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Involvement of miR-485-5p in hepatocellular carcinoma progression targeting EMMPRIN
Biomedicine & Pharmacotherapy 72 (2015) 58–65
Available online at
ScienceDirect www.sciencedirect.com
Original article
Involvement of miR-485-5p in hepatocellular carcinoma progression targeting EMMPRIN Xiangjun Sun a, Yonglei Liu b,c,*, Ming Li b, Mingchun Wang d, Yutong Wang c a
Department of Hepatobiliary Surgery, Linyi People’s Hospital, Linyi, Shangdong, China Research Center, Linyi People’s Hospital, Linyi, Shangdong, China c Liver Cancer Center, Zhongshan Hospital, Fudan University, Shanghai, China d Department of Surgery, Linyi People’s Hospital, Linyi, Shangdong, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 March 2015 Accepted 3 April 2015
EMMPRIN plays important roles in cancer development, which includes EMMPRIN 1, 2, 3, and 4 isoforms. EMMPRIN2 is the main component in human cancers, but its regulation by miRNAs is still unclear. In this study, we will investigate the mechanism of EMMPRIN regulation in hepatocellular carcinoma (HCC) by miRNAs. Through RT-PCR, we found that EMMPRIN2 was the main isoform in HCC cells. EMMPRIN2 was down-regulated significantly by predicted miRNAs and miR-485-5p was one of the miRNA that regulated EMMPRIN in HCC cell lines. It was verified that EMMPRIN was a target gene of miR-485-5p by using luciferase analysis assay. We found that miR-485-5p was significantly downregulated in HCC tissues and that its expression was inversely correlated with the TNM stage and metastasis in HCC samples. Results of cellular functions in HCC showed that miR-485-5p could inhibit cell proliferation and metastasis. Additionally, miR-485-5p overexpression suppressed HCC growth in vivo by downregulation of EMMPRIN. Our study for the first time demonstrated that miR-485-5p represses HCC invasive and metastatic capacities by targeting EMMPRIN expression. ß 2015 Published by Elsevier Masson SAS.
Keywords: EMMPRIN miR-485-5p Hepatocellular carcinoma Metastasis
1. Introduction Hepatocellular carcinoma (HCC) is the third cause for cancerrelated death in the world [1]. The incidence of HCC is much higher in China. Despite advances in HCC treatment, the 5-year overall survival rate remains very poor [2]. The molecular mechanism of HCC is very complicated and remains unclear [3,4]. It is needed to elucidate the mechanism of HCC carcinogenesis to find new targets or methods for developing effective strategies of HCC. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a widely expressed transmembrane glycoprotein which belongs to the immunoglobulin superfamily including four isoforms: EMMPRIN1, EMMPRIN2, EMMPRIN3 and EMMPRIN4 [5,6]. EMMPRIN involves in various physiological processes such as fetal development, reproduction, T cell differentiation, and neural and retinal functions [7–10]. EMMPRIN1 is a tissue specific protein expressed on retina tissue; EMMPRIN2 is the most predominant isoform, encoding two Ig-like extracellular domains and being well-known
* Corresponding author at: 27 Jiefang Road, Linyi People’s Hospital, Linyi 276000, Shangdong, China. Tel.: +86 05398226999. E-mail address: [email protected] (Y. Liu). http://dx.doi.org/10.1016/j.biopha.2015.04.008 0753-3322/ß 2015 Published by Elsevier Masson SAS.
as basigin/CD147/EMMPRIN; the other two isoforms were rarely reported which showed that EMMPRIN3 and EMMPRIN4 were increased in HCC tissues and EMMPRIN3 acted as an inhibitor of proliferation and invasion of HCC [11–13]. EMMPRIN expresses widely in many tumors, including liver cancer, lung cancer, breast cancer, bladder cancer, lymphoma, oral squamous cell carcinoma, glioma, melanoma, and others [14– 16]. Accordingly, EMMPRIN expression has been associated with known risk factors for breast cancer and with poor prognosis of breast cancer patients [14–16]. These suggest that EMMPRIN plays important roles in carcinogenesis. Moreover, it has been shown that higher expression of EMMPRIN was associated with poor prognosis of cancer patients and could serve as an independent predictor of poor survival in cancer. The most life-threatening aspects of the oncogenic process are invasion and metastasis. MicroRNAs (miRNAs) are small RNAs and its noncoding mRNA sequences containing about 22–29 nucleotides that act as important regulators of gene expression [17,18]. miRNAs inhibit gene expression on transcriptional or post-transcriptional levels by specifically binding the seed sequence of target genes, cleaving mRNAs or inhibiting their translation [19,20]. Most of human miRNAs are located in cancer-associated genomic regions in the
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chromosome, which function as inhibitor or onco-miRNAs depending on their targets in tumor. miRNAs have also been associated with HCC formation and growth [21,22]. In this study, we predicted that miR-485-5p was a regulator of EMMPRIN. Then we investigated the potential involvement of miR485-5p in HCC. We examined the expression of miR-485-5p in human HCC cells and tissues and tested its effects on cell growth, cell-cycle distribution and metastasis. We also investigated a potential role of miR-485-5p on tumorigenesis in a murine model. We identified that miR-485-5p was a tumor suppressor and negatively regulated EMMPRIN in progression of HCC.
2. Material and methods
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2.6. RNA isolation and real-time RT-PCR Total RNA, following the manufacturer’s instructions, was isolated from the cells using Trizol reagent (Invitrogen). Briefly, the cells were lysed in TRIzol and then mixed with chloroform. The lysate was centrifuged to separate RNA, DNA and protein, total RNA recovered, precipitated with isopropanol, washed in 75% ethanol to remove impurities before dissolved in water. After that, 2 mg of RNA was taken and treated with DNase to remove contaminating DNA prior to the reverse transcription to cDNA using SYBR1 PCR Kit (Takara, Japan). To measure mRNA expression, real-time RTPCR was performed using a sequence detector (ABI-Prism, Applied Biosystems). The primers were ordered from Kang-Chen Biotech (Kangcheng, Shanghai, China).
