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threonine protein kinase, PIM1, in melanoma
Biochemical and Biophysical Research Communications 474 (2016) 315e320
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
miR-542-3p suppresses invasion and metastasis by targeting the proto-oncogene serine/threonine protein kinase, PIM1, in melanoma Zhen Rang, Ge Yang, You-wei Wang, Fan Cui* Institute of Dermatology, Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, Chengdu, Sichuan, China
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Article history: Received 5 April 2016 Accepted 17 April 2016 Available online 20 April 2016
Aberrant microRNAs (miRNAs) contribute to metastasis of various cancer types, including melanoma. miR-542-3p has been characterized as a tumor suppressor in several cancers. However, the exact expression patterns of miR-542-3p and the precise molecular mechanisms underlying its role in melanoma require further exploration. In the current study, we demonstrated that miR-542-3p is significantly downregulated in melanoma cell lines and clinical specimens. Exogenous expression of miR-542-3p resulted in marked inhibition of melanoma cell migration, invasion and epithelial-mesenchymal transition (EMT) in vitro and lung metastasis in vivo. The proto-oncogene serine/threonine protein kinase, PIM1, was identified as a direct target of miR-542-3p using luciferase reporter assay, qRT-PCR and western blot analyses. Overexpression of PIM1 partially rescued miR-542-3p-mediated suppression of cell migration, invasion and EMT. Our results collectively indicate that miR-542-3p serves as a metastasis suppressor in melanoma, supporting its utility as a promising therapeutic candidate. © 2016 Elsevier Inc. All rights reserved.
Keywords: miR-542-3p Melanoma Migration Invasion PIM1
1. Introduction Melanoma is the most aggressive form of skin malignancy originating from neural crest-derived melanocytes [1,2]. In 2014, an estimated 76,100 new cases of melanoma were diagnosed in United States, among which 9710 died of the disease [3]. The prognosis for melanoma is excellent in cases of early diagnosis and surgical resection [4]. However, when metastatic disease is present, the 5year survival rate is less than 10% [5]. Therefore, elucidation of the molecular mechanisms underlying melanoma invasion and metastasis is crucial to develop novel and effective therapeutic strategies and targets. MicroRNAs (miRNAs), a class of small, non-coding endogenous RNA molecules, can negatively regulate gene expression at the post-transcriptional level by pairing to the 30 untranslated regions (30 UTR) of target mRNAs [6,7]. A single miRNA may repress multiple target genes either directly or indirectly. Several miRNAs are involved in diverse critical physiological and pathological processes, such as cell proliferation, differentiation, migration and invasion [8]. miRNAs are deregulated in many types of human cancer,
* Corresponding author. Institute of Dermatology, Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, No. 32, Western 2nd Section, 1st Ring Road, Chengdu, Sichuan, China. E-mail address: [email protected] (F. Cui). http://dx.doi.org/10.1016/j.bbrc.2016.04.093 0006-291X/© 2016 Elsevier Inc. All rights reserved.
and can serve as either oncogenes or tumor suppressors [9]. Interestingly, miRNAs have been implicated in both suppression and promotion of melanoma metastasis [10]. For example, miR-382 and miR-516b significantly suppress melanoma cell invasion in vitro and lung metastasis in vivo, while miR-455-5p promotes melanoma metastasis through inhibition of the tumor suppressor gene CPEB1 [11,12]. Recent studies have reported that miR-542-3p is suppressed and displays anti-oncogenic activity in several cancer types, including astrocytoma, neuroblastoma, gastric cancer, colon cancer and bladder cancer [13e17]. However, the expression patterns and functions of miR-542-3p in melanoma remain largely unknown. Data from the current study clearly demonstrate that miR-542-3p is downregulated in melanoma cell lines and clinical tissues. Moreover, miR-542-3p inhibits cell migration, invasion and epithelialmesenchymal transition (EMT) in vitro, and delays metastasis in vivo via downregulating the proto-oncogene serine/threonine protein kinase, PIM1. 2. Materials and methods 2.1. Cell lines and tissue samples Melanoma cell lines, A375, SK-MEL-19, SK-MEL-28, WM451 and B16F10 were obtained from the American Type Culture Collection,
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and cultured in Dulbecco's modified Eagle medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) at 37 C with 5% humidified CO2. Melanoma tissues were obtained from 24 patients subjected to radical resection for melanomas at Sichuan Provincial People's Hospital (Chengdu, China) and 12 non-neoplastic skin specimens from patients with chronic inflammatory skin diseases. All sample tissues were frozen and stored at 80 C until RNA extraction. The research protocols were approved by Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, and informed consent obtained from each patient.
