATCC (Manassas, VA, USA). These cells were maintained in high-glucose DMEM (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal calf serum (Hyclone, Logan, UT, USA), 100 units/ml penicillin, 100 lg/ml streptomycin and 1% glutamine (Invitrogen, Grand Island, NY, USA) and incubated in a humidified atmosphere with 5% CO2 at 37 °C. To further investigate the ability of cancer cell inoculation and mimic cancer cell detachment and metastasis in animal model, MHCC-97H and SMMC-7721 were injected into the abdominal cavity of BALB/c (nu/nu) mice, 3 mice in each group, 2.5 106 cells per mouse. Two weeks later, the colonization of cancer cells in mice was monitored by anatomy.
2.2. Vectors and inhibitor The miR-21 expression vector of pGL3-miR21-EGFP and its negative control vector pGL3-Ctrl-EGFP containing the enhanced green fluorescent protein (EGFP) gene were constructed and preserved. The plasmid pSUPER-shSulf1 containing the hSulf-1 shRNA (19 oligonucleotide pairs targeting hSulf-1 cDNA positions 294–312: 50 -GTATGTGCACAATCACAAT-30 ) was kindly gifted from Viji Shridhar (Department of Experimental Pathology, Mayo Clinic Cancer Center, Rochester, MN, USA). The plasmid pGenesil-shPTEN containing the PTEN shRNA (19 oligonucleotide pairs targeting PTEN cDNA positions 1026–1044: 50 -GGTGAAGCTGTACTTCACA-30 ), the miR-21 inhibitor, and the negative control plasmid pGenesil-shNC (19 oligonucleotide pairs: 50 -gacttcataaggcgcatgc-30 ) were purchased from GenePharma Inc., Shanghai, China.
2.3. Transfection Totally 5 105 cells were seeded in six-well plates and grown to 80% confluence. Transfection with the miR-21 and shRNA vectors into HCC cells was performed at a concentration of 2 lg/well using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer’s protocol. miR-21 inhibitor was directly worked on MHCC-97H cells at a final concentration of 100 nmol/L. All groups were performed in triplicate. Twenty-four h after transfection, the transfected cells were observed under a fluorescent microscope to judge the transfection efficiency and the morphological change, followed by G418 selection and cultured for another 24 h. Finally, the transfected cells were harvested for total RNA and protein extraction, or miRNAs isolation.
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2.4. Real time qRT-PCR analysis The miR-21 reverse transcription (RT) primer (50 -CGTCGCTACATCGAGTGT AGCATATGCGACGTCAACATC-30 ) and qPCR primers (Forward: 50 -TAGCTTATCAGACTGATG-30 ; Reverse: 50 -ACATCGAGTGTAGCATA-30 ) were synthesized by Sangon Biotech., Inc. (Shanghai, China). miRNAs was extracted from cells using a mirVana miRNA Isolation Kit (Applied Biosystems, Foster City, CA, USA). The real time qRT-PCR was performed to measure the expression of miR-21 by a MiniOpticon™ Two-Color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). U6 was used as the inner control. 2.5. Western blotting analysis Cells were washed twice with PBS and lysed on the culture dishes using RIPA lysis buffer (1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 150 mmol/L NaCl and 10 mmol/L Tris–HCl) containing 1/100 phenylmethanesulfonyl fluoride (PMSF) solution. The total protein concentration was determined using the BCA method and 30 lg of each sample was separated by SDS-PAGE (8, 10, or 12%) and transferred to PVDF membrane. Non-specific binding sites were blocked by incubating with TBST containing 5% (w/v) non-fat dried milk for 1 h at room temperature. The membrane was immunoblotted overnight at 4 °C and HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibody was incubated with the membrane for 1 h after three washes with TBST. The primary antibodies included rabbit anti-human hSulf-1, mouse anti-human AKT, rabbit anti-human p-AKT (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-human PTEN, ERK1/2 and p-ERK1/2 (Cell Signaling Technology, Danvers, MA, USA); rabbit anti-human E-cadherin, mouse antihuman b-catenin, mouse anti-human Vimentin (Bioworld Technology Inc., St. Louis Park, MN, USA) and mouse anti-human glyceraldehydes 3-phosphate dehydrogenase (GAPDH) antibodies (Kangchen Bio-tech, Shanghai, China). The secondary antibodies included horseradish peroxidase-conjugated sheep anti-mouse IgG and sheep anti-rabbit IgG (R&D Systems China, Shanghai, China). Signals were visualized by ECL chemiluminescence. Equal protein loading was assessed by the expression of GAPDH. The bands were semi-quantified using ImageJ software. 2.6. Cell proliferation assay Cell proliferation assay was performed by 3-(4,5-dimethylthiazol-2-yl)-2 5diphenyltetrazolium bromide (MTT) assay. After transfected with the indicated plasmid or inhibitor, cells were plated in 96-well plates at 5 103 per well in 200 lL culture medium. After 48 h, 20 lL of 5 mg/mL MTT was added to each well, then the cells were incubated for 4 h before 150 lL DMSO was added. Once the insoluble crystals were completely dissolved, the absorbance values at 570 nm were measured by micro-enzyme-linked immunosorbent assay plate reader. 2.7. Migration and invasion assays Cell migration and invasion were evaluated using transwell chamber assay (Millipore, Billerica, MA, USA) according to the manufacturer’s instruction. For invasion assay, totally 5 104 cells were seeded on an 8-lm pore size transwell insert coated with extracellular matrix (ECM) (1:6) (BD Biosciences, China), while cell migration assay did not coat with ECM. After incubated at 37 °C for 48 h, the cells adherent to the upper surface of the filter were removed using a cotton applicator, then stained with crystal violet, and the values obtained were calculated by averaging the total numbers of cells from triplicate determinations. 2.8. Wound healing assay Cells were seeded in six-well plates at a density of 4 105 cells per well and cultured for 24 h at 37 °C. Wounds were created in the cell monolayer by a pipette tip and the indicated plasmids or inhibitor were transfected into cells. The dead cells were washed away with PBS. Images were taken at 0 and 48 h. Migration distance was detected by the ImageJ software. 2.9. Soft agar colony formation assay To test the capacity of cell colony growth, cells were collected after treated, and 1 104 cells were mixed with a 0.6% agar solution in DMEM containing 20% fetal bovine serum (FBS) and layered on top of a 1.2% agar layer in six-well tissue culture plates. After the gel solidified, cells were incubated for 2 to 3 weeks in a 5% CO2 humidified incubator at 37 °C until colonies were formed. Cultural medium of DMEM supplemented with 10% FBS was added every 3 to 4 days. The colonies containing more than 50 cells were counted under microscope. 2.10. Animal HCC xenograft experiments Thirty male BALB/C nude mice at 4 weeks of age (Shanghai SLAC Laboratory Animal Center of Chinese Academy of Sciences, Shanghai, China) were randomly divided into three groups (n = 10 per group) and subcutaneously injected the parental, pGL3-miR21-EGFP-transfected and pGL3-Ctrl-EGFP-transfected SMMC-7721
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cells, respectively, into mouse right flank at 1 106 cells/100 lL per mouse. On day 14 post-inoculation, the tumorigenic rate in every group was 100%. The tumor volume was measured every week and calculated using the formula ‘‘a b2 0.5’’, in which a and b represent the maximal and minimal diameters, and the tumor inhibition rate was calculated using the formula ‘‘(tumor volume of the control group – tumor volume of the treatment group)/tumor volume of the control group 100%. On days 35 after cell implantation, the tumor volume in the miR21-transfected group was overload beyond the standard approved by the Animal Care Committee of Second Military Medical University, and the mice were sacrificed by anesthetization, the tumor specimens were removed and weighed. The fresh specimens were prepared for examining miR-21 expression by qRT-PCR and the paraffin-embedded consecutive sections were cut for examining the expression of hSulf-1, PTEN, p-AKT, p-ERK and Ki67 (mouse anti-Ki67 antibody: GeneTex Inc., Irvine, CA, USA) by immunohistochemistry with the streptavdin–peroxidase (S–P) kit (Fuzhou Maixin Biotechnology Development Co., Fuzhou, China). Each slice was enumerated under 5 fields of medium magnification (200) to determine the proportion of positive cells. 2.11. Statistical analysis All data are expressed as mean ± standard deviation (SD). Statistical evaluation of the data was performed with one-way ANOVA. Pair-wise comparisons were conducted by Student’s t test. The P value less than 0.05 was considered statistically significant. All analyses in the study were evaluated with SPSS version 17.0 software.