2.1. Cell culture BEL-7405, SMMC7721, HepG2 and Huh7 human liver cancer cell lines were primary purchased from ATCC (Manassas, VA, USA). BEL-7404 cells were stored in our lab. The cells were maintained in the Dulbecco’s Modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% fatal bovine serum (Gibco, USA) in a humidified incubator at 37 8C and 5% CO2. 2.2. Tissue samples Primary HCC and their adjacent tissues were obtained from the patients with HCC of our hospital (Linyi People’s Hospital, Shandong, China). Both tumor and normal tissues were histologically confirmed by H&E (hematoxylin and eosin) staining. The study was approved by the ethics committee of the hospital. 2.3. Antibodies and reagents EMMPRIN, MMP9, E-cadherin, Vimentin, FSP1 and a-SMA primary antibody were ordered from Santa Cruz (Dallas, TX, USA). Anti-GAPDH antibody and secondary antibodies conjugated with HRP were purchased from Kang-Chen Biotech (Kangcheng, Shanghai, China). EMMPRIN plasmid was ordered from Origene (Rockville, MD, USA). 2.4. Plasmid vector construction and lentivirus transduction Lentivirus vector expressing miR-485-5p was constructed using the BLOCK-iT pol II miR RNAi Expression Vector Kit with EmGFP (Invitrogen, Carlsbad, CA, USA). The primary miRNA sequence of miR-485-5p with flanking regions was obtained by PCR and inserted into the Block-iT Pol II miR RNAi Expression Vector, pcDNA6.2-GW/EmGFP-miR. For transfection, cells were seeded in antibiotic-free medium at a density of 30–40% with lipofectamine2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. 2.5. Transfection and dual luciferase assay The pGL3 plasmid was modified by adding the human 30 -UTR or the 30 -UTR with mutations in regions complementary to miR-4855p seed regions behind the firefly luciferase gene. Cells were plated on 24-well Plates 24 h before transfection. Transient transfection was carried out in Opti-MEM medium using lipofectamine-2000 according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). The medium was changed after transfection for 5 h, and the cells incubated at 37 8C for the indicated time. Cells were washed once with phosphate-buffered saline and luciferase assays were performed using the Dual Luciferase Assay System (Promega, WI, USA) according to the supplier’s protocol.
Fig. 1. miR-485-5p down-regulated EMMPRN expression in HCC cells. (A) The expression of EMMPRIN mRNA in five HCC cell lines. EMMPRIN 1 (NM_001728), 2 (NM_198589), 3 (NM_198590) and 4 (NM_198591) mRNA were examined by RT-PCR. (B) The most potential miRNAs targeted EMMPRIN2 in SMMC771 cells. The predicted miRNAs with high scores were verified by real time RT-PCR. (C) miR-485-5p inhibited EMMPRIN2 mRNA expression in HCC cell lines. The data presented are shown as means SD collected from three independent experiments. *P < 0.05, **P < 0.01.
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2.7. CCK8 The Cell Counting Kit-8 (CCK-8) (Beyotime, China) was used to determine cell viability according to the manufacturer’s instructions. Cells were plated at a density of 1–2 104 cells per well in 96-well microtiter plates and cultured overnight at 37 8C in a humidified incubator containing 5% CO2. Next, at different time points, the culture medium was replaced with 100 ml of fresh medium followed by the addition of 10 ml of CCK-8 solution. The cells were further incubated for 2 h at 37 8C, and the optical density at 450 nm was recorded. All experiments were performed in triplicate.
secondary antibody for about 3 h at RT. Washed in PBST (3, 10 min each wash), the proteins were finally visualized using ECL based on the manufacturer’s instructions. 2.9. Cell cycle analysis In 2 ml culture medium 2 105 cells/well (6-well plate) were seeded, and cultured for 16 h or added Nocodazole (5 mg/ml) for the indicated time before collection. The cells were stabilized with 75% ethanol for 24 h, and dyed with PI, and analyzed with ModFit of flow cytometry. 2.10. Colony formation assay
2.8. Western blotting The cells were scraped from the dishes, cellular protein extracts prepared by homogenization in an ice-cold lysis buffer and their lysates obtained by centrifugation at 12,000 g for 20 min, and the total protein concentration determined using Lowry method. Equal amounts of protein, separated by SDS-PAGE, were electrophoretically transferred to a PVDF membrane at 320 mA for 2 h at a low temperature and the membrane was blocked with 5% fat-free milk with 0.05% Tween 20 in PBS. Subsequently, the membrane was probed with the primary antibodies. The blots were washed in PBST and then incubated in anti-mouse IgG or anti-rabbit IgG
The cells were harvested, sparsely plated, and were cultured under the normal condition. The medium underwent the replacement at three-day intervals. And then the cells were fixed in 90% ethanol, stained with crystal violet and colonies consisting of at least 50 cells so they were counted ten days later. 2.11. Migration and invasion assay The migration assay was conducted as follows: 2 104 cells in 0.1 ml of media added to the upper chamber (Millpore, MA, USA), followed by an addition of 0.6 ml medium in lower chamber; those
Fig. 2. EMMPRIN is a target gene of miR-485-5p in human HCC cells. (A) The 30 -UTR of the EMMPRIN gene contains binding sites for miR-485-5p according to bioinformatic analysis. (B) miR-485-5p suppressed the expression of a luciferase reporter gene with the 30 -UTR of EMMPRIN. SMMC7721 and BEL-7404 cells were transiently co-transfected with the control or miR-485-5p together with the indicated luciferase constructs, and luciferase activity was analyzed 48 h later. Data are presented as relative firefly luciferase activity normalized to Renilla luciferase activity from the same construct. (C) miR-485-5p down-regulated EMMPRIN protein in SMMC7721 and BEL-7404 cells. Cells were transfected with miR-485-5p or its control for 48 h, and then collected for Western blot. (D) miR-485-5p down-regulated EMMPRIN mRNA in SMMC7721 and BEL7404 cells. Cells were transfected with anti-miR-485-5p or its control for 48 h, and then collected for real time RT-PCR. (E) miR-485-5p inhibition induced up-regulation of EMMPRIN in SMMC7721 and BEL-7404 cells. Cells were transfected with anti-miR-485-5p or its control for 48 h, and then collected for EMMPRIN immunoblotting. The data presented are shown as means SD collected from three independent experiments. **P < 0.01.
