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MiR-769 promoted cell proliferation in human melanoma by suppressing GSK3B expression
Biomedicine & Pharmacotherapy 82 (2016) 117–123
ScienceDirect
MiR-769 promoted cell proliferation in human melanoma by suppressing GSK3B expression Hai-jiang Qiua,b , Xiao-he Lua,* , Sha-sha Yangb , Chen-yin Wengb , E-keng Zhangb , Fang-chao Chenb a
Department of Ophthalmology, Zhujiang Hospital, Southern Medical University, Guangzhou 510515, People’s Republic of China, People’s Republic of China Department of ophthalmology, Guangzhou First People’s Hospital, Guangzhou Medical University, Guangzhou 510180, People’s Republic of China, People’s Republic of China b
A R T I C L E I N F O
Article history: Received 30 March 2016 Received in revised form 24 April 2016 Accepted 25 April 2016 Keywords: miR-769 Human melanoma GSK3B Cell proliferation
A B S T R A C T
MicroRNAs (miRNAs) are short, non-coding RNAs with post-transcriptional regulatory function, playing crucial roles in cancer development and progression of human melanoma. Previous studies have indicated that miR-769 was implicated in diverse biological processes. However, the underlying mechanism of miR-769 in human melanoma has not been intensively investigated. In this present study, we aimed to investigate the role of miR-769 and its target genes in human melanoma. We found that miR769 expression was strongly increased in human melanoma cells and clinical tissues compared with their corresponding controls. Overexpression of miR-769 promoted cell proliferation in human melanoma cell line A375, whereas miR-769-in reverses the function. Glycogen synthase kinase-3 Beta (GSK3B), a potential target gene of miR-769, and was validated by luciferase assay. Further studies revealed that miR-769 regulated cell proliferation of human melanoma by directly suppressing GSK3B expression and the knockdown of GSK3B expression reversed the effect of miR-769-in on human melanoma cell proliferation. In summary, our data demonstrated that miR-769 might act as a tumor promoter by targeting GSK3B during development of human melanoma. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Human melanoma is one of the deadliest and most aggressive forms of skin cancer in humans [1]. Recent years, although advanced treatment of surgery, chemotherapy and immunotherapy, the median survival of patients with advanced metastatic melanoma is limited, it is urgent to understand the progression of human melanoma. MicroRNAs (miRNAs) are non-coding RNAs that regulated gene function by direct binding of target mRNAs and then playing important roles in nearly all the biological functions, such as cell proliferation, cell migration, cell invasion, cell apoptosis and cell angiogenesis [2–8]. MiRNAs acted as potential therapeutic targets for treatment of human cancers, including human melanoma [9– 13]. It is reported that miR-26b suppressed TRAF5-mediated MAPK activation and decreased melanoma cell proliferation [14]. Report by Ding et al. revealed that miR-664 identified as a tumor suppressor and loss of miR-664 expression increased human
* Corresponding author. E-mail address: [email protected] (X.-h. Lu). http://dx.doi.org/10.1016/j.biopha.2016.04.052 0753-3322/ ã 2016 Elsevier Masson SAS. All rights reserved.
melanoma proliferation by upregulating expression of PLP2 [15]. Finding of Zhou et al. indicated that miR-33a functioned as a tumor suppressor by targeting HIF-1a in human melanoma proliferation [16]. MiR-3646 was reported to contribute to docetaxel resistance in human breast cancer cells by GSK-3b/b-Catenin signaling pathway [17]. However, the functional role of miR-769 in Human melanoma has remained unknown. In the current study, our result revealed that miR-769 was up-regulated in malignant melanoma tissue and cell lines. Ectopic expression of miR-769 in malignant melanoma cells led to the promotion of cell growth. Furthermore, we found that miR769 might regulate Human melanoma cell proliferation by targeting GSK3B through bioinformatics analyses. Therefore, the findings of this study revealed that overexpression of miR769 could promote cell proliferation in malignant melanoma by repressing GSK3B.