2.4. Wound healing assay The wound healing assay was performed to measure cell migration. MM cells were seeded into six-well plates. After serum starvation via incubation in serum-free medium for 24 h, an artificial wound was created on the confluent cell monolayer using a standard 200 mL plastic pipette tip. Cells migrated into the scratch area as single cells from the confluent sides. The width of the scratch gap was measured and photographed at 0 and 24 h. Three replicate wells from a six-well plate were used for the experiment. 2.5. Cell invasion assay
2.2. Oligonucleotide, plasmid transfection and lentivirus transduction miR-542-3p mimics and PIM1 siRNA were purchased from RiboBio (Guangzhou, China) and transfected into A375 and SKMEL-28 cells using Lipofectamine™ 2000 (Invitrogen). To generate luciferase reporter plasmids, a fragment of the PIM1 wild-type (WT) 30 UTR containing the miR-542-3p binding site was cloned downstream of the firefly luciferase gene in the pGL3 vector (Promega, Madison, WI, USA), and the corresponding mutant (MUT) 30 UTR plasmid subsequently created using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). The pcDNA3.1-PIM1 (lacking 30 UTR) plasmid was constructed by inserting the full-length PIM1 coding region into pcDNA3.1 vector (Invitrogen). Lentiviral constructs expressing miR-542-3p were purchased from GeneChem Management Inc. (Shanghai, China). B16F10 cells were infected with the recombinant lentivirus transducing units in the presence of 5 mg/mL Polybrene (Sigma, St. Louis, MO, USA). 2.3. RNA extraction and quantitative RT-PCR (qRT-PCR) For mRNA detection, total RNA was extracted from cell lines and clinical samples using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized from total RNA using the PrimeScript RT reagent Kit (TaKaRa, Dalian, China). miRNAs were extracted using a mirVana™ miRNA Isolation Kit (Ambion, Austin, TX, USA) and a One-Step PrimeScript miRNA cDNA Synthesis Kit (Takara) used to convert miRNA into cDNA. qRT-PCR was performed with the SYBR green Premix Ex Taq II (TaKaRa) to detect the relative transcript levels of miR-542-3p and PIM1. RNU6B (U6) or b-actin were used as the endogenous control for normalization. Relative expression levels were calculated using the 2DDCT method. All experiments were performed in triplicate.
Cell invasion was assessed using a Transwell chamber (Corning, Steuben County, NY, USA) coated with Matrigel (BD Biosciences, San Jose, CA, USA). Transfected cells were harvested and resuspended in serum-free medium. For the invasion assay, 1 105 cells were seeded into the upper chamber and medium supplemented with 10% FBS placed into the lower chamber. After 24 h of incubation, cells remaining on the upper surface of the membrane were removed, and those that had invaded the lower surface were fixed, stained and counted using a light microscope. Five random fields of view were analyzed for each chamber. 2.6. In vivo lung metastasis assay The animal experiment was performed using a classic in vivo model of B16F10 melanoma lung metastasis as described previously [11]. B16F10 cells (1.5 105/100 ml) infected with miR-542-3p or control lentivirus were injected into the lateral tail veins of each C57BL/6J mouse (6 weeks old, n ¼ 6) under anesthesia. Ten days after inoculation, mice were killed and their lungs removed and fixed for HE staining. Lung metastasis was quantified by calculating the number of tumor foci in eight random fields. All experiments involving animals were undertaken in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of Second Military Medical University (Shanghai, China). 2.7. Luciferase reporter assay The luciferase reporter assay was performed as described previously [18]. HEK-293T cells were seeded in triplicate in 24-well plates and allowed to settle for 24 h. Cells were transfected using Lipofectamine 2000 reagent with the PIM1 WT or MUT 30 UTR reporter vector and miR-542-3p or NC mimics plus pRL-TK Renilla
Fig. 1. miR-542-3p is downregulated in melanoma cell lines and tissues. (A) Relative miR-542-3p expression in primary melanocytes and four melanoma cell lines was detected using qRT-PCR, with U6 as a control. (B) qRT-PCR analysis of miR-542-3p expression in 24 melanoma specimens and 12 non-neoplastic skin tissues. *P < 0.05.
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Fig. 2. Ectopic expression of miR-542-3p inhibits melanoma cell invasion and tumor metastasis in vitro and in vivo. A375 and SK-MEL-28 cells were transfected with miR-542-3p or NC mimics. (A) Assessment of cell migration with the wound healing assay. (B) Analysis of cell invasion using Matrigel Transwell invasion assay. (C) Western blot analysis for the EMT markers, E-cadherin and Vimentin. (D) B16F10 mouse melanoma cells transduced with miR-542-3p or control lentiviruses were injected intravenously into C57BL/6J mice (n ¼ 6). Ten days after inoculation, mice were killed, and lung metastatic nodules were photographed and counted. HE-stained sections of lung metastases (Middle; magnification 5). *P < 0.05.
plasmid (Promega). At 48 h after transfection, Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega). 2.8. Western blotting Cells were lysed using RIPA buffer, and the protein concentrations in lysates assessed using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Each protein sample (30 mg) was denatured in SDS sample buffer and separated via 10% SDSePAGE. Separated proteins were transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) blocked with 5% non-fat milk and incubated overnight with primary antibodies. Detection was carried out with horseradish peroxidase-conjugated to goat anti-rabbit IgG (Sigma), followed by chemiluminescent reaction (Pierce, Rockford, IL, USA). GAPDH was used as a loading control. Antibodies against PIM1, E-cadherin, Vimentin and GAPDH were purchased from Proteintech (Chicago, IL, USA). 2.9. Statistical analysis Data are presented as means ± standard deviation of at least three independent experiments. Statistical analyses were
performed using SPSS 16.0 statistical software (SPSS, Chicago, IL, USA). The two-tailed student's t-test was used to determine significant differences between two groups. The relationship between miR-542-3p and PIM1 expression was explored using Spearman's correlation. Differences were considered significant at P < 0.05.