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on the surface of the polycarbonate membrane, non-invading cells, removed 16 h later by scraping with a cotton swab; those migrated to the pores of the underlying membrane fixed in 90% ethanol and stained with 0.1% crystal violet and counted under fluorescence microscopy (100), the mean and SD were calculated from three independent experiments. For invasion assay, there was Matrigel (BD Biosciences, San Jose, CA, USA) in the up-chamber. Invaded or migrated cells = Cells migrated through the membrane 100%/ Cells seeded in the well. 2.12. Statistical analysis Each experiment was repeated at least three times, Student’s t tests performed to determine the statistical significance for the assays of promoter activity, migration, FACS and colony formation, error bars representing SE. 3. Results 3.1. miR-485-5p down-regulates EMMPRN expression in HCC cells To investigate EMMPRIN regulation by miRNAs, firstly, we examined EMMPRIN expression in HCC cells by RT-PCR. It was found that EMMPRIN2 was the main isoform, EMMPRIN3 and EMMPRIN4 were very weak and there was no expression of EMMPRIN1 in HCC cells including HepG2, BEL-7404, BEL-7405 SMMC7721, Huh7 (Fig. 1A). So, the following research only focuses on EMMPRIN isoform 2. miRNA database was used to predict miRNAs targeting EMMPRIN. We selected miRNAs with high scores and verified them by real time RT-PCR (Fig. 1B). miR-485-5p was one of the potential regulator of EMMPRIN. When the above HCC cells were transfected with miR-485-5p, EMMPRIN2 mRNA inhibition was the most significant in the above HCC cell lines (Fig. 1C). EMMPRIN3 and 4 mRNA levels were inhibited by
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miR-485-5p; however, their expression was very weak, so further research will be focused on EMMPRIN2 (EMMPRIN in the following). 3.2. EMMPRIN is a target gene of miR-485-5p in human HCC cells We then investigated whether EMMPRIN is the direct target gene of miR-485-5p. EMMPRIN is predicted as the target gene of miR-485-5p (Fig. 2A). Assay of luciferase activity showed that the luciferase activity of pGL3-EMMPRIN-WT in the cells was much lower than in control cells. The luciferase activity of pGL3EMMPRIN-Mut was rescued in SMMC7721 and BEL-7404 cells (Fig. 2B). We next examined whether miR-485-5p could regulate EMMPRIN protein in the two HCC lines. It was found that EMMPRIN protein was suppressed greatly by miR-485-5p in SMMC7721 and BEL-7404 cells (Fig. 2C). EMMPRIN mRNA and protein levels increased greatly in SMMC7721 and BEL-7404 cells with anti-miR-485-5p compared with the control (Fig. 2D and E). 3.3. miR-485-5p expression is down-regulated and reversely associated with EMMPRIN in HCC tissues To investigate the possible role of miR-485-5p in HCC, we detected the expression of miR-485-5p in human HCC specimens by real time RT-PCR. We examined miR-485-5p expression in 32 HCC tissues, and found that it was reduced in most of the samples compared with the adjacent ones (Fig. 3A). The HCC tissues were classified according to the clinic stage and it was found that miR-485-5p expression decreased significantly with the stage becoming higher (Fig. 3B). The data also showed that miR485-5p expression decreased significantly in metastatic tissues than the non-metastatic ones (Fig. 3C). It was analyzed that miR485-5p expression was reversely associated with EMMPRIN mRNA in cancer cells tissues than the normal ones (Fig. 3D). The above
Fig. 3. miR-485-5p expression is down-regulated and reversely associated with EMMPRIN in HCC tissues. (A) Average expression level of miR-485-5p in human HCC specimens (n = 32) and normal tissues (n = 32). miRNA abundance was assessed by real time RT-PCR and normalized to U6 RNA. (B) miR-485-5p expression decreased in HCC tissues with advanced stage. (C) miR-485-5p expression decreased much more in metastatic HCC tissues than the control. (D) miR-485-5p is negatively related to EMMPRIN in HCC tissues (r = 0.6751, P < 0.001). The data presented are shown as means SD collected from three independent experiments. **P < 0.01.
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Fig. 4. Enforced expression of miR-485-5p induced growth inhibition in HCC in vitro. Effect of miR-485-5p on cell proliferation was measured by CCK8 assay after miR-485-5p infection in (A) SMMC7721 and (B) BEL-7404 cells. Effect of miR-485-5p on cell proliferation was measured by colony formation assay after miR-485-5p infection in (C) SMMC7721 and (D) BEL-7404 cells. Effect of miR-485-5p on cell cycle was measured by flow cytometry after miR-485-5p infection in (E) SMMC7721 and (F) BEL-7404 cells. The data presented are shown as means SD collected from three independent experiments. **P < 0.01 and * P < 0.05.