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2. Materials and methods 2.1. Clinical specimens Skin tissues were obtained from patients and histopathologically diagnosed at Guangzhou First People's Hospital, Guangzhou Medical University (Guangzhou, People’s Republic of China). Ethical approval for the study was approved by the ethics committee of Guangzhou First People's Hospital, Guangzhou Medical University (Guangzhou, People’s Republic of China). All samples were collected and analyzed with prior written, informed consent of the patients. 2.2. Cell culture Human melanoma cells lines, including MHEM, SK-MEL-28, WM-115, UACC257, A375, A7 and MeWo were purchased from National Rodent Laboratory Animal Resource (Shanghai, People’s Republic of China) and were grown in RPMI-1640 medium (Gbico, USA) supplemented with 10% fetal bovine serum (FBS, Sigma, USA), 100units/ml of penicillin-streptomycin (Invitrogen, Carlsbad, CA), and primary Human Epidermal Melanocytes (PEM, PromoCell, Germany) from adult skin were maintained in serum- and PMAfree melanocyte growth medium M2 (PromoCell, Germany).All cells were cultured in a humidified incubator at 37 C with 5% CO2. 2.3. Plasmids, small interfering RNA and transfection
(Applied Biosystems). The expression of miRNA was defined based on the threshold cycle (Ct), and relative expression levels were calculated as 2[(CtofmiR769)(CtofU6)] after normalization to U6 small nuclear RNA. Real-time PCR was performed using the Applied Biosystems 7500 Sequence Detection system. The following PCR primers were synthesized by GeneCopoeiaTM as followed: Cyclin D1 (HQP016204), MYC (HQP011597) and GAPDH (HQP006940). Expression levels of genes were normalized to that of the housekeeping gene GAPDH as the control and calculated as 2[(CtofCyclinD1orMYC)(CtofGAPDH)]. 2.5. MTT assays and colony formation The viability of A375 cells was measured by MTT assay (Sigma-Aldrich). Five thousand A375 cells were seeded in 96-well plates for each transfection groups: miR-769, miR-769-in and the relative control mimics. Cells were incubated under conditions of 37 C at 5% CO2. After 1, 2, 3, 4 and 5 day, 20 ml of 5 mg/ml MTT (Sigma, USA) was added to each well and incubated for 4 h, and then the generated formazan was dissolved in 150 ml dimethyl sulfoxide (DMSO) for 10 min, and then measured in terms of optical density (OD) at a wavelength of 495 nm. For the colony formation assay, A375 cells (500 cells per well) after transfection with miR-769, miR-769-in or the relative controls were plated into 6-cm plates and incubated for 14 days, the cells were stained using crystal violet (0.5% w/v, Sigma) for 1 min after fixed with frozen methanol. Colonies were counted only if they contained more than 50 cells.
MiR-769, miR-769-inhibitor (miR-769-in), miR-769-mut and relative control miRNAs were synthesized by RiboBio Corporation (Guangzhou, China), and transfection was performed using Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA) as recommended by the manufacturer's instructions. For GSK3B depletion, small interfering RNA (siRNA) was synthesized and purified by purchased from GeneCopoeia (Guangzhou, China). Transfection of siRNAs were performed using lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol.
A375 cells were trypsinized, and 1000 cells were resuspended in 2 ml complete medium plus 0.3% agar (Sigma), and were plated on top of the agar layer. Cells were incubated for two weeks at 37 C until colony formation and colonies were stained with 1% Crystal Violet for counting under microscope and cell colonies were photographed at an original magnification of 100. Colonies greater than 0.1 mm in diameter were counted.
2.4. RNA extraction and real-time quantitative PCR
2.7. Luciferase assays
Total RNA from cultured cells and fresh surgical liver cancer tissues was extracted using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. The expression levels of miR-769 were quantified using miRNA-specific TaqMan MiRNA Assay Kit
The wild type 30 UTR of GSK3B were cloned into the pGL3 luciferase assays vector (Promega, USA), and then cotransfected into the cells with miR-769, miR-769-in or miR-769-mut using Lipofectamine 2000 Reagent (Invitrogen). Lysates were
2.6. Anchorage-independent growth assay
Fig. 1. Expression of miR-769 in human melanoma cell lines and tissues. (A) Real-time PCR analysis of miR-769 expression in primary Human Epidermal Melanocytes PEM cell lines and human melanoma cell lines, including MHEM, SK-MEL-28, WM-115, UACC257, A375, A7 and MeWo. (B) Relative miR-769 expression levels in 8 paired primary HCC tissues (T) and the tumor adjacent normal tissues (TAT) from the same patient were detected by PCR analysis. Each bar represents the mean of three independent experiments. *P < 0.05.
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collected 48 h after transfection and measured using a DualLuciferase Reporter System (Promega, Madison, WI, USA) according to the manufacturer protocol. 2.8. Western blotting Cells were transfected and protein lysates were prepared, the equivalent aliquots of proteins were electrophoresed by SDSPAGE on 10% gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membrane was incubated overnight with anti-TNIP2, anti-Cyclin D1, anti-c-Myc, anti-Rb and anti-p-Rb (1:1000; Cell Signaling Technology). To control sample loading, the blotting membranes were stripped and re-probed with an anti-atubulin antibody (Sigma-Aldrich), and followed by incubation with an HRP-conjugated secondary antibody (Santa Cruz Biotechnology).And signals were visualized by enhanced chemiluminescence (Pierce, Rockford, IL, USA) 2.9. Bromodeoxyuridine labeling and immunofluorescence Cells after transfection and grown on cover slips (Fisher, Pittsburgh, PA, USA) were incubated with bromodeoxyuridine (BrdU) for 1 h and then stained with an anti-BrdU antibody (Upstate, Temecula, CA) according to the manufacturer’s instructions. Gray level images were acquired using a laser scanning microscope (Axioskop 2 plus; Carl Zeiss Co., Ltd., Jena, Germany).