3. Results 3.1. miR-542-3p is downregulated in melanoma cell lines and tissues miR-542-3p expression was initially evaluated in four human melanoma cell lines using qRT-PCR. miR-542-3p was expressed at markedly lower levels in A375, SK-MEL-28 and SK-MEL-19, particularly in the first two cell lines, compared with primary melanocytes (Fig. 1A). Additionally, examination of miR-542-3p expression in 24 primary melanoma and 12 non-neoplastic skin specimens disclosed significant downregulation of miR-542-3p in melanoma tissues, compared with normal skin tissues (Fig. 1B).
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Fig. 3. miR-542-3p directly targets PIM1. (A) The wild-type (WT) binding site and corresponding mutated sequences for miR-542-3p within PIM1 30 UTR are shown. (B) Luciferase reporter assays in A375 and SK-MEL-28 cells. A firefly luciferase vector containing WT or mutant target site (MUT) of PIM1 30 UTR was generated and transfected as specified in Materials and methods. Luciferase activities were analyzed as the relative activity of firefly to Renilla. qRT-PCR (C) and western blot (D) analysis of PIM1 mRNA and protein expression in the indicated cells. (E) qRT-PCR analysis of PIM1 mRNA expression in melanoma tissues and normal skin tissues. (F) Spearman's correlation analysis of miR-542-3p and PIM1 expression in melanoma tissues (n ¼ 24); significant inverse correlation. *P < 0.05.
3.2. Ectopic expression of miR-542-3p inhibits melanoma cell migration, invasion and EMT in vitro To determine whether miR-542-3p affects melanoma cell invasion and metastasis, A375 and SK-MEL-28 cells were transiently transfected with miR-542-3p or control mimics. In the wound healing assay, ectopic expression of miR-542-3p reduces the ability of both melanoma cell lines to close the scratch to an extent (Fig. 2A). Similarly, data obtained with the Matrigel Transwell invasion assay demonstrated that miR-542-3p overexpression decreases the invasive ability of melanoma cells (Fig. 2B). Given that EMT is a key event in the initiation and maintenance of invasion and metastasis capacity in diverse malignancies [19], western blot was further performed to evaluate EMT marker expression in transfected cells. As shown in Fig. 2C, miR-542-3p overexpression led to upregulation of the epithelial marker, E-cadherin, and downregulation of the mesenchymal marker, Vimentin, suggestive of inhibition of EMT in melanoma cells. 3.3. miR-542-3p suppresses lung metastasis in vivo To further determine the in vivo effects of miR-542-3p on tumor metastasis, we constructed a B16F10 cell line stably overexpressing
miR-542-3p via lentiviral-mediated transduction and used a classic in vivo model of melanoma lung metastasis in which infected cells were injected into the tail vein of C57BL/6J mice. Ten days after injection, mice were sacrificed and the lungs recovered for macroand microscopic histology assessment. The number of metastatic nodules in the lung was dramatically decreased in miR-542-3p groups, compared with the negative control group (Fig. 2D), clearly demonstrating that miR-542-3p suppresses the ability of melanoma cells to extravasate or seed at a distant site. 3.4. miR-542-3p directly targets PIM1 We used the online publicly available databases (miRanda and TargetScan) to predict candidate target genes of miR-542-3p. PIM1 attracted our interest, owing to the findings that a binding site for miR-542-3p is present in its 30 UTR region (Fig. 3A) and the protein plays positive roles in cancer cell proliferation and invasion. To determine whether PIM1 is a direct target of miR-542-3p, wildtype or mutant PIM1 30 UTR binding sites were inserted into luciferase reporter vector and co-transfected with miR-542-3p or control mimics into HEK-293T cells. In the luciferase reporter assay, miR-542-3p overexpression induced a decrease in the luciferase activity of PIM1 WT 30 UTR, but had no obvious effect on PIM1
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Fig. 4. miR-542-3p suppresses cell motility by directly targeting PIM1 in melanoma cells. (A) Western blot analysis of PIM1, E-cadherin and Vimentin protein expression in A375 cells transfected with siPIM1 or negative control. Cell migration and invasion were determined with the wound healing and Transwell invasion assays. (B) Western blot analysis of the indicated proteins in A375 cells transfected with NC, miR-542-3p mimics or miR-542-3p mimics plus PIM1 plasmid. Cell migration and invasion are shown. *P < 0.05 vs. NC mimics, $P < 0.05 vs. miR-542-3p.