3. Results 3.1. miR-21 expression is up-regulated in highly metastatic cancer cells miR-21 is widely over-expressed in tumorigenesis including HCC cell lines [14]. Furthermore, miR-21 was closely connected
with motility, migration, and invasion of human cancers. To explore the expression of miR-21 in cancer cell lines, different cancer cell lines varied in their metastatic potentials, including MHCC-97H, MHCC-97L and SMCC-7721, were selected and measured the relative expression of miR-21 by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), compared with normal cell lines WRL-68 and MRC-5. The relative expression of miR-21 was significantly up-regulated in MHCC97H and MHCC-97L cell lines (6.73 ± 1.01 in MHCC-97H, 4.35 ± 0.53 in MHCC-97L; P < 0.01 versus normal cell lines), but not in SMMC-7721 cells (1.10 ± 0.13; P = 0.582 versus normal cell lines; Fig. 1A). To confirm the metastatic potential of miR-21 high expression cells (MHCC-97H) and miR-21 low expression cells (SMCC-7721), the harvested cells were injected into the abdominal cavity of BALB/c (nu/nu) mice at 2.5 106 cells per mouse. Two weeks later, mice were dissected under body vision microscope and the cancer nodules were monitored and counted. The results showed that MHCC-97H cells formed more cancer nodules with larger size than SMCC-7721 cells (Fig. 1B). 3.2. Over-expression of miR-21 induced EMT by regulating the activity of PTEN- and hSulf-1-mediated AKT and ERK signaling in HCC cells To further confirm the role of miR-21 in regulating cancer cell EMT, we constructed an expression vector of GFP-tagged miR-21 and transfected into SMMC-7721 cells. Totally 5 105 SMMC7721 cells were transfected with miR-21 vectors at a concentration
Fig. 1. Expression of miR-21 in HCC cells and its relation to cellular metastatic ability. (A) miR-21 expression was detected by real time qRT-PCR, the results were normalized to U6 expression; P<0.01 versus the average value of two normal cell lines. (B) MHCC-97H and SMMC-7721 were injected into the abdominal cavity of BALB/c (nu/nu) mice (n = 3 in every group) at 2.5 106 cells per mouse. Two weeks later, mice were dissected under body vision microscope and the number of cancer nodules and total weight of cancer nodules in every mouse were monitored and counted; P < 0.05, P < 0.01.
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Fig. 2. Re-expression of miR-21 induced EMT by down-expression of PTEN and hSulf-1 and activation of AKT and ERK signaling. (A) SMMC-7721 cells were transfected with the expression vector of GFP-tagged miR-21, and miR-21 expression was detected by real time qRT-PCR. The results were normalized to U6 expression; P < 0.01 versus the parental cell group (Blank) and the negative vector control group (Negative). (B), Expression levels of EMT markers (E-cadherin, vimentin and b-catenin) were measured by Western blotting, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control, and bands were semi-quantitatively analyzed by using ImageJ software, normalized to GAPDH density; P < 0.05, P < 0.01 versus the blank and the negative control groups. (C) Mesenchymal morphological change was induced by overexpression of miR-21 tagged with GFP in SMMC-7721 cells, typical images were showed by normal light field (original magnification 100) and fluorescent field (original magnification 200). (D) Expression levels of PTEN, hSulf-1, p-AKT and AKT, as well as p-ERK and ERK were semi-quantitatively analyzed by using ImageJ software, normalized to GAPDH density; P < 0.05, P < 0.01 versus the blank and the negative control groups. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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of 2 lg/well, the transfection efficiency was (65.10 ± 2.00)% 24 h after transfection. Followed by G418 selection, the transfection efficiency was (92.32 ± 15.22)%. In the miR-21-transfected cells, miR-21 expression was increased to more than 18-fold, as compared with the parental cells without transfection (Fig. 2A), indicating that transfection of miR-21 expression vector could increase the relative expression of miR-21. To examine whether the forced re-expression of miR-21 could induce EMT, the protein expression of EMT biomarkers, the phosphorylation levels of EMT-associated AKT and ERK signaling and the expression of their upstream signal regulatory factors, PTEN and hSulf-1, were measured by Western blotting assay. Compared with the negative vector control group, over-expression of miR-21 increased the expression levels of b-catenin and vimentin, while decreased the expression of E-cadherin (Fig. 2B), suggesting that over-expression of miR-21 could induce EMT in SMMC-7721 cells. After overexpression of miR-21, SMMC-7721 cells were induced a striking EMT-like transformation, which was morphologically characterized by a spindle-shaped change and loss of cell-cell contacts (Fig. 2C). Meanwhile, over-expression of miR-21 decreased the expression of PTEN and hSulf-1 and increased the expression of p-AKT and p-ERK in SMMC-7721 cells (Fig. 2D), suggesting that over-expression of miR-21 could activate AKT and ERK pathways. These results supported that miR-21 could regulate EMT by modifying the activity of both PTEN- and hSulf-1-mediated AKT/ERK pathways.
3.3. Inhibition of miR-21 could reverse EMT by inactivating AKT and ERK signal activity through up-regulation of PTEN and hSulf-1 expression in HCC cells MHCC-97H is a high-metastatic HCC cell line and has a high expression level of miR-21 and low expression of PTEN and hSulf-1. To further investigate the effect of miR-21 on EMT process of HCC cells, the inhibitor of miR-21 was transfected into MHCC97H cells and the expression of miR-21 was measured by real time qRT-PCR. The expression of miR-21 was decreased to more than 6fold after treated with miR-21 inhibitor, as compared to the parental cell control (Fig. 3A). The relative expression levels of EMT biomarkers were measured by Western blotting analysis. The results demonstrated that inhibition of miR-21 increased the protein expression of E-cadherin and decreased the expression of b-catenin and vimentin (Fig. 3B). Meanwhile, the relative expression of pAKT and p-ERK was decreased accompany by an increase of PTEN and hSulf-1 (Fig. 3C), indicating that inhibition of miR-21 could reverse EMT by inactivating AKT and ERK pathways through up-regulation of PTEN and hSulf-1. 3.4. miR-21 can separately regulate the hSulf-1-mediated and PTENmediated AKT and ERK signal pathways Both PTEN and hSulf-1 can regulate the activity of AKT and ERK pathways [19,26–28]. It was reported that miR-21 can inactivate
Fig. 3. Inhibition of miR-21 induced an increased expression of PTEN and hSulf-1 and inactivated AKT and ERK signaling to reverse EMT. (A) MHCC-97H was transfected with miR-21 inhibitor to inhibit miR-21 expression; P < 0.01 versus the parental cell control group. (B) EMT markers (E-cadherin, vimentin and b-catenin) were detected by Western blot analysis, GAPDH was used as loading control, and bands were semi-quantitatively analyzed by using ImageJ software, normalized to GAPDH density; P < 0.05, P < 0.01 versus the parental cell group (Blank) and negative vector control group (Negative). (C) Protein levels of PTEN, hSulf-1, p-AKT, AKT, p-ERK and ERK were detected by Western blot analysis, and bands were semi-quantitatively analyzed by using ImageJ software, normalized to GAPDH density; P < 0.05, P < 0.01 versus the blank and negative control groups.