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2.10. Statistical analysis Statistical analysis was performed using the SPSS 17.0 (SPSS Inc, Chicago, IL, USA) and done by analysis of variance (ANOVA) or Student’s t-test. Statistical significance was defined as a value of P < 0.05. 3. Result 3.1. MiR-769 expression was upregulated in melanoma tissues and melanoma cell lines To explore the potential impact of miR-769 upon human melanoma development, we analyzed the expression of miR769 in different human melanoma cell lines and clinical tissues by qRT-PCR. Our results showed that expression of miR-769 was significantly upregulated in human melanoma cell lines (MHEM, SK-MEL-28, WM-115, UACC257, A375, A7 and MeWo), as compared with Primary Human Epidermal Melanocytes PEM cells (Fig. 1A). Next, we determined whether miR-769 expression was similarly correlated with the malignant melanoma. MiR-769 was significantly upregulated in malignant melanoma tissues, as compared with that detected in tumor adjacent normal tissues (TAT) (Fig. 1B). Taken together, these data indicated that upregulation of miR769 might be involved in human malignant melanoma development.
Fig. 2. Upregulation of miR-769 promoted human melanoma cell proliferation. (A) Validation of miR-769 expression levels after transfection by PCR analysis. (B)MTT assays revealed that inhibition of miR-769 promoted growth of A375 cell line. (C) Representative quantification of crystal violet-stained cell colonies. (D) Upregulation of miR769 promoted the anchorage-independent growth of A375 cells. Representative micrographs (left) and quantification of colonies that were >0.1 mm (right). (E) Representative micrographs (left) and quantification (right) of the BrdUrd incorporation assay in A375 cells. Each bar represents the mean of three independent experiments. *P < 0.05.
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3.2. MiR-769 promoted, while miR-769-in suppressed melanoma cell proliferation
3.3. MiR-769 directly targets GSK3B by binding to its 30 -UTR and altered levels of proteins related to cell proliferation in A375 cells
To explore the role of miR-769 in human melanoma, A375 cells stably overexpressing miR-769 and downregulation of miR-769 were established for further experiment, and the result of PCR revealed that both of them showed great transfection efficiency (Figs. 2A and 3A). Results of MTT and colony formation assays revealed that ectopic expression of miR-769 dramatically enhanced the growth rate of A375 cells compared with that of control cell (Fig. 2B and C). Additionally, result of anchorage independent growth assay revealed that A375 cells stably overexpressing miR-769 showed more and largersized colonies than control cells (Fig. 2D). Result of BrdU incorporation assay revealed that the level of DNA synthesis was significantly increased in miR769 overexpressing A375 cells, whereas the control cells displayed relatively lower BrdUrd incorporation rates (Fig. 2E). Moreover, miR-769-in significantly decreased the growth rate of A375 cells as compared with that of NC transfected cells (Fig. 3B and C). The anchorage-independent growth assay indicated that miR-769-in led to fewer and smaller colonies than the control cells (Fig. 3D). BrdU incorporation assays demonstrated that the level of DNA synthesis was reduced by miR-769-in (Fig. 3E). These observations suggested that upregulation of miR-769 promoted the proliferation and tumorigenicity of malignant melanoma cells in vitro.
To investigate the molecular mechanism of miR-769 in human melanoma cells, the potential binding sites of miR-769 in the 30 -UTR mRNA of GSK3B was predicted using the publicly available algorithms (TargetScan, http://www.targetscan.org/) (Fig. 4A). And the protein expression of GSK3B was further found to be decreased in A375 cells transfection with miR-769, while miR-769-in led to higher GSK3B expression (Fig. 4B). Further, to examine if miR-769 could bind to 30 -UTR of GSK3B by luciferase assays. Indeed, overexpression of miR-769 led to a remarkable decrease of luciferase activity of GSK3B 30 -UTR, whereas miR-769-in led to increased luciferase activity. However, luciferase activity was not affected by miR-769-mut (Fig. 4C). As miR-769 promoted cell proliferation, we further investigated the effect of miR-769 on the expression of the genes which regulate cell proliferation, including Cyclin D1 and c-myc. Using RT-PCR and Western bolting, compared to NC transfected cells, we observed that Cyclin D1 expression and MYC expression was up-regulated by miR-769, while Cyclin D1 expression and MYC expression was down-regulated after transfected with miR-769-in (Fig. 4D and E). Collectively, all these findings suggested that miR-769 probably regulated cell growth through GSK3B.
Fig. 3. Inhibition of miR-769 inhibited human melanoma cell proliferation. (A) Validation of miR-769 expression levels after transfection by PCR analysis. (B)MTT assays revealed that miR-769-in suppressed growth of A375 cells. (C) Representative quantification of crystal violet-stained cell colonies. (D) Inhibition of miR-769 inhibited the anchorage-independent growth of A375 cells. Representative micrographs (left) and quantification of colonies that were >0.1 mm (right). (E) Representative micrographs (left) and quantification (right) of the BrdUrd incorporation assay in A375 cells. Each bar represents the mean of three independent experiments. *P < 0.05.