mutant 30 UTR (Fig. 3B). PIM1 mRNA and protein levels were decreased in response to miR-542-3p treatment, as shown in Fig. 3C and D. Furthermore, qRT-PCR measurement of PIM1 mRNA levels in primary melanoma and normal tissues revealed higher PIM1 expression in melanoma tissues, compared with normal tissues (Fig. 3E). A statistically significant inverse correlation between miR-542-3p and PIM1 expression in melanoma tissues was observed with Spearman's correlation analysis (Fig. 3F). Based on these results, we suggest that miR-542-3p modulates PIM1 expression by directly targeting its 30 UTR region. 3.5. PIM1 is involved in miR-542-3p-induced tumor suppressive effects In view of the finding that PIM1 is a direct protein target of miR542-3p, we hypothesized that PIM1 contributes to miR-542-3pmediated A375 cell motility suppression. To examine this theory, we eliminated PIM1 expression via RNA interference and subsequently performed loss-of-function analyses. The results showed that silencing of PIM1 inhibits EMT via upregulation of E-cadherin and downregulation of Vimentin expression, as well as cell migration and invasion (Fig. 4A). To further confirm the role of PIM1 in inhibition of miR-542-3p-induced melanoma cell malignant behavior, we constructed a PIM1-overexpressing plasmid (lacking the 30 UTR) and performed gain-of-function analyses. As shown in Fig. 4B, PIM1 overexpression reversed the inhibitory effects of EMT, cell migration and invasion induced by miR-542-3p in A375 cells. Our data collectively indicate that miR-542-3p suppresses cell
migration and invasion by directly targeting PIM1 in melanoma cells. 4. Discussion In this study, we investigated the biological role of miR-542-3p in human melanoma metastasis. miR-542-3p levels were significantly lower in melanoma cell lines and tissues. Forced expression of miR-542-3p suppressed cell migration, invasion and EMT in vitro and prohibited lung metastasis in vivo. Furthermore, PIM1 was identified as a direct target of miR-542-3p. PIM1 knockdown recapitulated the effects of miR-542-3p overexpression, while PIM1 overexpression partially but significantly abrogated miR-542-3pmediated metastasis suppression. Dysregulation of miRNA frequently occurs in many types of malignancies, and aberrant miRNA expression contributes to human carcinogenesis and development via directly downregulating multiple target genes. Thus, comprehensive understanding of the relationship between specific miRNAs and cancer development is valuable for improving cancer diagnosis and therapy. Several studies have shown that miR-542-3p is decreased in a variety of human cancers, and associated with tumor growth and metastasis [13,14,20]. For example, miR-542-3p is downregulated in neuroblastoma, and suppresses cell viability and proliferation and induces apoptosis [14]. miR-542-3p is additionally decreased in glioblastoma cell lines and astrocytoma tissues, and exogenous miR-542-3p inhibits glioblastoma cell invasion [13]. Moreover, miR-542-3p has been shown to reduce tumor growth, angiogenesis
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and metastasis in breast cancer [20]. Consistent with these results, our experiments disclosed marked downregulation of miR-542-3p in melanoma clinical tissues and cell lines. Ectopic miR-542-3p expression robustly attenuated migration and invasion of melanoma cells in vitro and suppressed EMT via enhancing the E-cadherin level and reducing expression of Vimentin. Furthermore, miR-543-3p suppressed pulmonary metastases in vivo, suggesting that this miRNA can control melanoma cell metastasis and invasion, both in vitro and in vivo. To determine the mechanism by which miR-542-3p exerts inhibitory effects on melanoma metastasis, we used two public prediction algorithms for target prediction and consequently identified PIM1 as a candidate direct target gene of miR-542-3p. PIM1 belongs to a serine/threonine kinase family composed of three members (PIM1, 2 and 3), and is highly conserved between species [21,22]. PIM1 and PIM2 kinase sequences share 61% homology whereas PIM1 and PIM3 are 71% identical at the amino acid level [23]. Owing to their high homology, the three PIM kinases display functional redundancy in vitro and in vivo [24e26]. Notably, PIM1 has been linked to various cancers. The protein is overexpressed in hepatocellular carcinoma, and facilitates cell proliferation, invasion as well as tumor growth and metastasis [27]. In a study on human salivary gland adenoid cystic carcinoma, overexpression of PIM1 promoted cell growth and invasion [28]. These results were confirmed in the U2OS human osteosarcoma cell line by Liao [26], suggesting a crucial role of PIM1 in the development and progression of cancer. In the current study, PIM1 expression was upregulated and inversely correlated with the miR-542-3p level in melanoma tissues. Knockdown of PIM1 via RNA interference replicated the phenotype of miR-542-3p expression in melanoma cell lines, while forced expression of PIM1 partially rescued the phenotype mediated by miR-542-3p. Our results clearly demonstrate that miR-542-3p suppresses the migration and invasion of melanoma cells through PIM1 downregulation. In summary, miR-542-3p, a miRNA downregulated in melanoma, markedly inhibits cell migration and invasion by directly targeting PIM1. The newly identified miR-542-3p/PIM1 axis may provide novel insights into the mechanisms underlying the pathogenesis of melanoma and serve as a potential therapeutic target.
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Conflict of interest All authors including Zhen Rang, Ge Yang, You-wei Wang and Fan Cui declare no conflict of interest.