results suggested that miR-485-5p may function as a negative regulator in HCC progression. 3.4. miR-485-5p inhibits proliferation and metastasis in HCC cells To explore the suppressing effect of miR-485-5p on HCC cell growth, SMMC7721 and BEL-7404 cells were infected with LVmiR-485-5a (miR-485-5p) or its control. The results of CCK8 assay indicated that in the SMMC7721, and BEL-7404 cells with miR485-5p restoration, cell survival rate was much lower than the control (Fig. 4A and B). The colony formation assay displayed that miR-485-5p inhibited colony formation rate in SMMC7721 and BEL-7404 cells (Fig. 4C and D). To further observe miR-485-5p
mediating growth inhibition, cells were examined for cell-cycle distribution. Compared with miR-control, cells infected with miR485-5p restoration displayed an increased percentage of cells in G1 phase and fewer cells in S phase (Fig. 4E and F). These results suggested that the growth-suppressive effect of miR-485-5p was partly due to a G1-phase arrest. 3.5. miR-485-5p inhibits metastasis and EMT in HCC cells Above data indicated that miR-485-5p was related to HCC metastasis suppression. To verify the role of miR-485-5p in HCC metastasis, SMMC7721 and BEL-7404 cells were re-expressed with miR-485-5p, and the results showed that up-regulation of
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Fig. 5. miR-485-5p suppresses HCC metastasis and EMT. (A) Migration of SMMC7721 and BEL-7404 cells with miR-485-5p or miR-control was assayed using transwell chamber. (B) Invasion of SMMC7721 and BEL-7404 cells with miR-485-5p or miR-control was assayed using transwell chamber. (C) miR-485-5p suppressed metastasis of HCC cells. EMT markers such as E-cadherin and a-SMA were assayed by immunofluorescence. The data presented are shown as means SD collected from three independent experiments. **P < 0.01 and * P < 0.05.
miR-485-5p could significantly decrease migration (Fig. 5A) and invasion (Fig. 5B). Metastasis associated markers were detected by Western blot and the results showed that MMP9 decreased in the SMMC7721 and BEL-7404 cells with miR-485-5p restoration (Fig. 5C). Epithelial–mesenchymal transition (EMT) is a characteristic of tumor metastasis, here, and the markers of it including Ecadherin was increased and Vimentin, FSP1, a-SMA decreased in cells with miR-485-5p restoration (Fig. 5C). The finding of EMT suppression by miR-485-5p in HCC cells was confirmed by immunofluorescence (Fig. 5C). 3.6. miR-485-5p inhibits HCC by targeting EMMPRIN in vitro and in vivo Since EMMPRIN is the target gene of miR-485-5p, whether miR485-5p regulates proliferation, migration and invasion in HCC cells by targeting EMMPRIN? To answer the question, cell proliferation and metastasis were assayed in BEL-7404 cells. We found that miR485-5p could inhibit cell proliferation in BEL-7404 cells with EMMPRIN overexpression (Fig. 6A). Invasion assay showed that miR-485-5p in BEL-7404 cells inhibited the invasion of the cells with EMMPRIN overexpression (Fig. 6B). In order to investigate miR-485-5p mediating growth inhibition in vivo, HCC nude mice were set up using BEL-7404-miR-485-5p and its control, and the results showed that miR-485-5p suppressed HCC growth with or without EMMPRIN treatment (Fig. 6C).
4. Discussion EMMPRIN is a known oncogene in many types of cancer, which play critical roles in tumor progression including HCC. The
predicted data indicated that many potential miRNAs could regulate EMMPRIN in HCC cells like miR-125b, miR-125a-5p, miR. In the present study, we investigated the molecular mechanism of EMMPRIN regulation by miR-485-5p. Our data for the first time demonstrated that miR-485-5p inhibited HCC progression by targeting EMMPRIN. It was also found that miR485-5p was significantly downregulated in HCC tissues compared with that of normal ones and negatively associated with EMMPRIN. EMMPRIN is a glycosylated protein and overexpressed in many types of tumor and its function in tumor includes inducing apoptosis resistance, promoting cell proliferation, angiogenesis and metastasis [23–28,16,29]. In this study, we carried out further research to investigate EMMPRIN regulation by miRNAs. It was verified that EMMPRIN was a new target gene of miR485-5p and miR-485-5p could inhibit cell proliferation and induce G1 arrest and metastasis by down-regulation of EMMPRIN. Previous reports showed that down-regulation of miR-485-5p was found in ovarian cancer [30] and ependymoma [31]. However, there are no reports showing the role of miR-485-5p in hepatocellular carcinoma progression. Our study showed that miR-485-5p was significantly decreased in HCC tissues and cell lines. Our data indicated that loss of miR-485-5p in HCC tissues was related to tumor progression such as TNM stage and metastasis, which suggests that miR-485-5p was tumor suppressor. We found that miR-485-5p induced G1 arrest of HCC cells, inhibited cell proliferation and promoted cell apoptosis, which were consistent with the reports from other tumors like gastric cancer. These results indicate that miR-485-5p regulates tumor growth as a tumor suppressor, which could partially explain the correlation between low expression of miR-485-5p and poor prognosis.
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Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgement The study was funded by Linyi People’s Hospital. References
Fig. 6. miR-485-5p inhibits progression of HCC cells targeting EMMPRIN. (A) miR485-5p inhibited proliferation of HCC cells with EMMPRIN overexpression in BEL7404. Cell proliferation was assayed by colony formation. (B) miR-485-5p inhibited invasion of HCC cells with EMMPRIN overexpression. (C) miR-485-5p inhibited HCC with EMMPRIN overexpression in vivo. Tumor growth on nude mice set up using HCC cells LV-miR-485-5p or its control. The data presented are shown as means SD collected from three independent experiments. **P < 0.01 and *P < 0.05.
In summary, we identified miR-485-5p to be a tumor suppressor miRNA in HCC, and low miR-485-5p expression was an unfavorable prognostic factor in patients with HCC. miR-485-5p partially influences human HCC through the regulation of EMMPRIN. These results suggest that miR-485-5p is a potential target for treating liver cancer and the critical roles of miR-485-5p in liver cancer tumorigenesis may aid patient prognosis and diagnosis. Our findings provide basic information to better understand the pathogenesis of liver cancer and its possible therapeutic strategies.