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Fig. 4. miR-769 suppressed GSK3B expression by directly targeting the TNIP2 30 -UTR and altered levels of proteins related to cell proliferation in A375 cells. (A) Predicted miR-769 target sequence in the 30 -UTR of GSK3B (GSK3B 30 -UTR) and positions of three mutated nucleotides (green) in the 30 -UTR of miR-769 (miR-769-mut). (B) Western blotting analysis of GSK3B expression in A375 cells transfected with miR-769 or the miR-769-in, a-Tubulin served as the loading control. (C) Luciferase reporter assay of the indicated cells transfected with the pGL3- GSK3B 30 -UTR reporter and miR-769 or miR-769-in or miR-769-mut.*P < 0.05. (D) Real-time PCR analysis of expression of Cyclin D1 and c-myc in indicated A375 cells. (E) Western blotting analysis of expression of Cyclin D1 and c-myc protein in A375 cells. a-Tubulin served as the loading control. *P < 0.05.
3.4. GSK3B downregulation counteracted the proliferation arrest by miR-769-in To further confirm the effect of GSK3B on human melanoma cell proliferation, specific siRNAs of GSK3B were used to suppress GSK3B expression in miR-769-in-transfected A375 cells, respectively (Fig. 5A). The results of colony formation assays and anchorage-independent growth assay both revealed that suppression of GSK3B in cells transfected with miR-769-in dramatically increased cell proliferation of human melanoma A375 cells (Fig. 5B and C).Taken together, our results demonstrated that GSK3B was a functionally important target of miR-769 and was involved in miR769 regulated human melanoma cell proliferation. 4. Discussion In the current study, we found that miR-769 was significantly upregulated in human melanoma cell lines and clinical tissues. Functional tests performed by transfecting miR-769 or miR-769-in
into A375 cells revealed that miR-769 increased human melanoma cell proliferation and tumorigenicity. Furthermore, to explore the mechanism of miR-769-induced cell proliferation, we investigated that GSK3B was identified as a direct target of miR-769 and are essential for the bio-function of miR- miR-769 in human melanoma. Taken together, our results suggested that miR769 played an essential role in the development and progression of human melanoma. Recently, the roles of miRNAs in the regulation of cancer progression and development are attracting increasing attention. Lots of reports have focused on the biological properties and progression of human melanoma. MiR-365 is reported to be strongly downregulated and inhibited cell growth, invasion and metastasis in malignant melanoma [18]. MiR-454 was found to functions as an oncogene by regulating PTEN in melanoma [19]. Furthermore, miR-137 targeted PAK2 and then inhibited proliferation of melanoma cells [20]. Sun et al. demonstrated that miR-144 suppressed cell proliferation and invasion of human melanoma [9]. But, the biological function of miR-769 and its
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Fig. 5. GSK3B downregulation counteracted the proliferation arrest by miR-769-in. (A) Western blot analysis verified that silencing GSK3B effectively decreased the expression of GSK3B in miR-769-in-transfected A375 cells. (B) miR-769-in-transfected A375 cells after transfection with GSK3B-siRNAs promoted cell colonies formation. (C) miR-769-in-transfected A375 cells after transfection with GSK3B-siRNAs promoted the anchorage-independent growth. Representative quantification of colonies that were >0.1 mm. Each bar represents the mean of three independent experiments. *P < 0.05.
mechanism in development of human melanoma has not been elucidated. In this study, we provided compelling biological evidence that miR-769 expression was significantly upregulated in human melanoma clinical tissues and cell lines, and miR769 promoted cell proliferation of human melanoma, suggesting miR-769 acting as an onco-miR and promoting cell proliferation in human melanoma. Growing reports have indicated that miRNAs play essential roles in translational repression by targeting the 30 untranslated region (30 -UTR) of its targeted mRNAs. MiR-524-5p was reported to suppress the growth of oncogenic BRAF melanoma by targeting BRAF and ERK2 [21]. MiR-203 was reported to target the polycomb group gene BMI1 and then suppressed melanoma invasive and proliferative abilities [22]. Currently, we demonstrated that GSK3B was the direct targets of miR-769 and that silencing GSK3B counteracted the proliferation arrest by miR-769-in. And further investigation indicated that altered levels of proteins (Cyclin D1 and c-myc) related to cell proliferation in A375 cells. Upregulation of Cyclin D1 and c-myc in A375 cells transfected with miR-769, indicating that miR-769 plays an essential role in human melanoma progression. In conclusion, the current study showed that miR-769 is significantly upregulated and promoted cell proliferation in human melanoma. MiR-769 suppressed 30 -UTR of GSK3B, and followed with dysregulation of CyclinD1 and c-myc, played critical roles during miR-769-mediated human melanoma cells proliferation. Our results presented novel insights into understanding the molecular mechanism of human melanoma tumorigenesis and provided a future direction for interventional therapeutic target of human melanoma. Conflict of interest The authors do not have any conflict of interest about this paper. Acknowledgments All authors designed the study together and performed the experiment together; all authors analyzed the data and wrote the paper; all authors approved the final manuscript. References [1] R. Siegel, D. Naishadham, A. Jemal, Cancer statistics, 2013, CA: Cancer J. Clin. 63 (2013) 11–30.