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Acknowledgements This work was supported by the National Natural Science Foundation of China (81101231).
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
miR-542-3p suppresses invasion and metastasis by targeting the proto-oncogene serine/threonine protein kinase, PIM1, in melanoma Zhen Rang, Ge Yang, You-wei Wang, Fan Cui* Institute of Dermatology, Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, Chengdu, Sichuan, China
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a b s t r a c t
Article history: Received 5 April 2016 Accepted 17 April 2016 Available online 20 April 2016
Aberrant microRNAs (miRNAs) contribute to metastasis of various cancer types, including melanoma. miR-542-3p has been characterized as a tumor suppressor in several cancers. However, the exact expression patterns of miR-542-3p and the precise molecular mechanisms underlying its role in melanoma require further exploration. In the current study, we demonstrated that miR-542-3p is significantly downregulated in melanoma cell lines and clinical specimens. Exogenous expression of miR-542-3p resulted in marked inhibition of melanoma cell migration, invasion and epithelial-mesenchymal transition (EMT) in vitro and lung metastasis in vivo. The proto-oncogene serine/threonine protein kinase, PIM1, was identified as a direct target of miR-542-3p using luciferase reporter assay, qRT-PCR and western blot analyses. Overexpression of PIM1 partially rescued miR-542-3p-mediated suppression of cell migration, invasion and EMT. Our results collectively indicate that miR-542-3p serves as a metastasis suppressor in melanoma, supporting its utility as a promising therapeutic candidate. © 2016 Elsevier Inc. All rights reserved.
Keywords: miR-542-3p Melanoma Migration Invasion PIM1
1. Introduction Melanoma is the most aggressive form of skin malignancy originating from neural crest-derived melanocytes [1,2]. In 2014, an estimated 76,100 new cases of melanoma were diagnosed in United States, among which 9710 died of the disease [3]. The prognosis for melanoma is excellent in cases of early diagnosis and surgical resection [4]. However, when metastatic disease is present, the 5year survival rate is less than 10% [5]. Therefore, elucidation of the molecular mechanisms underlying melanoma invasion and metastasis is crucial to develop novel and effective therapeutic strategies and targets. MicroRNAs (miRNAs), a class of small, non-coding endogenous RNA molecules, can negatively regulate gene expression at the post-transcriptional level by pairing to the 30 untranslated regions (30 UTR) of target mRNAs [6,7]. A single miRNA may repress multiple target genes either directly or indirectly. Several miRNAs are involved in diverse critical physiological and pathological processes, such as cell proliferation, differentiation, migration and invasion [8]. miRNAs are deregulated in many types of human cancer,
* Corresponding author. Institute of Dermatology, Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, No. 32, Western 2nd Section, 1st Ring Road, Chengdu, Sichuan, China. E-mail address: [email protected] (F. Cui). http://dx.doi.org/10.1016/j.bbrc.2016.04.093 0006-291X/© 2016 Elsevier Inc. All rights reserved.
and can serve as either oncogenes or tumor suppressors [9]. Interestingly, miRNAs have been implicated in both suppression and promotion of melanoma metastasis [10]. For example, miR-382 and miR-516b significantly suppress melanoma cell invasion in vitro and lung metastasis in vivo, while miR-455-5p promotes melanoma metastasis through inhibition of the tumor suppressor gene CPEB1 [11,12]. Recent studies have reported that miR-542-3p is suppressed and displays anti-oncogenic activity in several cancer types, including astrocytoma, neuroblastoma, gastric cancer, colon cancer and bladder cancer [13e17]. However, the expression patterns and functions of miR-542-3p in melanoma remain largely unknown. Data from the current study clearly demonstrate that miR-542-3p is downregulated in melanoma cell lines and clinical tissues. Moreover, miR-542-3p inhibits cell migration, invasion and epithelialmesenchymal transition (EMT) in vitro, and delays metastasis in vivo via downregulating the proto-oncogene serine/threonine protein kinase, PIM1. 2. Materials and methods 2.1. Cell lines and tissue samples Melanoma cell lines, A375, SK-MEL-19, SK-MEL-28, WM451 and B16F10 were obtained from the American Type Culture Collection,
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and cultured in Dulbecco's modified Eagle medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) at 37 C with 5% humidified CO2. Melanoma tissues were obtained from 24 patients subjected to radical resection for melanomas at Sichuan Provincial People's Hospital (Chengdu, China) and 12 non-neoplastic skin specimens from patients with chronic inflammatory skin diseases. All sample tissues were frozen and stored at 80 C until RNA extraction. The research protocols were approved by Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, and informed consent obtained from each patient.