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PTEN-mediated signaling [14,29], but the effect of miR-21 on hSulf-1-mediated signaling was not reported previously. To further differentiate the respective effect of PTEN and hSulf-1 on AKT and ERK pathways under the regulation of miR-21, MHCC-97H cells were transfected with PTEN-small hairpin RNA (shRNA) vector (pGenesil-shPTEN) and hSulf-1-shRNA (pSUPER-shSulf1), respectively, to block the expression of PTEN and hSulf-1, then generated the cell lines, MHCC-97HPTEN— and MHCC-97HhSulf-1—. When the cells were down-regulated miR-21 expression by miR-21 inhibitor, hSulf-1 expression was increased only in MHCC-97HPTEN— cells and PTEN expression was increased only in MHCC-97HhSulf-1— cells. More interestingly, whether the increase of hSulf-1 or PTEN expression, the phosphorylation levels of AKT and ERK were decreased both in MHCC-97HPTEN— and MHCC-97HhSulf-1— cell lines (Fig. 4). 3.5. miR-21 enhances cellular motility in HCC cells To investigate the effect of miR-21 on cellular motility, HCC cells with different expression status of miR-21, including SMMC-7721 and MHCC-97H cell lines, were measured by wound healing, transwell migration and invasion assays after transfected miR-21 expression vector or treated with miR-21 inhibitor. Transfection of miR-21 expression vector significantly enhanced the capacity of wound healing in SMMC-7721 cells, as compared with the parental control cells without transfection (Fig. 5A). However, the capacity of wound healing in MHCC-97H cells was significantly attenuated after treated with miR-21 inhibitor, as compared to the parental control cells without treatment (Fig. 5B).
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Meanwhile, after transfected with miR-21 expression vector, the relative migrated and invaded cell number of SMMC-7721 cells was significantly more than that of the parental control cells (Fig. 5C and D), and the relative migrated and invaded cell number was significantly decreased in the miR-21 inhibitor-treated MHCC97H cells, as compared with the parental control cells (Fig. 5E and F), suggesting that miR-21 plays an important role in the regulation of HCC cellular motility, including the cancer cell invasive and metastatic capacity. 3.6. miR-21 influences the proliferation and neoplastic capacity of HCC cells To corroborate the effect of miR-21 on proliferation and neoplastic capacity in HCC cells, we measured the cancer cell viability and neoplastic capacity of SMMC-7721 cells and MHCC-97H cells after modification of miR-21 expression by MTT assay and anchorage-independent growth assay. The results demonstrated that the cell viability of SMMC-7721 cells transfected with miR-21 expression vector was significantly increased compared with the parental cell and negative control groups (Fig. 6A), and the cell viability of MHCC-97H cells transfected with miR-21 inhibitor was significantly lower than that of the control groups (Fig. 6B). After 2 weeks post-treatment, we also examined the capacity of colony formation of SMMC-7721 cells and MHCC-97H cells. The results suggested that SMMC-7721 cells displayed more colonies when transfected with over-expression vector of miR-21, compared with the control groups (Fig. 6C and D), and MHCC-97H that transfected with inhibitor of miR-21 displayed less colonies, compared with the control
Fig. 4. miR-21 regulated separately both hSulf-1-mediated and PTEN-mediated AKT and ERK signal pathways. MHCC-97H cells were transfected with PTEN-shRNA (pGenesilshPTEN) and hSulf-1-shRNA (pSUPER-shSulf1) vectors, respectively, at a vector concentration of 20 lg/well to 5 105 cells in six-well plates to establish cell sublines MHCC97HPTEN— (A) and MHCC-97HhSulf-1— (B). These cell sublines were treated with miR-21 inhibitor at a final concentration of 100 nmol/L, and the expression of miR-21, hSulf-1, PTEN, AKT and ERK was examined by qRT-PCR or Western blotting. The band densitometry was analyzed normalized to GAPDH density; P < 0.05, P < 0.01.
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Fig. 5. Effect of miR-21 on cellular motility in HCC cells. (A and B) Motility capacity of SMMC-7721 cells transfected with miR-21 expression vector (A) and MHCC-97H treated with miR-21 inhibitor (B) was assessed by wound healing assay (original magnification 100). (C–F) Migratory and invasive properties of SMMC-7721 cells transfected with miR-21 expression vector (C and D) and MHCC-97H treated with miR-21 inhibitor (E and F) were tested by migration and invasion assay in transwell plates (original magnification 100). The penetrated cells were counted and analyzed in histogram; P < 0.01 versus the parental cell (Blank) and negative vector control groups (Negative).
groups (Fig. 6E and F). The results suggested that miR-21 can influence the proliferation and neoplastic capacity of HCC cells. 3.7. miR-21 promotes the growth of HCC cell xenografts in nude mice The parental, pGL3-miR21-EGFP-transfected and pGL3-CtrlEGFP-transfected SMMC-7721 cells were used to establish HCC xenograft models in nude mice. Three weeks after cell transplantation, tumors in the miR21-transfected group grew more quickly compared with the parental and negative control groups, and the difference was further intensified on day 35 (Fig. 7A). Tumor weight in the miR21-transfected group was higher than that in the other two groups (Fig. 7B). In the miR21-transfected tumor cells, the expression of miR-21 was positively increased (Fig. 7C), the expression of both PTEN and hSulf-1 was down-regulated, and the expression of p-AKT, p-ERK and Ki67 was up-regulated, compared with the other two groups (Fig. 7D).
4. Discussion Cancer metastasis is a multistep and multifactorial process, it is a major cause of cancer-related death. EMT is a crucial event in the invasion and migration of tumor cells, which contributes to cancer cells invading through basement membrane and stroma into blood or lymph vessels, facilitates anoikis resistance and avoids immune destruction, and may ensure tumor cells to survive the journey to the metastatic site [4,30]. A growing number of evidence has demonstrated that the process of EMT is tightly linked with the sequences and compositions of miRNA molecules. miRNAs are thought to interact with multiple mRNAs which are involved in the EMT process [31]. miR-21, as a representative oncogene in the family of miRNAs, is an important component of the cellular signaling circuitry that regulates the EMT program [3,13]. PTEN, as a typical target gene of miR-21, controls a variety of biological processes including cell proliferation, growth, metastasis, and
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Fig. 6. Influences of miR-21 on proliferation and neoplastic capacity of HCC cells. (A and B) Cell viability of SMMC-7721 cells transfected with expression vector of miR-21 (A) and MHCC-97H cells treated with inhibitor of miR-21 (B) was measured by MTT assay; P < 0.01 versus the parental cell group (Blank) and negative vector control group (Negative). (C–F), Neoplastic capacity of SMMC-7721 cells transfected with over-expression vector of miR-21 (C and D) and MHCC-97H treated with inhibitor of miR-21 (E and F) was assessed by anchorage-independent growth assay. Cell colonies were counted and analyzed in histogram; P < 0.01 versus the blank and negative groups.
death. Therefore, PTEN may also be subjected to deliberated regulation in EMT process. To investigate the role of miR-21 in regulating EMT and effect on metastasis, growth and neoplastic capacity of HCC, we selected two HCC cell lines, SMMC-7721 and MHCC-97H, with obvious different expression levels of miR-21, which were demonstrated to have different metastatic potentials. MHCC-97H, a highly metastatic HCC cells, expressed a higher level of miR-21. Conversely, SMMC-7721 is a low metastatic HCC cells and was demonstrated to have a low level of miR-21. By up-regulating miR-21 expression
in SMMC-7721 cells, and down-regulating miR-21 expression in MHCC-97H cells, it was found that miR-21 not only regulates the expression of PTEN, but also influences the expression of hSulf-1. PTEN as miR-21 target gene was reported previously [14], and this is the first evidence to find that the hSulf-1 gene is also target of miR-21. We could not find the corresponding miR-21 binding sites within 30 -UTR of hSulf-1. Consequently, we guess that the hSulf-1 is an indirect target gene of miR-21. As we know, hSulf-1 is a heparin-degrading endosulfatase that can hydrolyze the sulfate ester bonds of heparan sulfate proteogly-
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Fig. 7. Effect of miR-21 on HCC xenograft tumor growth in nude mice. (A) The parental, pGL3-miR21-EGFP-transfected and pGL3-Ctrl-EGFP-transfected SMMC-7721 cells were subcutaneously injected into mouse right flank at 106 cells to establish HCC xenograft models, n = 10 per group. Tumor volume was measured regularly; P < 0.01 versus the parental cell group. (B) Comparison of tumor weight on day 35 after cell transplantation; P < 0.05 versus the parental cell group. (C) miR-21 expressions in xenograft tumors were examined by qRT-PCR; P < 0.01 versus the parental cell group. (D) Expression of PTEN, hSulf-1, p-AKT, p-ERK and Ki67 in xenograft tumors were quantified by immunohistochemistry in percentages of positive cells within 5 medium-power fields under microscope and showed in histograms; P < 0.05, P < 0.01 versus the parental cell group; original magnification 200.