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ScienceDirect
MiR-769 promoted cell proliferation in human melanoma by suppressing GSK3B expression Hai-jiang Qiua,b , Xiao-he Lua,* , Sha-sha Yangb , Chen-yin Wengb , E-keng Zhangb , Fang-chao Chenb a
Department of Ophthalmology, Zhujiang Hospital, Southern Medical University, Guangzhou 510515, People’s Republic of China, People’s Republic of China Department of ophthalmology, Guangzhou First People’s Hospital, Guangzhou Medical University, Guangzhou 510180, People’s Republic of China, People’s Republic of China b
A R T I C L E I N F O
Article history: Received 30 March 2016 Received in revised form 24 April 2016 Accepted 25 April 2016 Keywords: miR-769 Human melanoma GSK3B Cell proliferation
A B S T R A C T
MicroRNAs (miRNAs) are short, non-coding RNAs with post-transcriptional regulatory function, playing crucial roles in cancer development and progression of human melanoma. Previous studies have indicated that miR-769 was implicated in diverse biological processes. However, the underlying mechanism of miR-769 in human melanoma has not been intensively investigated. In this present study, we aimed to investigate the role of miR-769 and its target genes in human melanoma. We found that miR769 expression was strongly increased in human melanoma cells and clinical tissues compared with their corresponding controls. Overexpression of miR-769 promoted cell proliferation in human melanoma cell line A375, whereas miR-769-in reverses the function. Glycogen synthase kinase-3 Beta (GSK3B), a potential target gene of miR-769, and was validated by luciferase assay. Further studies revealed that miR-769 regulated cell proliferation of human melanoma by directly suppressing GSK3B expression and the knockdown of GSK3B expression reversed the effect of miR-769-in on human melanoma cell proliferation. In summary, our data demonstrated that miR-769 might act as a tumor promoter by targeting GSK3B during development of human melanoma. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Human melanoma is one of the deadliest and most aggressive forms of skin cancer in humans [1]. Recent years, although advanced treatment of surgery, chemotherapy and immunotherapy, the median survival of patients with advanced metastatic melanoma is limited, it is urgent to understand the progression of human melanoma. MicroRNAs (miRNAs) are non-coding RNAs that regulated gene function by direct binding of target mRNAs and then playing important roles in nearly all the biological functions, such as cell proliferation, cell migration, cell invasion, cell apoptosis and cell angiogenesis [2–8]. MiRNAs acted as potential therapeutic targets for treatment of human cancers, including human melanoma [9– 13]. It is reported that miR-26b suppressed TRAF5-mediated MAPK activation and decreased melanoma cell proliferation [14]. Report by Ding et al. revealed that miR-664 identified as a tumor suppressor and loss of miR-664 expression increased human
* Corresponding author. E-mail address: [email protected] (X.-h. Lu). http://dx.doi.org/10.1016/j.biopha.2016.04.052 0753-3322/ ã 2016 Elsevier Masson SAS. All rights reserved.
melanoma proliferation by upregulating expression of PLP2 [15]. Finding of Zhou et al. indicated that miR-33a functioned as a tumor suppressor by targeting HIF-1a in human melanoma proliferation [16]. MiR-3646 was reported to contribute to docetaxel resistance in human breast cancer cells by GSK-3b/b-Catenin signaling pathway [17]. However, the functional role of miR-769 in Human melanoma has remained unknown. In the current study, our result revealed that miR-769 was up-regulated in malignant melanoma tissue and cell lines. Ectopic expression of miR-769 in malignant melanoma cells led to the promotion of cell growth. Furthermore, we found that miR769 might regulate Human melanoma cell proliferation by targeting GSK3B through bioinformatics analyses. Therefore, the findings of this study revealed that overexpression of miR769 could promote cell proliferation in malignant melanoma by repressing GSK3B.