2.4. Wound healing assay The wound healing assay was performed to measure cell migration. MM cells were seeded into six-well plates. After serum starvation via incubation in serum-free medium for 24 h, an artificial wound was created on the confluent cell monolayer using a standard 200 mL plastic pipette tip. Cells migrated into the scratch area as single cells from the confluent sides. The width of the scratch gap was measured and photographed at 0 and 24 h. Three replicate wells from a six-well plate were used for the experiment. 2.5. Cell invasion assay
2.2. Oligonucleotide, plasmid transfection and lentivirus transduction miR-542-3p mimics and PIM1 siRNA were purchased from RiboBio (Guangzhou, China) and transfected into A375 and SKMEL-28 cells using Lipofectamine™ 2000 (Invitrogen). To generate luciferase reporter plasmids, a fragment of the PIM1 wild-type (WT) 30 UTR containing the miR-542-3p binding site was cloned downstream of the firefly luciferase gene in the pGL3 vector (Promega, Madison, WI, USA), and the corresponding mutant (MUT) 30 UTR plasmid subsequently created using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). The pcDNA3.1-PIM1 (lacking 30 UTR) plasmid was constructed by inserting the full-length PIM1 coding region into pcDNA3.1 vector (Invitrogen). Lentiviral constructs expressing miR-542-3p were purchased from GeneChem Management Inc. (Shanghai, China). B16F10 cells were infected with the recombinant lentivirus transducing units in the presence of 5 mg/mL Polybrene (Sigma, St. Louis, MO, USA). 2.3. RNA extraction and quantitative RT-PCR (qRT-PCR) For mRNA detection, total RNA was extracted from cell lines and clinical samples using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized from total RNA using the PrimeScript RT reagent Kit (TaKaRa, Dalian, China). miRNAs were extracted using a mirVana™ miRNA Isolation Kit (Ambion, Austin, TX, USA) and a One-Step PrimeScript miRNA cDNA Synthesis Kit (Takara) used to convert miRNA into cDNA. qRT-PCR was performed with the SYBR green Premix Ex Taq II (TaKaRa) to detect the relative transcript levels of miR-542-3p and PIM1. RNU6B (U6) or b-actin were used as the endogenous control for normalization. Relative expression levels were calculated using the 2DDCT method. All experiments were performed in triplicate.
Cell invasion was assessed using a Transwell chamber (Corning, Steuben County, NY, USA) coated with Matrigel (BD Biosciences, San Jose, CA, USA). Transfected cells were harvested and resuspended in serum-free medium. For the invasion assay, 1 105 cells were seeded into the upper chamber and medium supplemented with 10% FBS placed into the lower chamber. After 24 h of incubation, cells remaining on the upper surface of the membrane were removed, and those that had invaded the lower surface were fixed, stained and counted using a light microscope. Five random fields of view were analyzed for each chamber. 2.6. In vivo lung metastasis assay The animal experiment was performed using a classic in vivo model of B16F10 melanoma lung metastasis as described previously [11]. B16F10 cells (1.5 105/100 ml) infected with miR-542-3p or control lentivirus were injected into the lateral tail veins of each C57BL/6J mouse (6 weeks old, n ¼ 6) under anesthesia. Ten days after inoculation, mice were killed and their lungs removed and fixed for HE staining. Lung metastasis was quantified by calculating the number of tumor foci in eight random fields. All experiments involving animals were undertaken in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of Second Military Medical University (Shanghai, China). 2.7. Luciferase reporter assay The luciferase reporter assay was performed as described previously [18]. HEK-293T cells were seeded in triplicate in 24-well plates and allowed to settle for 24 h. Cells were transfected using Lipofectamine 2000 reagent with the PIM1 WT or MUT 30 UTR reporter vector and miR-542-3p or NC mimics plus pRL-TK Renilla
Fig. 1. miR-542-3p is downregulated in melanoma cell lines and tissues. (A) Relative miR-542-3p expression in primary melanocytes and four melanoma cell lines was detected using qRT-PCR, with U6 as a control. (B) qRT-PCR analysis of miR-542-3p expression in 24 melanoma specimens and 12 non-neoplastic skin tissues. *P < 0.05.
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Fig. 2. Ectopic expression of miR-542-3p inhibits melanoma cell invasion and tumor metastasis in vitro and in vivo. A375 and SK-MEL-28 cells were transfected with miR-542-3p or NC mimics. (A) Assessment of cell migration with the wound healing assay. (B) Analysis of cell invasion using Matrigel Transwell invasion assay. (C) Western blot analysis for the EMT markers, E-cadherin and Vimentin. (D) B16F10 mouse melanoma cells transduced with miR-542-3p or control lentiviruses were injected intravenously into C57BL/6J mice (n ¼ 6). Ten days after inoculation, mice were killed, and lung metastatic nodules were photographed and counted. HE-stained sections of lung metastases (Middle; magnification 5). *P < 0.05.
plasmid (Promega). At 48 h after transfection, Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega). 2.8. Western blotting Cells were lysed using RIPA buffer, and the protein concentrations in lysates assessed using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Each protein sample (30 mg) was denatured in SDS sample buffer and separated via 10% SDSePAGE. Separated proteins were transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) blocked with 5% non-fat milk and incubated overnight with primary antibodies. Detection was carried out with horseradish peroxidase-conjugated to goat anti-rabbit IgG (Sigma), followed by chemiluminescent reaction (Pierce, Rockford, IL, USA). GAPDH was used as a loading control. Antibodies against PIM1, E-cadherin, Vimentin and GAPDH were purchased from Proteintech (Chicago, IL, USA). 2.9. Statistical analysis Data are presented as means ± standard deviation of at least three independent experiments. Statistical analyses were
performed using SPSS 16.0 statistical software (SPSS, Chicago, IL, USA). The two-tailed student's t-test was used to determine significant differences between two groups. The relationship between miR-542-3p and PIM1 expression was explored using Spearman's correlation. Differences were considered significant at P < 0.05.