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Original article
Involvement of miR-485-5p in hepatocellular carcinoma progression targeting EMMPRIN Xiangjun Sun a, Yonglei Liu b,c,*, Ming Li b, Mingchun Wang d, Yutong Wang c a
Department of Hepatobiliary Surgery, Linyi People’s Hospital, Linyi, Shangdong, China Research Center, Linyi People’s Hospital, Linyi, Shangdong, China c Liver Cancer Center, Zhongshan Hospital, Fudan University, Shanghai, China d Department of Surgery, Linyi People’s Hospital, Linyi, Shangdong, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 March 2015 Accepted 3 April 2015
EMMPRIN plays important roles in cancer development, which includes EMMPRIN 1, 2, 3, and 4 isoforms. EMMPRIN2 is the main component in human cancers, but its regulation by miRNAs is still unclear. In this study, we will investigate the mechanism of EMMPRIN regulation in hepatocellular carcinoma (HCC) by miRNAs. Through RT-PCR, we found that EMMPRIN2 was the main isoform in HCC cells. EMMPRIN2 was down-regulated significantly by predicted miRNAs and miR-485-5p was one of the miRNA that regulated EMMPRIN in HCC cell lines. It was verified that EMMPRIN was a target gene of miR-485-5p by using luciferase analysis assay. We found that miR-485-5p was significantly downregulated in HCC tissues and that its expression was inversely correlated with the TNM stage and metastasis in HCC samples. Results of cellular functions in HCC showed that miR-485-5p could inhibit cell proliferation and metastasis. Additionally, miR-485-5p overexpression suppressed HCC growth in vivo by downregulation of EMMPRIN. Our study for the first time demonstrated that miR-485-5p represses HCC invasive and metastatic capacities by targeting EMMPRIN expression. ß 2015 Published by Elsevier Masson SAS.
Keywords: EMMPRIN miR-485-5p Hepatocellular carcinoma Metastasis
1. Introduction Hepatocellular carcinoma (HCC) is the third cause for cancerrelated death in the world [1]. The incidence of HCC is much higher in China. Despite advances in HCC treatment, the 5-year overall survival rate remains very poor [2]. The molecular mechanism of HCC is very complicated and remains unclear [3,4]. It is needed to elucidate the mechanism of HCC carcinogenesis to find new targets or methods for developing effective strategies of HCC. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a widely expressed transmembrane glycoprotein which belongs to the immunoglobulin superfamily including four isoforms: EMMPRIN1, EMMPRIN2, EMMPRIN3 and EMMPRIN4 [5,6]. EMMPRIN involves in various physiological processes such as fetal development, reproduction, T cell differentiation, and neural and retinal functions [7–10]. EMMPRIN1 is a tissue specific protein expressed on retina tissue; EMMPRIN2 is the most predominant isoform, encoding two Ig-like extracellular domains and being well-known
* Corresponding author at: 27 Jiefang Road, Linyi People’s Hospital, Linyi 276000, Shangdong, China. Tel.: +86 05398226999. E-mail address: [email protected] (Y. Liu). http://dx.doi.org/10.1016/j.biopha.2015.04.008 0753-3322/ß 2015 Published by Elsevier Masson SAS.
as basigin/CD147/EMMPRIN; the other two isoforms were rarely reported which showed that EMMPRIN3 and EMMPRIN4 were increased in HCC tissues and EMMPRIN3 acted as an inhibitor of proliferation and invasion of HCC [11–13]. EMMPRIN expresses widely in many tumors, including liver cancer, lung cancer, breast cancer, bladder cancer, lymphoma, oral squamous cell carcinoma, glioma, melanoma, and others [14– 16]. Accordingly, EMMPRIN expression has been associated with known risk factors for breast cancer and with poor prognosis of breast cancer patients [14–16]. These suggest that EMMPRIN plays important roles in carcinogenesis. Moreover, it has been shown that higher expression of EMMPRIN was associated with poor prognosis of cancer patients and could serve as an independent predictor of poor survival in cancer. The most life-threatening aspects of the oncogenic process are invasion and metastasis. MicroRNAs (miRNAs) are small RNAs and its noncoding mRNA sequences containing about 22–29 nucleotides that act as important regulators of gene expression [17,18]. miRNAs inhibit gene expression on transcriptional or post-transcriptional levels by specifically binding the seed sequence of target genes, cleaving mRNAs or inhibiting their translation [19,20]. Most of human miRNAs are located in cancer-associated genomic regions in the
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chromosome, which function as inhibitor or onco-miRNAs depending on their targets in tumor. miRNAs have also been associated with HCC formation and growth [21,22]. In this study, we predicted that miR-485-5p was a regulator of EMMPRIN. Then we investigated the potential involvement of miR485-5p in HCC. We examined the expression of miR-485-5p in human HCC cells and tissues and tested its effects on cell growth, cell-cycle distribution and metastasis. We also investigated a potential role of miR-485-5p on tumorigenesis in a murine model. We identified that miR-485-5p was a tumor suppressor and negatively regulated EMMPRIN in progression of HCC.
2. Material and methods
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2.6. RNA isolation and real-time RT-PCR Total RNA, following the manufacturer’s instructions, was isolated from the cells using Trizol reagent (Invitrogen). Briefly, the cells were lysed in TRIzol and then mixed with chloroform. The lysate was centrifuged to separate RNA, DNA and protein, total RNA recovered, precipitated with isopropanol, washed in 75% ethanol to remove impurities before dissolved in water. After that, 2 mg of RNA was taken and treated with DNase to remove contaminating DNA prior to the reverse transcription to cDNA using SYBR1 PCR Kit (Takara, Japan). To measure mRNA expression, real-time RTPCR was performed using a sequence detector (ABI-Prism, Applied Biosystems). The primers were ordered from Kang-Chen Biotech (Kangcheng, Shanghai, China).