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2. Materials and methods 2.1. Clinical specimens Skin tissues were obtained from patients and histopathologically diagnosed at Guangzhou First People's Hospital, Guangzhou Medical University (Guangzhou, People’s Republic of China). Ethical approval for the study was approved by the ethics committee of Guangzhou First People's Hospital, Guangzhou Medical University (Guangzhou, People’s Republic of China). All samples were collected and analyzed with prior written, informed consent of the patients. 2.2. Cell culture Human melanoma cells lines, including MHEM, SK-MEL-28, WM-115, UACC257, A375, A7 and MeWo were purchased from National Rodent Laboratory Animal Resource (Shanghai, People’s Republic of China) and were grown in RPMI-1640 medium (Gbico, USA) supplemented with 10% fetal bovine serum (FBS, Sigma, USA), 100units/ml of penicillin-streptomycin (Invitrogen, Carlsbad, CA), and primary Human Epidermal Melanocytes (PEM, PromoCell, Germany) from adult skin were maintained in serum- and PMAfree melanocyte growth medium M2 (PromoCell, Germany).All cells were cultured in a humidified incubator at 37 C with 5% CO2. 2.3. Plasmids, small interfering RNA and transfection
(Applied Biosystems). The expression of miRNA was defined based on the threshold cycle (Ct), and relative expression levels were calculated as 2[(CtofmiR769)(CtofU6)] after normalization to U6 small nuclear RNA. Real-time PCR was performed using the Applied Biosystems 7500 Sequence Detection system. The following PCR primers were synthesized by GeneCopoeiaTM as followed: Cyclin D1 (HQP016204), MYC (HQP011597) and GAPDH (HQP006940). Expression levels of genes were normalized to that of the housekeeping gene GAPDH as the control and calculated as 2[(CtofCyclinD1orMYC)(CtofGAPDH)]. 2.5. MTT assays and colony formation The viability of A375 cells was measured by MTT assay (Sigma-Aldrich). Five thousand A375 cells were seeded in 96-well plates for each transfection groups: miR-769, miR-769-in and the relative control mimics. Cells were incubated under conditions of 37 C at 5% CO2. After 1, 2, 3, 4 and 5 day, 20 ml of 5 mg/ml MTT (Sigma, USA) was added to each well and incubated for 4 h, and then the generated formazan was dissolved in 150 ml dimethyl sulfoxide (DMSO) for 10 min, and then measured in terms of optical density (OD) at a wavelength of 495 nm. For the colony formation assay, A375 cells (500 cells per well) after transfection with miR-769, miR-769-in or the relative controls were plated into 6-cm plates and incubated for 14 days, the cells were stained using crystal violet (0.5% w/v, Sigma) for 1 min after fixed with frozen methanol. Colonies were counted only if they contained more than 50 cells.
MiR-769, miR-769-inhibitor (miR-769-in), miR-769-mut and relative control miRNAs were synthesized by RiboBio Corporation (Guangzhou, China), and transfection was performed using Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA) as recommended by the manufacturer's instructions. For GSK3B depletion, small interfering RNA (siRNA) was synthesized and purified by purchased from GeneCopoeia (Guangzhou, China). Transfection of siRNAs were performed using lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol.
A375 cells were trypsinized, and 1000 cells were resuspended in 2 ml complete medium plus 0.3% agar (Sigma), and were plated on top of the agar layer. Cells were incubated for two weeks at 37 C until colony formation and colonies were stained with 1% Crystal Violet for counting under microscope and cell colonies were photographed at an original magnification of 100. Colonies greater than 0.1 mm in diameter were counted.
2.4. RNA extraction and real-time quantitative PCR
2.7. Luciferase assays
Total RNA from cultured cells and fresh surgical liver cancer tissues was extracted using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. The expression levels of miR-769 were quantified using miRNA-specific TaqMan MiRNA Assay Kit
The wild type 30 UTR of GSK3B were cloned into the pGL3 luciferase assays vector (Promega, USA), and then cotransfected into the cells with miR-769, miR-769-in or miR-769-mut using Lipofectamine 2000 Reagent (Invitrogen). Lysates were
2.6. Anchorage-independent growth assay
Fig. 1. Expression of miR-769 in human melanoma cell lines and tissues. (A) Real-time PCR analysis of miR-769 expression in primary Human Epidermal Melanocytes PEM cell lines and human melanoma cell lines, including MHEM, SK-MEL-28, WM-115, UACC257, A375, A7 and MeWo. (B) Relative miR-769 expression levels in 8 paired primary HCC tissues (T) and the tumor adjacent normal tissues (TAT) from the same patient were detected by PCR analysis. Each bar represents the mean of three independent experiments. *P < 0.05.
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collected 48 h after transfection and measured using a DualLuciferase Reporter System (Promega, Madison, WI, USA) according to the manufacturer protocol. 2.8. Western blotting Cells were transfected and protein lysates were prepared, the equivalent aliquots of proteins were electrophoresed by SDSPAGE on 10% gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membrane was incubated overnight with anti-TNIP2, anti-Cyclin D1, anti-c-Myc, anti-Rb and anti-p-Rb (1:1000; Cell Signaling Technology). To control sample loading, the blotting membranes were stripped and re-probed with an anti-atubulin antibody (Sigma-Aldrich), and followed by incubation with an HRP-conjugated secondary antibody (Santa Cruz Biotechnology).And signals were visualized by enhanced chemiluminescence (Pierce, Rockford, IL, USA) 2.9. Bromodeoxyuridine labeling and immunofluorescence Cells after transfection and grown on cover slips (Fisher, Pittsburgh, PA, USA) were incubated with bromodeoxyuridine (BrdU) for 1 h and then stained with an anti-BrdU antibody (Upstate, Temecula, CA) according to the manufacturer’s instructions. Gray level images were acquired using a laser scanning microscope (Axioskop 2 plus; Carl Zeiss Co., Ltd., Jena, Germany).