3. Results 3.1. miR-542-3p is downregulated in melanoma cell lines and tissues miR-542-3p expression was initially evaluated in four human melanoma cell lines using qRT-PCR. miR-542-3p was expressed at markedly lower levels in A375, SK-MEL-28 and SK-MEL-19, particularly in the first two cell lines, compared with primary melanocytes (Fig. 1A). Additionally, examination of miR-542-3p expression in 24 primary melanoma and 12 non-neoplastic skin specimens disclosed significant downregulation of miR-542-3p in melanoma tissues, compared with normal skin tissues (Fig. 1B).
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Fig. 3. miR-542-3p directly targets PIM1. (A) The wild-type (WT) binding site and corresponding mutated sequences for miR-542-3p within PIM1 30 UTR are shown. (B) Luciferase reporter assays in A375 and SK-MEL-28 cells. A firefly luciferase vector containing WT or mutant target site (MUT) of PIM1 30 UTR was generated and transfected as specified in Materials and methods. Luciferase activities were analyzed as the relative activity of firefly to Renilla. qRT-PCR (C) and western blot (D) analysis of PIM1 mRNA and protein expression in the indicated cells. (E) qRT-PCR analysis of PIM1 mRNA expression in melanoma tissues and normal skin tissues. (F) Spearman's correlation analysis of miR-542-3p and PIM1 expression in melanoma tissues (n ¼ 24); significant inverse correlation. *P < 0.05.
3.2. Ectopic expression of miR-542-3p inhibits melanoma cell migration, invasion and EMT in vitro To determine whether miR-542-3p affects melanoma cell invasion and metastasis, A375 and SK-MEL-28 cells were transiently transfected with miR-542-3p or control mimics. In the wound healing assay, ectopic expression of miR-542-3p reduces the ability of both melanoma cell lines to close the scratch to an extent (Fig. 2A). Similarly, data obtained with the Matrigel Transwell invasion assay demonstrated that miR-542-3p overexpression decreases the invasive ability of melanoma cells (Fig. 2B). Given that EMT is a key event in the initiation and maintenance of invasion and metastasis capacity in diverse malignancies [19], western blot was further performed to evaluate EMT marker expression in transfected cells. As shown in Fig. 2C, miR-542-3p overexpression led to upregulation of the epithelial marker, E-cadherin, and downregulation of the mesenchymal marker, Vimentin, suggestive of inhibition of EMT in melanoma cells. 3.3. miR-542-3p suppresses lung metastasis in vivo To further determine the in vivo effects of miR-542-3p on tumor metastasis, we constructed a B16F10 cell line stably overexpressing
miR-542-3p via lentiviral-mediated transduction and used a classic in vivo model of melanoma lung metastasis in which infected cells were injected into the tail vein of C57BL/6J mice. Ten days after injection, mice were sacrificed and the lungs recovered for macroand microscopic histology assessment. The number of metastatic nodules in the lung was dramatically decreased in miR-542-3p groups, compared with the negative control group (Fig. 2D), clearly demonstrating that miR-542-3p suppresses the ability of melanoma cells to extravasate or seed at a distant site. 3.4. miR-542-3p directly targets PIM1 We used the online publicly available databases (miRanda and TargetScan) to predict candidate target genes of miR-542-3p. PIM1 attracted our interest, owing to the findings that a binding site for miR-542-3p is present in its 30 UTR region (Fig. 3A) and the protein plays positive roles in cancer cell proliferation and invasion. To determine whether PIM1 is a direct target of miR-542-3p, wildtype or mutant PIM1 30 UTR binding sites were inserted into luciferase reporter vector and co-transfected with miR-542-3p or control mimics into HEK-293T cells. In the luciferase reporter assay, miR-542-3p overexpression induced a decrease in the luciferase activity of PIM1 WT 30 UTR, but had no obvious effect on PIM1
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Fig. 4. miR-542-3p suppresses cell motility by directly targeting PIM1 in melanoma cells. (A) Western blot analysis of PIM1, E-cadherin and Vimentin protein expression in A375 cells transfected with siPIM1 or negative control. Cell migration and invasion were determined with the wound healing and Transwell invasion assays. (B) Western blot analysis of the indicated proteins in A375 cells transfected with NC, miR-542-3p mimics or miR-542-3p mimics plus PIM1 plasmid. Cell migration and invasion are shown. *P < 0.05 vs. NC mimics, $P < 0.05 vs. miR-542-3p.