2.1. Cell culture BEL-7405, SMMC7721, HepG2 and Huh7 human liver cancer cell lines were primary purchased from ATCC (Manassas, VA, USA). BEL-7404 cells were stored in our lab. The cells were maintained in the Dulbecco’s Modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% fatal bovine serum (Gibco, USA) in a humidified incubator at 37 8C and 5% CO2. 2.2. Tissue samples Primary HCC and their adjacent tissues were obtained from the patients with HCC of our hospital (Linyi People’s Hospital, Shandong, China). Both tumor and normal tissues were histologically confirmed by H&E (hematoxylin and eosin) staining. The study was approved by the ethics committee of the hospital. 2.3. Antibodies and reagents EMMPRIN, MMP9, E-cadherin, Vimentin, FSP1 and a-SMA primary antibody were ordered from Santa Cruz (Dallas, TX, USA). Anti-GAPDH antibody and secondary antibodies conjugated with HRP were purchased from Kang-Chen Biotech (Kangcheng, Shanghai, China). EMMPRIN plasmid was ordered from Origene (Rockville, MD, USA). 2.4. Plasmid vector construction and lentivirus transduction Lentivirus vector expressing miR-485-5p was constructed using the BLOCK-iT pol II miR RNAi Expression Vector Kit with EmGFP (Invitrogen, Carlsbad, CA, USA). The primary miRNA sequence of miR-485-5p with flanking regions was obtained by PCR and inserted into the Block-iT Pol II miR RNAi Expression Vector, pcDNA6.2-GW/EmGFP-miR. For transfection, cells were seeded in antibiotic-free medium at a density of 30–40% with lipofectamine2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. 2.5. Transfection and dual luciferase assay The pGL3 plasmid was modified by adding the human 30 -UTR or the 30 -UTR with mutations in regions complementary to miR-4855p seed regions behind the firefly luciferase gene. Cells were plated on 24-well Plates 24 h before transfection. Transient transfection was carried out in Opti-MEM medium using lipofectamine-2000 according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). The medium was changed after transfection for 5 h, and the cells incubated at 37 8C for the indicated time. Cells were washed once with phosphate-buffered saline and luciferase assays were performed using the Dual Luciferase Assay System (Promega, WI, USA) according to the supplier’s protocol.
Fig. 1. miR-485-5p down-regulated EMMPRN expression in HCC cells. (A) The expression of EMMPRIN mRNA in five HCC cell lines. EMMPRIN 1 (NM_001728), 2 (NM_198589), 3 (NM_198590) and 4 (NM_198591) mRNA were examined by RT-PCR. (B) The most potential miRNAs targeted EMMPRIN2 in SMMC771 cells. The predicted miRNAs with high scores were verified by real time RT-PCR. (C) miR-485-5p inhibited EMMPRIN2 mRNA expression in HCC cell lines. The data presented are shown as means SD collected from three independent experiments. *P < 0.05, **P < 0.01.
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2.7. CCK8 The Cell Counting Kit-8 (CCK-8) (Beyotime, China) was used to determine cell viability according to the manufacturer’s instructions. Cells were plated at a density of 1–2 104 cells per well in 96-well microtiter plates and cultured overnight at 37 8C in a humidified incubator containing 5% CO2. Next, at different time points, the culture medium was replaced with 100 ml of fresh medium followed by the addition of 10 ml of CCK-8 solution. The cells were further incubated for 2 h at 37 8C, and the optical density at 450 nm was recorded. All experiments were performed in triplicate.
secondary antibody for about 3 h at RT. Washed in PBST (3, 10 min each wash), the proteins were finally visualized using ECL based on the manufacturer’s instructions. 2.9. Cell cycle analysis In 2 ml culture medium 2 105 cells/well (6-well plate) were seeded, and cultured for 16 h or added Nocodazole (5 mg/ml) for the indicated time before collection. The cells were stabilized with 75% ethanol for 24 h, and dyed with PI, and analyzed with ModFit of flow cytometry. 2.10. Colony formation assay
2.8. Western blotting The cells were scraped from the dishes, cellular protein extracts prepared by homogenization in an ice-cold lysis buffer and their lysates obtained by centrifugation at 12,000 g for 20 min, and the total protein concentration determined using Lowry method. Equal amounts of protein, separated by SDS-PAGE, were electrophoretically transferred to a PVDF membrane at 320 mA for 2 h at a low temperature and the membrane was blocked with 5% fat-free milk with 0.05% Tween 20 in PBS. Subsequently, the membrane was probed with the primary antibodies. The blots were washed in PBST and then incubated in anti-mouse IgG or anti-rabbit IgG
The cells were harvested, sparsely plated, and were cultured under the normal condition. The medium underwent the replacement at three-day intervals. And then the cells were fixed in 90% ethanol, stained with crystal violet and colonies consisting of at least 50 cells so they were counted ten days later. 2.11. Migration and invasion assay The migration assay was conducted as follows: 2 104 cells in 0.1 ml of media added to the upper chamber (Millpore, MA, USA), followed by an addition of 0.6 ml medium in lower chamber; those
Fig. 2. EMMPRIN is a target gene of miR-485-5p in human HCC cells. (A) The 30 -UTR of the EMMPRIN gene contains binding sites for miR-485-5p according to bioinformatic analysis. (B) miR-485-5p suppressed the expression of a luciferase reporter gene with the 30 -UTR of EMMPRIN. SMMC7721 and BEL-7404 cells were transiently co-transfected with the control or miR-485-5p together with the indicated luciferase constructs, and luciferase activity was analyzed 48 h later. Data are presented as relative firefly luciferase activity normalized to Renilla luciferase activity from the same construct. (C) miR-485-5p down-regulated EMMPRIN protein in SMMC7721 and BEL-7404 cells. Cells were transfected with miR-485-5p or its control for 48 h, and then collected for Western blot. (D) miR-485-5p down-regulated EMMPRIN mRNA in SMMC7721 and BEL7404 cells. Cells were transfected with anti-miR-485-5p or its control for 48 h, and then collected for real time RT-PCR. (E) miR-485-5p inhibition induced up-regulation of EMMPRIN in SMMC7721 and BEL-7404 cells. Cells were transfected with anti-miR-485-5p or its control for 48 h, and then collected for EMMPRIN immunoblotting. The data presented are shown as means SD collected from three independent experiments. **P < 0.01.