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2.10. Statistical analysis Statistical analysis was performed using the SPSS 17.0 (SPSS Inc, Chicago, IL, USA) and done by analysis of variance (ANOVA) or Student’s t-test. Statistical significance was defined as a value of P < 0.05. 3. Result 3.1. MiR-769 expression was upregulated in melanoma tissues and melanoma cell lines To explore the potential impact of miR-769 upon human melanoma development, we analyzed the expression of miR769 in different human melanoma cell lines and clinical tissues by qRT-PCR. Our results showed that expression of miR-769 was significantly upregulated in human melanoma cell lines (MHEM, SK-MEL-28, WM-115, UACC257, A375, A7 and MeWo), as compared with Primary Human Epidermal Melanocytes PEM cells (Fig. 1A). Next, we determined whether miR-769 expression was similarly correlated with the malignant melanoma. MiR-769 was significantly upregulated in malignant melanoma tissues, as compared with that detected in tumor adjacent normal tissues (TAT) (Fig. 1B). Taken together, these data indicated that upregulation of miR769 might be involved in human malignant melanoma development.
Fig. 2. Upregulation of miR-769 promoted human melanoma cell proliferation. (A) Validation of miR-769 expression levels after transfection by PCR analysis. (B)MTT assays revealed that inhibition of miR-769 promoted growth of A375 cell line. (C) Representative quantification of crystal violet-stained cell colonies. (D) Upregulation of miR769 promoted the anchorage-independent growth of A375 cells. Representative micrographs (left) and quantification of colonies that were >0.1 mm (right). (E) Representative micrographs (left) and quantification (right) of the BrdUrd incorporation assay in A375 cells. Each bar represents the mean of three independent experiments. *P < 0.05.
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3.2. MiR-769 promoted, while miR-769-in suppressed melanoma cell proliferation
3.3. MiR-769 directly targets GSK3B by binding to its 30 -UTR and altered levels of proteins related to cell proliferation in A375 cells
To explore the role of miR-769 in human melanoma, A375 cells stably overexpressing miR-769 and downregulation of miR-769 were established for further experiment, and the result of PCR revealed that both of them showed great transfection efficiency (Figs. 2A and 3A). Results of MTT and colony formation assays revealed that ectopic expression of miR-769 dramatically enhanced the growth rate of A375 cells compared with that of control cell (Fig. 2B and C). Additionally, result of anchorage independent growth assay revealed that A375 cells stably overexpressing miR-769 showed more and largersized colonies than control cells (Fig. 2D). Result of BrdU incorporation assay revealed that the level of DNA synthesis was significantly increased in miR769 overexpressing A375 cells, whereas the control cells displayed relatively lower BrdUrd incorporation rates (Fig. 2E). Moreover, miR-769-in significantly decreased the growth rate of A375 cells as compared with that of NC transfected cells (Fig. 3B and C). The anchorage-independent growth assay indicated that miR-769-in led to fewer and smaller colonies than the control cells (Fig. 3D). BrdU incorporation assays demonstrated that the level of DNA synthesis was reduced by miR-769-in (Fig. 3E). These observations suggested that upregulation of miR-769 promoted the proliferation and tumorigenicity of malignant melanoma cells in vitro.
To investigate the molecular mechanism of miR-769 in human melanoma cells, the potential binding sites of miR-769 in the 30 -UTR mRNA of GSK3B was predicted using the publicly available algorithms (TargetScan, http://www.targetscan.org/) (Fig. 4A). And the protein expression of GSK3B was further found to be decreased in A375 cells transfection with miR-769, while miR-769-in led to higher GSK3B expression (Fig. 4B). Further, to examine if miR-769 could bind to 30 -UTR of GSK3B by luciferase assays. Indeed, overexpression of miR-769 led to a remarkable decrease of luciferase activity of GSK3B 30 -UTR, whereas miR-769-in led to increased luciferase activity. However, luciferase activity was not affected by miR-769-mut (Fig. 4C). As miR-769 promoted cell proliferation, we further investigated the effect of miR-769 on the expression of the genes which regulate cell proliferation, including Cyclin D1 and c-myc. Using RT-PCR and Western bolting, compared to NC transfected cells, we observed that Cyclin D1 expression and MYC expression was up-regulated by miR-769, while Cyclin D1 expression and MYC expression was down-regulated after transfected with miR-769-in (Fig. 4D and E). Collectively, all these findings suggested that miR-769 probably regulated cell growth through GSK3B.
Fig. 3. Inhibition of miR-769 inhibited human melanoma cell proliferation. (A) Validation of miR-769 expression levels after transfection by PCR analysis. (B)MTT assays revealed that miR-769-in suppressed growth of A375 cells. (C) Representative quantification of crystal violet-stained cell colonies. (D) Inhibition of miR-769 inhibited the anchorage-independent growth of A375 cells. Representative micrographs (left) and quantification of colonies that were >0.1 mm (right). (E) Representative micrographs (left) and quantification (right) of the BrdUrd incorporation assay in A375 cells. Each bar represents the mean of three independent experiments. *P < 0.05.