cans (HSPG) and modulate the sulfation status of HSPG. The sulfation of N-acetylglucosamine residues of HSPG on cellular surface is critical for heparin binding growth factor signaling [23,32]. Once the sulfation of HSPG was desulfated by hSulf-1, many heparin binding growth factor signal pathways are negatively modulated with a decreased activity of the growth factor receptor tyrosine kinases, such as the receptors of fibroblast growth factor (FGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) [33]. Much substantial evidence indicated that hSulf-1 expresses at low levels in several types of cancers, including HCC, ovarian and breast cancer
[25,34], which is associated with the processes of carcinogenesis, angiogenesis, tumor growth and invasion [32–35]. Re-expression of hSulf-1 in cancer cells can diminish the cascade phosphorylation of a series of kinases including the ERK, MEK and AKT, and followed by inactivation of downstream signaling pathways [34,35]. Therefore, hSulf-1 plays an important role in the regulation of cancer cell microenviroment construction. miR-21 was reported to over-express in some cancers and mediate tumor growth [17,36,37]. For further understanding the function of miR-21 and its relation to HCC cellular migration, invasion and neoplastic capacity, we modified miR-21 expression with
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miR-21 expression vector or miR-21 inhibitor. The increase of miR21 expression could decrease PTNE and hSulf-1 expression levels, followed by increase of AKT and ERK phosphorylation levels. Concurrently, HCC cells were induced to show EMT phenomenon and their capacity of proliferation and motility was enhanced. The decrease of miR-21 expression in HCC cells resulted in opposite changes. Importantly, whatever in the PTEN-knocked down HCC cells or hSulf-1-knocked down HCC cells, inhibition of miR-21 expression could suppress the activity of AKT and ERK signaling, demonstrating that miR-21 can separately regulate the hSulf-1mediated and PTEN-mediated signal pathways. During the invasion and metastasis of cancers, it is important for tumor cells to acquire a movement ability and anoikis-resistence behavior. EMT is a multistage process that contributes to cancer cells with dramatic changes in cellular morphology, loss and remodeling of cell–cell and cell–matrix adhesions, and gain of migratory and invasive capabilities [38]. In the past years, several signal pathways have been identified that are critical for EMT in cancer progression and metastasis. Activation of AKT and ERK signaling by growth factors notably triggers EMT and endows cancer cells with strong ability to survive or to initiate metastatic tumors [39]. PTEN is a dual protein/lipid phosphatase inside the cells, inactivation of PTEN in cancer cells leads to activated downstream signaling including AKT and/or ERK. Therefore, it is not difficult to understand that miR-21-mediated decrease of PTEN expression can activate downstream AKT and ERK signaling, and enhance cancer cell proliferation and movement ability by inducing EMT in HCC cells. Moreover, this study also found that miR21 can suppress hSulf-1 and PTEN expression in HCC xenograft models in nude mice and increase the phosphorylation of AKT and ERK. The nuclear proliferation antigen Ki67 was up-regulated in miR-21-transfected HCC xenografts, which demonstrated that miR-21 increased the proliferation activity of HCC cells, and promote tumor growth. This new insight into miR-21 regulatory function on HCC cells may help us to design strategies for cancer target biotherapy. In summary, miR-21 can regulate HCC cellular proliferation, migration, invasion and tumor growth by inducing EMT through AKT/ERK pathways. Interestingly, miR-21 separately suppresses the expression of hSulf-1 and PTEN in HCC cells, which may be considered the main mechanism for activating AKT/ERK pathways. In particular, hSulf-1 expression is important in the regulation of signal transduction form outside into inside the cells, and miR-21mediated the suppression of hSulf-1 expression and the cascade activation of many growth factor signal pathways are helpful for cancer cells to construct the favorable metastatic microenviroment. These findings may provide candidate targets in prevention and treatment of HCC recurrence and metastasis. Conflict of Interest None. Acknowledgments This work was supported by the Plan Project of Shanghai Outstanding Academic Leaders (13XD1400300 to C. Su), the National Natural Scientific Foundation of China (Nos. 81071866 to C. Su, 81170499 to J. Chen and 81172308 to C. Xu), and the Opening Research Subject from Jiangsu Key Laboratory of Tumor Biological Therapy (ZL1204 to Y. Yan).
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Contents lists available at SciVerse ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet
MicroRNA-21 suppresses PTEN and hSulf-1 expression and promotes hepatocellular carcinoma progression through AKT/ERK pathways Longlong Bao a,1, Yan Yan a,1, Can Xu b,1, Weidan Ji a, Shuwen Shen a, Gaoya Xu a, Yong Zeng a, Bin Sun a, Haihua Qian a, Lei Chen a, Mengchao Wu a, Changqing Su a,⇑, Jie Chen b,⇑ a b
Department of Molecular Oncology, Eastern Hepatobiliary Surgical Hospital & Institute, Second Military Medical University, Shanghai 200438, China Changhai Hospital, Second Military Medical University, Shanghai 200168, China
a r t i c l e
i n f o
Article history: Received 1 April 2013 Received in revised form 27 April 2013 Accepted 4 May 2013
Keywords: Hepatocellular carcinoma MicroRNA Signaling Epithelial–mesenchymal transition Human sulfatase-1
a b s t r a c t MicroRNAs (miRNAs) have been believed to associate with malignant progression including cancer cell proliferation, apoptosis, differentiation, angiogenesis, invasion and metastasis. However, the functions of miRNAs are intricate, one miRNA can directly or indirectly target multiple genes and function as oncogene or tumor suppressor gene. In this study, we found that miR-21 inhibits PTEN and human sulfatase-1 (hSulf-1) expression in hepatocellular carcinoma (HCC) cells. The hSulf-1 is a heparin-degrading endosulfatase, which can inhibit the heparin binding growth factor-mediated signaling transduction into cells. Therefore, miR-21-mediated suppression of both hSulf-1 and PTEN led to activation of AKT/ERK pathways and epithelial–mesenchymal transition (EMT) in HCC cells, and finally enhance the activity of HCC cell proliferation and movement and promote HCC xenograft tumor growth in mouse models. These findings may provide candidate targets for prevention and treatment of HCC. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cancer is a complex multipronged physiological and pathological process that involved in the activation of oncogenic pathways and/or inactivation of tumor suppressor signals [1]. The proliferation and invasion of tumor cells are considered as a foundation of cancer survival and development. Accumulating evidences demonstrated that the induction of epithelial–mesenchymal transition (EMT) and aberrant expression of microRNAs (miRNAs) are associated with tumorigenesis, tumor progression, metastasis and relapse in cancers, including hepatocellular carcinoma (HCC) [2–4]. EMT describes the process which epithelial cell gradually lose polarity and typical epithelial characteristics, then show characteristics of mesenchymal cells [5]. EMT phenomenon endows epithelial cells with enhanced migratory and invasive properties, and Abbreviations: HCC, hepatocellular carcinoma; EMT, epithelial–mesenchymal transition; AKT, PI3 K-protein kinase B; ERK, extracellular signal regulated kinase; hSulf-1, human sulfatase-1; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; HSPG, heparan sulfate proteoglycans. ⇑ Corresponding authors. Addresses: Department of Molecular Oncology, Eastern Hepatobiliary Surgical Hospital & Institute, Second Military Medical University, Shanghai 200438, China. Tel./fax: +86 21 8187 5351 (C. Su), Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China. Tel./fax: +86 21 8187 3222 (J. Chen). E-mail addresses: [email protected] (C. Su), [email protected] (J. Chen). 1 These authors contributed equally to this work. 0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2013.05.007
increasing data are provided to show the existence of EMT phenotypes in the invasion and migration process of cancer cells [6]. Inducers of EMT include some protein polypeptides, transcription factors, growth factors and miRNAs. As a marker of epithelial cells, E-cadherin regulates homotypic cell–cell adhesion and binds to cytoskeletal components to mediate the morphology, polarity and behavior of epithelial cells [7]. Accompanied by the change of E-cadherin, the expression of mesenchymal markers such as N-cadherin, Vimentin, and alpha-SMA also have corresponding changes [8,9]. miRNAs are a family of small highly conserved endogenous non-coding RNAs which inhibit translation of target genes by base pairing to the 30 -UTR of mRNAs. Recently, a series of miRNAs have been shown to play critical roles in the processes of EMT of cancer cells to promote the progression and metastasis of human malignancies, including human HCC [2,4,10–12], in which miR-21 is an important component involved in the cellular signaling pathways that regulates EMT processes [3,13] and is always up-expressed in many kinds of malignancies. Recent studies showed that several tumor suppressors including PTEN, TPM1, PDCD4, maspin, and TIMP3 were targets of miR-21 [14–18], suggesting that miR-21 is an important oncogenic miRNA which is closely related to tumor growth and metastasis [2]. As a typical target gene of miR-21, PTEN negatively regulates the PI3K-protein kinase B (AKT) pathway [19] and mitogen-activated protein kinase/extracellular signal regulated kinase (ERK) pathway [20], but it always lowly expressed in
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some cancer cells [14], partly because of inactivation by miR-21. Antagonism of miR-21 could re-activate PTEN and inactivate AKT and ERK pathways through PTEN up-regulation and finally reverse EMT in MDA-MB-231 cells [2]. Up-regulation of miR-21 can inhibit PTEN expression and lead to an activation of AKT and ERK pathways, finally enhance HIF-1a and VEGF expression and induce tumor angiogenesis [21]. However, we found that, in PTEN-silenced cancer cells, manipulation of miR-21 expression also results in the changes of AKT and ERK pathway activity, demonstrating that there is another mechanism involved in the regulation of AKT and ERK signaling besides PTEN. In this study, we have revealed that up- or down-regulation of miR-21 expression in HCC cells could change the human sulfatase-1 (hSulf-1) expression and downstream AKT and ERK signaling activity, then regulate cancer cell migration and invasion by inducing cellular EMT. The hSulf-1 is always expressed at lower levels in several tumor types, including ovarian, breast, and hepatocellular cancers, compared with the expression levels in the corresponding normal tissues [22,23]. Over-expression of hSulf-1 could reduce the activity of ERK and AKT signaling and suppress cell growth, migration and invasion [24,25]. The results strongly suggested that miR-21 might regulate hSulf-1-mediated AKT and ERK pathways in HCC, finally influence cancer progression, invasion and metastasis. Anyway, we provided the first evidence that miR-21 promotes cancer progression, invasion and metastasis by hSulf-1-mediated AKT and ERK pathways in HCC, which may provide a candidate target for cancer gene therapy. 2. Materials and methods 2.1. Cell lines and cell culture The human HCC cell lines MHCC-97H and MHCC-97L were obtained from the Liver Cancer Institute of Fudan University (Shanghai, China). SMMC-7721 was purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). The human liver cell line WRL-68 and the fibroblast cell line MRC-5 were purchased from the ATCC (Manassas, VA, USA). These cells were maintained in high-glucose DMEM (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal calf serum (Hyclone, Logan, UT, USA), 100 units/ml penicillin, 100 lg/ml streptomycin and 1% glutamine (Invitrogen, Grand Island, NY, USA) and incubated in a humidified atmosphere with 5% CO2 at 37 °C. To further investigate the ability of cancer cell inoculation and mimic cancer cell detachment and metastasis in animal model, MHCC-97H and SMMC-7721 were injected into the abdominal cavity of BALB/c (nu/nu) mice, 3 mice in each group, 2.5 106 cells per mouse. Two weeks later, the colonization of cancer cells in mice was monitored by anatomy.
2.2. Vectors and inhibitor The miR-21 expression vector of pGL3-miR21-EGFP and its negative control vector pGL3-Ctrl-EGFP containing the enhanced green fluorescent protein (EGFP) gene were constructed and preserved. The plasmid pSUPER-shSulf1 containing the hSulf-1 shRNA (19 oligonucleotide pairs targeting hSulf-1 cDNA positions 294–312: 50 -GTATGTGCACAATCACAAT-30 ) was kindly gifted from Viji Shridhar (Department of Experimental Pathology, Mayo Clinic Cancer Center, Rochester, MN, USA). The plasmid pGenesil-shPTEN containing the PTEN shRNA (19 oligonucleotide pairs targeting PTEN cDNA positions 1026–1044: 50 -GGTGAAGCTGTACTTCACA-30 ), the miR-21 inhibitor, and the negative control plasmid pGenesil-shNC (19 oligonucleotide pairs: 50 -gacttcataaggcgcatgc-30 ) were purchased from GenePharma Inc., Shanghai, China.
2.3. Transfection Totally 5 105 cells were seeded in six-well plates and grown to 80% confluence. Transfection with the miR-21 and shRNA vectors into HCC cells was performed at a concentration of 2 lg/well using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer’s protocol. miR-21 inhibitor was directly worked on MHCC-97H cells at a final concentration of 100 nmol/L. All groups were performed in triplicate. Twenty-four h after transfection, the transfected cells were observed under a fluorescent microscope to judge the transfection efficiency and the morphological change, followed by G418 selection and cultured for another 24 h. Finally, the transfected cells were harvested for total RNA and protein extraction, or miRNAs isolation.