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on the surface of the polycarbonate membrane, non-invading cells, removed 16 h later by scraping with a cotton swab; those migrated to the pores of the underlying membrane fixed in 90% ethanol and stained with 0.1% crystal violet and counted under fluorescence microscopy (100), the mean and SD were calculated from three independent experiments. For invasion assay, there was Matrigel (BD Biosciences, San Jose, CA, USA) in the up-chamber. Invaded or migrated cells = Cells migrated through the membrane 100%/ Cells seeded in the well. 2.12. Statistical analysis Each experiment was repeated at least three times, Student’s t tests performed to determine the statistical significance for the assays of promoter activity, migration, FACS and colony formation, error bars representing SE. 3. Results 3.1. miR-485-5p down-regulates EMMPRN expression in HCC cells To investigate EMMPRIN regulation by miRNAs, firstly, we examined EMMPRIN expression in HCC cells by RT-PCR. It was found that EMMPRIN2 was the main isoform, EMMPRIN3 and EMMPRIN4 were very weak and there was no expression of EMMPRIN1 in HCC cells including HepG2, BEL-7404, BEL-7405 SMMC7721, Huh7 (Fig. 1A). So, the following research only focuses on EMMPRIN isoform 2. miRNA database was used to predict miRNAs targeting EMMPRIN. We selected miRNAs with high scores and verified them by real time RT-PCR (Fig. 1B). miR-485-5p was one of the potential regulator of EMMPRIN. When the above HCC cells were transfected with miR-485-5p, EMMPRIN2 mRNA inhibition was the most significant in the above HCC cell lines (Fig. 1C). EMMPRIN3 and 4 mRNA levels were inhibited by
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miR-485-5p; however, their expression was very weak, so further research will be focused on EMMPRIN2 (EMMPRIN in the following). 3.2. EMMPRIN is a target gene of miR-485-5p in human HCC cells We then investigated whether EMMPRIN is the direct target gene of miR-485-5p. EMMPRIN is predicted as the target gene of miR-485-5p (Fig. 2A). Assay of luciferase activity showed that the luciferase activity of pGL3-EMMPRIN-WT in the cells was much lower than in control cells. The luciferase activity of pGL3EMMPRIN-Mut was rescued in SMMC7721 and BEL-7404 cells (Fig. 2B). We next examined whether miR-485-5p could regulate EMMPRIN protein in the two HCC lines. It was found that EMMPRIN protein was suppressed greatly by miR-485-5p in SMMC7721 and BEL-7404 cells (Fig. 2C). EMMPRIN mRNA and protein levels increased greatly in SMMC7721 and BEL-7404 cells with anti-miR-485-5p compared with the control (Fig. 2D and E). 3.3. miR-485-5p expression is down-regulated and reversely associated with EMMPRIN in HCC tissues To investigate the possible role of miR-485-5p in HCC, we detected the expression of miR-485-5p in human HCC specimens by real time RT-PCR. We examined miR-485-5p expression in 32 HCC tissues, and found that it was reduced in most of the samples compared with the adjacent ones (Fig. 3A). The HCC tissues were classified according to the clinic stage and it was found that miR-485-5p expression decreased significantly with the stage becoming higher (Fig. 3B). The data also showed that miR485-5p expression decreased significantly in metastatic tissues than the non-metastatic ones (Fig. 3C). It was analyzed that miR485-5p expression was reversely associated with EMMPRIN mRNA in cancer cells tissues than the normal ones (Fig. 3D). The above
Fig. 3. miR-485-5p expression is down-regulated and reversely associated with EMMPRIN in HCC tissues. (A) Average expression level of miR-485-5p in human HCC specimens (n = 32) and normal tissues (n = 32). miRNA abundance was assessed by real time RT-PCR and normalized to U6 RNA. (B) miR-485-5p expression decreased in HCC tissues with advanced stage. (C) miR-485-5p expression decreased much more in metastatic HCC tissues than the control. (D) miR-485-5p is negatively related to EMMPRIN in HCC tissues (r = 0.6751, P < 0.001). The data presented are shown as means SD collected from three independent experiments. **P < 0.01.
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Fig. 4. Enforced expression of miR-485-5p induced growth inhibition in HCC in vitro. Effect of miR-485-5p on cell proliferation was measured by CCK8 assay after miR-485-5p infection in (A) SMMC7721 and (B) BEL-7404 cells. Effect of miR-485-5p on cell proliferation was measured by colony formation assay after miR-485-5p infection in (C) SMMC7721 and (D) BEL-7404 cells. Effect of miR-485-5p on cell cycle was measured by flow cytometry after miR-485-5p infection in (E) SMMC7721 and (F) BEL-7404 cells. The data presented are shown as means SD collected from three independent experiments. **P < 0.01 and * P < 0.05.