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Fig. 4. miR-769 suppressed GSK3B expression by directly targeting the TNIP2 30 -UTR and altered levels of proteins related to cell proliferation in A375 cells. (A) Predicted miR-769 target sequence in the 30 -UTR of GSK3B (GSK3B 30 -UTR) and positions of three mutated nucleotides (green) in the 30 -UTR of miR-769 (miR-769-mut). (B) Western blotting analysis of GSK3B expression in A375 cells transfected with miR-769 or the miR-769-in, a-Tubulin served as the loading control. (C) Luciferase reporter assay of the indicated cells transfected with the pGL3- GSK3B 30 -UTR reporter and miR-769 or miR-769-in or miR-769-mut.*P < 0.05. (D) Real-time PCR analysis of expression of Cyclin D1 and c-myc in indicated A375 cells. (E) Western blotting analysis of expression of Cyclin D1 and c-myc protein in A375 cells. a-Tubulin served as the loading control. *P < 0.05.
3.4. GSK3B downregulation counteracted the proliferation arrest by miR-769-in To further confirm the effect of GSK3B on human melanoma cell proliferation, specific siRNAs of GSK3B were used to suppress GSK3B expression in miR-769-in-transfected A375 cells, respectively (Fig. 5A). The results of colony formation assays and anchorage-independent growth assay both revealed that suppression of GSK3B in cells transfected with miR-769-in dramatically increased cell proliferation of human melanoma A375 cells (Fig. 5B and C).Taken together, our results demonstrated that GSK3B was a functionally important target of miR-769 and was involved in miR769 regulated human melanoma cell proliferation. 4. Discussion In the current study, we found that miR-769 was significantly upregulated in human melanoma cell lines and clinical tissues. Functional tests performed by transfecting miR-769 or miR-769-in
into A375 cells revealed that miR-769 increased human melanoma cell proliferation and tumorigenicity. Furthermore, to explore the mechanism of miR-769-induced cell proliferation, we investigated that GSK3B was identified as a direct target of miR-769 and are essential for the bio-function of miR- miR-769 in human melanoma. Taken together, our results suggested that miR769 played an essential role in the development and progression of human melanoma. Recently, the roles of miRNAs in the regulation of cancer progression and development are attracting increasing attention. Lots of reports have focused on the biological properties and progression of human melanoma. MiR-365 is reported to be strongly downregulated and inhibited cell growth, invasion and metastasis in malignant melanoma [18]. MiR-454 was found to functions as an oncogene by regulating PTEN in melanoma [19]. Furthermore, miR-137 targeted PAK2 and then inhibited proliferation of melanoma cells [20]. Sun et al. demonstrated that miR-144 suppressed cell proliferation and invasion of human melanoma [9]. But, the biological function of miR-769 and its
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Fig. 5. GSK3B downregulation counteracted the proliferation arrest by miR-769-in. (A) Western blot analysis verified that silencing GSK3B effectively decreased the expression of GSK3B in miR-769-in-transfected A375 cells. (B) miR-769-in-transfected A375 cells after transfection with GSK3B-siRNAs promoted cell colonies formation. (C) miR-769-in-transfected A375 cells after transfection with GSK3B-siRNAs promoted the anchorage-independent growth. Representative quantification of colonies that were >0.1 mm. Each bar represents the mean of three independent experiments. *P < 0.05.
mechanism in development of human melanoma has not been elucidated. In this study, we provided compelling biological evidence that miR-769 expression was significantly upregulated in human melanoma clinical tissues and cell lines, and miR769 promoted cell proliferation of human melanoma, suggesting miR-769 acting as an onco-miR and promoting cell proliferation in human melanoma. Growing reports have indicated that miRNAs play essential roles in translational repression by targeting the 30 untranslated region (30 -UTR) of its targeted mRNAs. MiR-524-5p was reported to suppress the growth of oncogenic BRAF melanoma by targeting BRAF and ERK2 [21]. MiR-203 was reported to target the polycomb group gene BMI1 and then suppressed melanoma invasive and proliferative abilities [22]. Currently, we demonstrated that GSK3B was the direct targets of miR-769 and that silencing GSK3B counteracted the proliferation arrest by miR-769-in. And further investigation indicated that altered levels of proteins (Cyclin D1 and c-myc) related to cell proliferation in A375 cells. Upregulation of Cyclin D1 and c-myc in A375 cells transfected with miR-769, indicating that miR-769 plays an essential role in human melanoma progression. In conclusion, the current study showed that miR-769 is significantly upregulated and promoted cell proliferation in human melanoma. MiR-769 suppressed 30 -UTR of GSK3B, and followed with dysregulation of CyclinD1 and c-myc, played critical roles during miR-769-mediated human melanoma cells proliferation. Our results presented novel insights into understanding the molecular mechanism of human melanoma tumorigenesis and provided a future direction for interventional therapeutic target of human melanoma. Conflict of interest The authors do not have any conflict of interest about this paper. Acknowledgments All authors designed the study together and performed the experiment together; all authors analyzed the data and wrote the paper; all authors approved the final manuscript. References [1] R. Siegel, D. Naishadham, A. Jemal, Cancer statistics, 2013, CA: Cancer J. Clin. 63 (2013) 11–30.
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