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2.4. Real time qRT-PCR analysis The miR-21 reverse transcription (RT) primer (50 -CGTCGCTACATCGAGTGT AGCATATGCGACGTCAACATC-30 ) and qPCR primers (Forward: 50 -TAGCTTATCAGACTGATG-30 ; Reverse: 50 -ACATCGAGTGTAGCATA-30 ) were synthesized by Sangon Biotech., Inc. (Shanghai, China). miRNAs was extracted from cells using a mirVana miRNA Isolation Kit (Applied Biosystems, Foster City, CA, USA). The real time qRT-PCR was performed to measure the expression of miR-21 by a MiniOpticon™ Two-Color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). U6 was used as the inner control. 2.5. Western blotting analysis Cells were washed twice with PBS and lysed on the culture dishes using RIPA lysis buffer (1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 150 mmol/L NaCl and 10 mmol/L Tris–HCl) containing 1/100 phenylmethanesulfonyl fluoride (PMSF) solution. The total protein concentration was determined using the BCA method and 30 lg of each sample was separated by SDS-PAGE (8, 10, or 12%) and transferred to PVDF membrane. Non-specific binding sites were blocked by incubating with TBST containing 5% (w/v) non-fat dried milk for 1 h at room temperature. The membrane was immunoblotted overnight at 4 °C and HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibody was incubated with the membrane for 1 h after three washes with TBST. The primary antibodies included rabbit anti-human hSulf-1, mouse anti-human AKT, rabbit anti-human p-AKT (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-human PTEN, ERK1/2 and p-ERK1/2 (Cell Signaling Technology, Danvers, MA, USA); rabbit anti-human E-cadherin, mouse antihuman b-catenin, mouse anti-human Vimentin (Bioworld Technology Inc., St. Louis Park, MN, USA) and mouse anti-human glyceraldehydes 3-phosphate dehydrogenase (GAPDH) antibodies (Kangchen Bio-tech, Shanghai, China). The secondary antibodies included horseradish peroxidase-conjugated sheep anti-mouse IgG and sheep anti-rabbit IgG (R&D Systems China, Shanghai, China). Signals were visualized by ECL chemiluminescence. Equal protein loading was assessed by the expression of GAPDH. The bands were semi-quantified using ImageJ software. 2.6. Cell proliferation assay Cell proliferation assay was performed by 3-(4,5-dimethylthiazol-2-yl)-2 5diphenyltetrazolium bromide (MTT) assay. After transfected with the indicated plasmid or inhibitor, cells were plated in 96-well plates at 5 103 per well in 200 lL culture medium. After 48 h, 20 lL of 5 mg/mL MTT was added to each well, then the cells were incubated for 4 h before 150 lL DMSO was added. Once the insoluble crystals were completely dissolved, the absorbance values at 570 nm were measured by micro-enzyme-linked immunosorbent assay plate reader. 2.7. Migration and invasion assays Cell migration and invasion were evaluated using transwell chamber assay (Millipore, Billerica, MA, USA) according to the manufacturer’s instruction. For invasion assay, totally 5 104 cells were seeded on an 8-lm pore size transwell insert coated with extracellular matrix (ECM) (1:6) (BD Biosciences, China), while cell migration assay did not coat with ECM. After incubated at 37 °C for 48 h, the cells adherent to the upper surface of the filter were removed using a cotton applicator, then stained with crystal violet, and the values obtained were calculated by averaging the total numbers of cells from triplicate determinations. 2.8. Wound healing assay Cells were seeded in six-well plates at a density of 4 105 cells per well and cultured for 24 h at 37 °C. Wounds were created in the cell monolayer by a pipette tip and the indicated plasmids or inhibitor were transfected into cells. The dead cells were washed away with PBS. Images were taken at 0 and 48 h. Migration distance was detected by the ImageJ software. 2.9. Soft agar colony formation assay To test the capacity of cell colony growth, cells were collected after treated, and 1 104 cells were mixed with a 0.6% agar solution in DMEM containing 20% fetal bovine serum (FBS) and layered on top of a 1.2% agar layer in six-well tissue culture plates. After the gel solidified, cells were incubated for 2 to 3 weeks in a 5% CO2 humidified incubator at 37 °C until colonies were formed. Cultural medium of DMEM supplemented with 10% FBS was added every 3 to 4 days. The colonies containing more than 50 cells were counted under microscope. 2.10. Animal HCC xenograft experiments Thirty male BALB/C nude mice at 4 weeks of age (Shanghai SLAC Laboratory Animal Center of Chinese Academy of Sciences, Shanghai, China) were randomly divided into three groups (n = 10 per group) and subcutaneously injected the parental, pGL3-miR21-EGFP-transfected and pGL3-Ctrl-EGFP-transfected SMMC-7721
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cells, respectively, into mouse right flank at 1 106 cells/100 lL per mouse. On day 14 post-inoculation, the tumorigenic rate in every group was 100%. The tumor volume was measured every week and calculated using the formula ‘‘a b2 0.5’’, in which a and b represent the maximal and minimal diameters, and the tumor inhibition rate was calculated using the formula ‘‘(tumor volume of the control group – tumor volume of the treatment group)/tumor volume of the control group 100%. On days 35 after cell implantation, the tumor volume in the miR21-transfected group was overload beyond the standard approved by the Animal Care Committee of Second Military Medical University, and the mice were sacrificed by anesthetization, the tumor specimens were removed and weighed. The fresh specimens were prepared for examining miR-21 expression by qRT-PCR and the paraffin-embedded consecutive sections were cut for examining the expression of hSulf-1, PTEN, p-AKT, p-ERK and Ki67 (mouse anti-Ki67 antibody: GeneTex Inc., Irvine, CA, USA) by immunohistochemistry with the streptavdin–peroxidase (S–P) kit (Fuzhou Maixin Biotechnology Development Co., Fuzhou, China). Each slice was enumerated under 5 fields of medium magnification (200) to determine the proportion of positive cells. 2.11. Statistical analysis All data are expressed as mean ± standard deviation (SD). Statistical evaluation of the data was performed with one-way ANOVA. Pair-wise comparisons were conducted by Student’s t test. The P value less than 0.05 was considered statistically significant. All analyses in the study were evaluated with SPSS version 17.0 software.
3. Results 3.1. miR-21 expression is up-regulated in highly metastatic cancer cells miR-21 is widely over-expressed in tumorigenesis including HCC cell lines [14]. Furthermore, miR-21 was closely connected
with motility, migration, and invasion of human cancers. To explore the expression of miR-21 in cancer cell lines, different cancer cell lines varied in their metastatic potentials, including MHCC-97H, MHCC-97L and SMCC-7721, were selected and measured the relative expression of miR-21 by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), compared with normal cell lines WRL-68 and MRC-5. The relative expression of miR-21 was significantly up-regulated in MHCC97H and MHCC-97L cell lines (6.73 ± 1.01 in MHCC-97H, 4.35 ± 0.53 in MHCC-97L; P < 0.01 versus normal cell lines), but not in SMMC-7721 cells (1.10 ± 0.13; P = 0.582 versus normal cell lines; Fig. 1A). To confirm the metastatic potential of miR-21 high expression cells (MHCC-97H) and miR-21 low expression cells (SMCC-7721), the harvested cells were injected into the abdominal cavity of BALB/c (nu/nu) mice at 2.5 106 cells per mouse. Two weeks later, mice were dissected under body vision microscope and the cancer nodules were monitored and counted. The results showed that MHCC-97H cells formed more cancer nodules with larger size than SMCC-7721 cells (Fig. 1B). 3.2. Over-expression of miR-21 induced EMT by regulating the activity of PTEN- and hSulf-1-mediated AKT and ERK signaling in HCC cells To further confirm the role of miR-21 in regulating cancer cell EMT, we constructed an expression vector of GFP-tagged miR-21 and transfected into SMMC-7721 cells. Totally 5 105 SMMC7721 cells were transfected with miR-21 vectors at a concentration
Fig. 1. Expression of miR-21 in HCC cells and its relation to cellular metastatic ability. (A) miR-21 expression was detected by real time qRT-PCR, the results were normalized to U6 expression; P<0.01 versus the average value of two normal cell lines. (B) MHCC-97H and SMMC-7721 were injected into the abdominal cavity of BALB/c (nu/nu) mice (n = 3 in every group) at 2.5 106 cells per mouse. Two weeks later, mice were dissected under body vision microscope and the number of cancer nodules and total weight of cancer nodules in every mouse were monitored and counted; P < 0.05, P < 0.01.
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Fig. 2. Re-expression of miR-21 induced EMT by down-expression of PTEN and hSulf-1 and activation of AKT and ERK signaling. (A) SMMC-7721 cells were transfected with the expression vector of GFP-tagged miR-21, and miR-21 expression was detected by real time qRT-PCR. The results were normalized to U6 expression; P < 0.01 versus the parental cell group (Blank) and the negative vector control group (Negative). (B), Expression levels of EMT markers (E-cadherin, vimentin and b-catenin) were measured by Western blotting, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control, and bands were semi-quantitatively analyzed by using ImageJ software, normalized to GAPDH density; P < 0.05, P < 0.01 versus the blank and the negative control groups. (C) Mesenchymal morphological change was induced by overexpression of miR-21 tagged with GFP in SMMC-7721 cells, typical images were showed by normal light field (original magnification 100) and fluorescent field (original magnification 200). (D) Expression levels of PTEN, hSulf-1, p-AKT and AKT, as well as p-ERK and ERK were semi-quantitatively analyzed by using ImageJ software, normalized to GAPDH density; P < 0.05, P < 0.01 versus the blank and the negative control groups. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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of 2 lg/well, the transfection efficiency was (65.10 ± 2.00)% 24 h after transfection. Followed by G418 selection, the transfection efficiency was (92.32 ± 15.22)%. In the miR-21-transfected cells, miR-21 expression was increased to more than 18-fold, as compared with the parental cells without transfection (Fig. 2A), indicating that transfection of miR-21 expression vector could increase the relative expression of miR-21. To examine whether the forced re-expression of miR-21 could induce EMT, the protein expression of EMT biomarkers, the phosphorylation levels of EMT-associated AKT and ERK signaling and the expression of their upstream signal regulatory factors, PTEN and hSulf-1, were measured by Western blotting assay. Compared with the negative vector control group, over-expression of miR-21 increased the expression levels of b-catenin and vimentin, while decreased the expression of E-cadherin (Fig. 2B), suggesting that over-expression of miR-21 could induce EMT in SMMC-7721 cells. After overexpression of miR-21, SMMC-7721 cells were induced a striking EMT-like transformation, which was morphologically characterized by a spindle-shaped change and loss of cell-cell contacts (Fig. 2C). Meanwhile, over-expression of miR-21 decreased the expression of PTEN and hSulf-1 and increased the expression of p-AKT and p-ERK in SMMC-7721 cells (Fig. 2D), suggesting that over-expression of miR-21 could activate AKT and ERK pathways. These results supported that miR-21 could regulate EMT by modifying the activity of both PTEN- and hSulf-1-mediated AKT/ERK pathways.