mutant 30 UTR (Fig. 3B). PIM1 mRNA and protein levels were decreased in response to miR-542-3p treatment, as shown in Fig. 3C and D. Furthermore, qRT-PCR measurement of PIM1 mRNA levels in primary melanoma and normal tissues revealed higher PIM1 expression in melanoma tissues, compared with normal tissues (Fig. 3E). A statistically significant inverse correlation between miR-542-3p and PIM1 expression in melanoma tissues was observed with Spearman's correlation analysis (Fig. 3F). Based on these results, we suggest that miR-542-3p modulates PIM1 expression by directly targeting its 30 UTR region. 3.5. PIM1 is involved in miR-542-3p-induced tumor suppressive effects In view of the finding that PIM1 is a direct protein target of miR542-3p, we hypothesized that PIM1 contributes to miR-542-3pmediated A375 cell motility suppression. To examine this theory, we eliminated PIM1 expression via RNA interference and subsequently performed loss-of-function analyses. The results showed that silencing of PIM1 inhibits EMT via upregulation of E-cadherin and downregulation of Vimentin expression, as well as cell migration and invasion (Fig. 4A). To further confirm the role of PIM1 in inhibition of miR-542-3p-induced melanoma cell malignant behavior, we constructed a PIM1-overexpressing plasmid (lacking the 30 UTR) and performed gain-of-function analyses. As shown in Fig. 4B, PIM1 overexpression reversed the inhibitory effects of EMT, cell migration and invasion induced by miR-542-3p in A375 cells. Our data collectively indicate that miR-542-3p suppresses cell
migration and invasion by directly targeting PIM1 in melanoma cells. 4. Discussion In this study, we investigated the biological role of miR-542-3p in human melanoma metastasis. miR-542-3p levels were significantly lower in melanoma cell lines and tissues. Forced expression of miR-542-3p suppressed cell migration, invasion and EMT in vitro and prohibited lung metastasis in vivo. Furthermore, PIM1 was identified as a direct target of miR-542-3p. PIM1 knockdown recapitulated the effects of miR-542-3p overexpression, while PIM1 overexpression partially but significantly abrogated miR-542-3pmediated metastasis suppression. Dysregulation of miRNA frequently occurs in many types of malignancies, and aberrant miRNA expression contributes to human carcinogenesis and development via directly downregulating multiple target genes. Thus, comprehensive understanding of the relationship between specific miRNAs and cancer development is valuable for improving cancer diagnosis and therapy. Several studies have shown that miR-542-3p is decreased in a variety of human cancers, and associated with tumor growth and metastasis [13,14,20]. For example, miR-542-3p is downregulated in neuroblastoma, and suppresses cell viability and proliferation and induces apoptosis [14]. miR-542-3p is additionally decreased in glioblastoma cell lines and astrocytoma tissues, and exogenous miR-542-3p inhibits glioblastoma cell invasion [13]. Moreover, miR-542-3p has been shown to reduce tumor growth, angiogenesis
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and metastasis in breast cancer [20]. Consistent with these results, our experiments disclosed marked downregulation of miR-542-3p in melanoma clinical tissues and cell lines. Ectopic miR-542-3p expression robustly attenuated migration and invasion of melanoma cells in vitro and suppressed EMT via enhancing the E-cadherin level and reducing expression of Vimentin. Furthermore, miR-543-3p suppressed pulmonary metastases in vivo, suggesting that this miRNA can control melanoma cell metastasis and invasion, both in vitro and in vivo. To determine the mechanism by which miR-542-3p exerts inhibitory effects on melanoma metastasis, we used two public prediction algorithms for target prediction and consequently identified PIM1 as a candidate direct target gene of miR-542-3p. PIM1 belongs to a serine/threonine kinase family composed of three members (PIM1, 2 and 3), and is highly conserved between species [21,22]. PIM1 and PIM2 kinase sequences share 61% homology whereas PIM1 and PIM3 are 71% identical at the amino acid level [23]. Owing to their high homology, the three PIM kinases display functional redundancy in vitro and in vivo [24e26]. Notably, PIM1 has been linked to various cancers. The protein is overexpressed in hepatocellular carcinoma, and facilitates cell proliferation, invasion as well as tumor growth and metastasis [27]. In a study on human salivary gland adenoid cystic carcinoma, overexpression of PIM1 promoted cell growth and invasion [28]. These results were confirmed in the U2OS human osteosarcoma cell line by Liao [26], suggesting a crucial role of PIM1 in the development and progression of cancer. In the current study, PIM1 expression was upregulated and inversely correlated with the miR-542-3p level in melanoma tissues. Knockdown of PIM1 via RNA interference replicated the phenotype of miR-542-3p expression in melanoma cell lines, while forced expression of PIM1 partially rescued the phenotype mediated by miR-542-3p. Our results clearly demonstrate that miR-542-3p suppresses the migration and invasion of melanoma cells through PIM1 downregulation. In summary, miR-542-3p, a miRNA downregulated in melanoma, markedly inhibits cell migration and invasion by directly targeting PIM1. The newly identified miR-542-3p/PIM1 axis may provide novel insights into the mechanisms underlying the pathogenesis of melanoma and serve as a potential therapeutic target.
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Conflict of interest All authors including Zhen Rang, Ge Yang, You-wei Wang and Fan Cui declare no conflict of interest.
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Acknowledgements This work was supported by the National Natural Science Foundation of China (81101231).
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