results suggested that miR-485-5p may function as a negative regulator in HCC progression. 3.4. miR-485-5p inhibits proliferation and metastasis in HCC cells To explore the suppressing effect of miR-485-5p on HCC cell growth, SMMC7721 and BEL-7404 cells were infected with LVmiR-485-5a (miR-485-5p) or its control. The results of CCK8 assay indicated that in the SMMC7721, and BEL-7404 cells with miR485-5p restoration, cell survival rate was much lower than the control (Fig. 4A and B). The colony formation assay displayed that miR-485-5p inhibited colony formation rate in SMMC7721 and BEL-7404 cells (Fig. 4C and D). To further observe miR-485-5p
mediating growth inhibition, cells were examined for cell-cycle distribution. Compared with miR-control, cells infected with miR485-5p restoration displayed an increased percentage of cells in G1 phase and fewer cells in S phase (Fig. 4E and F). These results suggested that the growth-suppressive effect of miR-485-5p was partly due to a G1-phase arrest. 3.5. miR-485-5p inhibits metastasis and EMT in HCC cells Above data indicated that miR-485-5p was related to HCC metastasis suppression. To verify the role of miR-485-5p in HCC metastasis, SMMC7721 and BEL-7404 cells were re-expressed with miR-485-5p, and the results showed that up-regulation of
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Fig. 5. miR-485-5p suppresses HCC metastasis and EMT. (A) Migration of SMMC7721 and BEL-7404 cells with miR-485-5p or miR-control was assayed using transwell chamber. (B) Invasion of SMMC7721 and BEL-7404 cells with miR-485-5p or miR-control was assayed using transwell chamber. (C) miR-485-5p suppressed metastasis of HCC cells. EMT markers such as E-cadherin and a-SMA were assayed by immunofluorescence. The data presented are shown as means SD collected from three independent experiments. **P < 0.01 and * P < 0.05.
miR-485-5p could significantly decrease migration (Fig. 5A) and invasion (Fig. 5B). Metastasis associated markers were detected by Western blot and the results showed that MMP9 decreased in the SMMC7721 and BEL-7404 cells with miR-485-5p restoration (Fig. 5C). Epithelial–mesenchymal transition (EMT) is a characteristic of tumor metastasis, here, and the markers of it including Ecadherin was increased and Vimentin, FSP1, a-SMA decreased in cells with miR-485-5p restoration (Fig. 5C). The finding of EMT suppression by miR-485-5p in HCC cells was confirmed by immunofluorescence (Fig. 5C). 3.6. miR-485-5p inhibits HCC by targeting EMMPRIN in vitro and in vivo Since EMMPRIN is the target gene of miR-485-5p, whether miR485-5p regulates proliferation, migration and invasion in HCC cells by targeting EMMPRIN? To answer the question, cell proliferation and metastasis were assayed in BEL-7404 cells. We found that miR485-5p could inhibit cell proliferation in BEL-7404 cells with EMMPRIN overexpression (Fig. 6A). Invasion assay showed that miR-485-5p in BEL-7404 cells inhibited the invasion of the cells with EMMPRIN overexpression (Fig. 6B). In order to investigate miR-485-5p mediating growth inhibition in vivo, HCC nude mice were set up using BEL-7404-miR-485-5p and its control, and the results showed that miR-485-5p suppressed HCC growth with or without EMMPRIN treatment (Fig. 6C).
4. Discussion EMMPRIN is a known oncogene in many types of cancer, which play critical roles in tumor progression including HCC. The
predicted data indicated that many potential miRNAs could regulate EMMPRIN in HCC cells like miR-125b, miR-125a-5p, miR. In the present study, we investigated the molecular mechanism of EMMPRIN regulation by miR-485-5p. Our data for the first time demonstrated that miR-485-5p inhibited HCC progression by targeting EMMPRIN. It was also found that miR485-5p was significantly downregulated in HCC tissues compared with that of normal ones and negatively associated with EMMPRIN. EMMPRIN is a glycosylated protein and overexpressed in many types of tumor and its function in tumor includes inducing apoptosis resistance, promoting cell proliferation, angiogenesis and metastasis [23–28,16,29]. In this study, we carried out further research to investigate EMMPRIN regulation by miRNAs. It was verified that EMMPRIN was a new target gene of miR485-5p and miR-485-5p could inhibit cell proliferation and induce G1 arrest and metastasis by down-regulation of EMMPRIN. Previous reports showed that down-regulation of miR-485-5p was found in ovarian cancer [30] and ependymoma [31]. However, there are no reports showing the role of miR-485-5p in hepatocellular carcinoma progression. Our study showed that miR-485-5p was significantly decreased in HCC tissues and cell lines. Our data indicated that loss of miR-485-5p in HCC tissues was related to tumor progression such as TNM stage and metastasis, which suggests that miR-485-5p was tumor suppressor. We found that miR-485-5p induced G1 arrest of HCC cells, inhibited cell proliferation and promoted cell apoptosis, which were consistent with the reports from other tumors like gastric cancer. These results indicate that miR-485-5p regulates tumor growth as a tumor suppressor, which could partially explain the correlation between low expression of miR-485-5p and poor prognosis.
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Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgement The study was funded by Linyi People’s Hospital. References
Fig. 6. miR-485-5p inhibits progression of HCC cells targeting EMMPRIN. (A) miR485-5p inhibited proliferation of HCC cells with EMMPRIN overexpression in BEL7404. Cell proliferation was assayed by colony formation. (B) miR-485-5p inhibited invasion of HCC cells with EMMPRIN overexpression. (C) miR-485-5p inhibited HCC with EMMPRIN overexpression in vivo. Tumor growth on nude mice set up using HCC cells LV-miR-485-5p or its control. The data presented are shown as means SD collected from three independent experiments. **P < 0.01 and *P < 0.05.
In summary, we identified miR-485-5p to be a tumor suppressor miRNA in HCC, and low miR-485-5p expression was an unfavorable prognostic factor in patients with HCC. miR-485-5p partially influences human HCC through the regulation of EMMPRIN. These results suggest that miR-485-5p is a potential target for treating liver cancer and the critical roles of miR-485-5p in liver cancer tumorigenesis may aid patient prognosis and diagnosis. Our findings provide basic information to better understand the pathogenesis of liver cancer and its possible therapeutic strategies.
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