3.3. Inhibition of miR-21 could reverse EMT by inactivating AKT and ERK signal activity through up-regulation of PTEN and hSulf-1 expression in HCC cells MHCC-97H is a high-metastatic HCC cell line and has a high expression level of miR-21 and low expression of PTEN and hSulf-1. To further investigate the effect of miR-21 on EMT process of HCC cells, the inhibitor of miR-21 was transfected into MHCC97H cells and the expression of miR-21 was measured by real time qRT-PCR. The expression of miR-21 was decreased to more than 6fold after treated with miR-21 inhibitor, as compared to the parental cell control (Fig. 3A). The relative expression levels of EMT biomarkers were measured by Western blotting analysis. The results demonstrated that inhibition of miR-21 increased the protein expression of E-cadherin and decreased the expression of b-catenin and vimentin (Fig. 3B). Meanwhile, the relative expression of pAKT and p-ERK was decreased accompany by an increase of PTEN and hSulf-1 (Fig. 3C), indicating that inhibition of miR-21 could reverse EMT by inactivating AKT and ERK pathways through up-regulation of PTEN and hSulf-1. 3.4. miR-21 can separately regulate the hSulf-1-mediated and PTENmediated AKT and ERK signal pathways Both PTEN and hSulf-1 can regulate the activity of AKT and ERK pathways [19,26–28]. It was reported that miR-21 can inactivate
Fig. 3. Inhibition of miR-21 induced an increased expression of PTEN and hSulf-1 and inactivated AKT and ERK signaling to reverse EMT. (A) MHCC-97H was transfected with miR-21 inhibitor to inhibit miR-21 expression; P < 0.01 versus the parental cell control group. (B) EMT markers (E-cadherin, vimentin and b-catenin) were detected by Western blot analysis, GAPDH was used as loading control, and bands were semi-quantitatively analyzed by using ImageJ software, normalized to GAPDH density; P < 0.05, P < 0.01 versus the parental cell group (Blank) and negative vector control group (Negative). (C) Protein levels of PTEN, hSulf-1, p-AKT, AKT, p-ERK and ERK were detected by Western blot analysis, and bands were semi-quantitatively analyzed by using ImageJ software, normalized to GAPDH density; P < 0.05, P < 0.01 versus the blank and negative control groups.
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PTEN-mediated signaling [14,29], but the effect of miR-21 on hSulf-1-mediated signaling was not reported previously. To further differentiate the respective effect of PTEN and hSulf-1 on AKT and ERK pathways under the regulation of miR-21, MHCC-97H cells were transfected with PTEN-small hairpin RNA (shRNA) vector (pGenesil-shPTEN) and hSulf-1-shRNA (pSUPER-shSulf1), respectively, to block the expression of PTEN and hSulf-1, then generated the cell lines, MHCC-97HPTEN— and MHCC-97HhSulf-1—. When the cells were down-regulated miR-21 expression by miR-21 inhibitor, hSulf-1 expression was increased only in MHCC-97HPTEN— cells and PTEN expression was increased only in MHCC-97HhSulf-1— cells. More interestingly, whether the increase of hSulf-1 or PTEN expression, the phosphorylation levels of AKT and ERK were decreased both in MHCC-97HPTEN— and MHCC-97HhSulf-1— cell lines (Fig. 4). 3.5. miR-21 enhances cellular motility in HCC cells To investigate the effect of miR-21 on cellular motility, HCC cells with different expression status of miR-21, including SMMC-7721 and MHCC-97H cell lines, were measured by wound healing, transwell migration and invasion assays after transfected miR-21 expression vector or treated with miR-21 inhibitor. Transfection of miR-21 expression vector significantly enhanced the capacity of wound healing in SMMC-7721 cells, as compared with the parental control cells without transfection (Fig. 5A). However, the capacity of wound healing in MHCC-97H cells was significantly attenuated after treated with miR-21 inhibitor, as compared to the parental control cells without treatment (Fig. 5B).
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Meanwhile, after transfected with miR-21 expression vector, the relative migrated and invaded cell number of SMMC-7721 cells was significantly more than that of the parental control cells (Fig. 5C and D), and the relative migrated and invaded cell number was significantly decreased in the miR-21 inhibitor-treated MHCC97H cells, as compared with the parental control cells (Fig. 5E and F), suggesting that miR-21 plays an important role in the regulation of HCC cellular motility, including the cancer cell invasive and metastatic capacity. 3.6. miR-21 influences the proliferation and neoplastic capacity of HCC cells To corroborate the effect of miR-21 on proliferation and neoplastic capacity in HCC cells, we measured the cancer cell viability and neoplastic capacity of SMMC-7721 cells and MHCC-97H cells after modification of miR-21 expression by MTT assay and anchorage-independent growth assay. The results demonstrated that the cell viability of SMMC-7721 cells transfected with miR-21 expression vector was significantly increased compared with the parental cell and negative control groups (Fig. 6A), and the cell viability of MHCC-97H cells transfected with miR-21 inhibitor was significantly lower than that of the control groups (Fig. 6B). After 2 weeks post-treatment, we also examined the capacity of colony formation of SMMC-7721 cells and MHCC-97H cells. The results suggested that SMMC-7721 cells displayed more colonies when transfected with over-expression vector of miR-21, compared with the control groups (Fig. 6C and D), and MHCC-97H that transfected with inhibitor of miR-21 displayed less colonies, compared with the control
Fig. 4. miR-21 regulated separately both hSulf-1-mediated and PTEN-mediated AKT and ERK signal pathways. MHCC-97H cells were transfected with PTEN-shRNA (pGenesilshPTEN) and hSulf-1-shRNA (pSUPER-shSulf1) vectors, respectively, at a vector concentration of 20 lg/well to 5 105 cells in six-well plates to establish cell sublines MHCC97HPTEN— (A) and MHCC-97HhSulf-1— (B). These cell sublines were treated with miR-21 inhibitor at a final concentration of 100 nmol/L, and the expression of miR-21, hSulf-1, PTEN, AKT and ERK was examined by qRT-PCR or Western blotting. The band densitometry was analyzed normalized to GAPDH density; P < 0.05, P < 0.01.
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Fig. 5. Effect of miR-21 on cellular motility in HCC cells. (A and B) Motility capacity of SMMC-7721 cells transfected with miR-21 expression vector (A) and MHCC-97H treated with miR-21 inhibitor (B) was assessed by wound healing assay (original magnification 100). (C–F) Migratory and invasive properties of SMMC-7721 cells transfected with miR-21 expression vector (C and D) and MHCC-97H treated with miR-21 inhibitor (E and F) were tested by migration and invasion assay in transwell plates (original magnification 100). The penetrated cells were counted and analyzed in histogram; P < 0.01 versus the parental cell (Blank) and negative vector control groups (Negative).
groups (Fig. 6E and F). The results suggested that miR-21 can influence the proliferation and neoplastic capacity of HCC cells. 3.7. miR-21 promotes the growth of HCC cell xenografts in nude mice The parental, pGL3-miR21-EGFP-transfected and pGL3-CtrlEGFP-transfected SMMC-7721 cells were used to establish HCC xenograft models in nude mice. Three weeks after cell transplantation, tumors in the miR21-transfected group grew more quickly compared with the parental and negative control groups, and the difference was further intensified on day 35 (Fig. 7A). Tumor weight in the miR21-transfected group was higher than that in the other two groups (Fig. 7B). In the miR21-transfected tumor cells, the expression of miR-21 was positively increased (Fig. 7C), the expression of both PTEN and hSulf-1 was down-regulated, and the expression of p-AKT, p-ERK and Ki67 was up-regulated, compared with the other two groups (Fig. 7D).
4. Discussion Cancer metastasis is a multistep and multifactorial process, it is a major cause of cancer-related death. EMT is a crucial event in the invasion and migration of tumor cells, which contributes to cancer cells invading through basement membrane and stroma into blood or lymph vessels, facilitates anoikis resistance and avoids immune destruction, and may ensure tumor cells to survive the journey to the metastatic site [4,30]. A growing number of evidence has demonstrated that the process of EMT is tightly linked with the sequences and compositions of miRNA molecules. miRNAs are thought to interact with multiple mRNAs which are involved in the EMT process [31]. miR-21, as a representative oncogene in the family of miRNAs, is an important component of the cellular signaling circuitry that regulates the EMT program [3,13]. PTEN, as a typical target gene of miR-21, controls a variety of biological processes including cell proliferation, growth, metastasis, and
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Fig. 6. Influences of miR-21 on proliferation and neoplastic capacity of HCC cells. (A and B) Cell viability of SMMC-7721 cells transfected with expression vector of miR-21 (A) and MHCC-97H cells treated with inhibitor of miR-21 (B) was measured by MTT assay; P < 0.01 versus the parental cell group (Blank) and negative vector control group (Negative). (C–F), Neoplastic capacity of SMMC-7721 cells transfected with over-expression vector of miR-21 (C and D) and MHCC-97H treated with inhibitor of miR-21 (E and F) was assessed by anchorage-independent growth assay. Cell colonies were counted and analyzed in histogram; P < 0.01 versus the blank and negative groups.
death. Therefore, PTEN may also be subjected to deliberated regulation in EMT process. To investigate the role of miR-21 in regulating EMT and effect on metastasis, growth and neoplastic capacity of HCC, we selected two HCC cell lines, SMMC-7721 and MHCC-97H, with obvious different expression levels of miR-21, which were demonstrated to have different metastatic potentials. MHCC-97H, a highly metastatic HCC cells, expressed a higher level of miR-21. Conversely, SMMC-7721 is a low metastatic HCC cells and was demonstrated to have a low level of miR-21. By up-regulating miR-21 expression
in SMMC-7721 cells, and down-regulating miR-21 expression in MHCC-97H cells, it was found that miR-21 not only regulates the expression of PTEN, but also influences the expression of hSulf-1. PTEN as miR-21 target gene was reported previously [14], and this is the first evidence to find that the hSulf-1 gene is also target of miR-21. We could not find the corresponding miR-21 binding sites within 30 -UTR of hSulf-1. Consequently, we guess that the hSulf-1 is an indirect target gene of miR-21. As we know, hSulf-1 is a heparin-degrading endosulfatase that can hydrolyze the sulfate ester bonds of heparan sulfate proteogly-
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Fig. 7. Effect of miR-21 on HCC xenograft tumor growth in nude mice. (A) The parental, pGL3-miR21-EGFP-transfected and pGL3-Ctrl-EGFP-transfected SMMC-7721 cells were subcutaneously injected into mouse right flank at 106 cells to establish HCC xenograft models, n = 10 per group. Tumor volume was measured regularly; P < 0.01 versus the parental cell group. (B) Comparison of tumor weight on day 35 after cell transplantation; P < 0.05 versus the parental cell group. (C) miR-21 expressions in xenograft tumors were examined by qRT-PCR; P < 0.01 versus the parental cell group. (D) Expression of PTEN, hSulf-1, p-AKT, p-ERK and Ki67 in xenograft tumors were quantified by immunohistochemistry in percentages of positive cells within 5 medium-power fields under microscope and showed in histograms; P < 0.05, P < 0.01 versus the parental cell group; original magnification 200.
cans (HSPG) and modulate the sulfation status of HSPG. The sulfation of N-acetylglucosamine residues of HSPG on cellular surface is critical for heparin binding growth factor signaling [23,32]. Once the sulfation of HSPG was desulfated by hSulf-1, many heparin binding growth factor signal pathways are negatively modulated with a decreased activity of the growth factor receptor tyrosine kinases, such as the receptors of fibroblast growth factor (FGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) [33]. Much substantial evidence indicated that hSulf-1 expresses at low levels in several types of cancers, including HCC, ovarian and breast cancer
[25,34], which is associated with the processes of carcinogenesis, angiogenesis, tumor growth and invasion [32–35]. Re-expression of hSulf-1 in cancer cells can diminish the cascade phosphorylation of a series of kinases including the ERK, MEK and AKT, and followed by inactivation of downstream signaling pathways [34,35]. Therefore, hSulf-1 plays an important role in the regulation of cancer cell microenviroment construction. miR-21 was reported to over-express in some cancers and mediate tumor growth [17,36,37]. For further understanding the function of miR-21 and its relation to HCC cellular migration, invasion and neoplastic capacity, we modified miR-21 expression with
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miR-21 expression vector or miR-21 inhibitor. The increase of miR21 expression could decrease PTNE and hSulf-1 expression levels, followed by increase of AKT and ERK phosphorylation levels. Concurrently, HCC cells were induced to show EMT phenomenon and their capacity of proliferation and motility was enhanced. The decrease of miR-21 expression in HCC cells resulted in opposite changes. Importantly, whatever in the PTEN-knocked down HCC cells or hSulf-1-knocked down HCC cells, inhibition of miR-21 expression could suppress the activity of AKT and ERK signaling, demonstrating that miR-21 can separately regulate the hSulf-1mediated and PTEN-mediated signal pathways. During the invasion and metastasis of cancers, it is important for tumor cells to acquire a movement ability and anoikis-resistence behavior. EMT is a multistage process that contributes to cancer cells with dramatic changes in cellular morphology, loss and remodeling of cell–cell and cell–matrix adhesions, and gain of migratory and invasive capabilities [38]. In the past years, several signal pathways have been identified that are critical for EMT in cancer progression and metastasis. Activation of AKT and ERK signaling by growth factors notably triggers EMT and endows cancer cells with strong ability to survive or to initiate metastatic tumors [39]. PTEN is a dual protein/lipid phosphatase inside the cells, inactivation of PTEN in cancer cells leads to activated downstream signaling including AKT and/or ERK. Therefore, it is not difficult to understand that miR-21-mediated decrease of PTEN expression can activate downstream AKT and ERK signaling, and enhance cancer cell proliferation and movement ability by inducing EMT in HCC cells. Moreover, this study also found that miR21 can suppress hSulf-1 and PTEN expression in HCC xenograft models in nude mice and increase the phosphorylation of AKT and ERK. The nuclear proliferation antigen Ki67 was up-regulated in miR-21-transfected HCC xenografts, which demonstrated that miR-21 increased the proliferation activity of HCC cells, and promote tumor growth. This new insight into miR-21 regulatory function on HCC cells may help us to design strategies for cancer target biotherapy. In summary, miR-21 can regulate HCC cellular proliferation, migration, invasion and tumor growth by inducing EMT through AKT/ERK pathways. Interestingly, miR-21 separately suppresses the expression of hSulf-1 and PTEN in HCC cells, which may be considered the main mechanism for activating AKT/ERK pathways. In particular, hSulf-1 expression is important in the regulation of signal transduction form outside into inside the cells, and miR-21mediated the suppression of hSulf-1 expression and the cascade activation of many growth factor signal pathways are helpful for cancer cells to construct the favorable metastatic microenviroment. These findings may provide candidate targets in prevention and treatment of HCC recurrence and metastasis. Conflict of Interest None. Acknowledgments This work was supported by the Plan Project of Shanghai Outstanding Academic Leaders (13XD1400300 to C. Su), the National Natural Scientific Foundation of China (Nos. 81071866 to C. Su, 81170499 to J. Chen and 81172308 to C. Xu), and the Opening Research Subject from Jiangsu Key Laboratory of Tumor Biological Therapy (ZL1204 to Y. Yan).
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