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MiR-454 inhibited cell proliferation of human glioblastoma cells by suppressing PDK1 expression
G Model BIOPHA 3716 No. of Pages 5
Biomedicine & Pharmacotherapy xxx (2015) xxx–xxx
Available online at
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Original article
MiR-454 inhibited cell proliferation of human glioblastoma cells by suppressing PDK1 expression Baojun Fanga,b , Jianxin Zhua,b , Yunhua Wangb , Fengyang Gengb , Gang Lia,* a b
Department of Neurosurgery, Qilu Hospital of Shandong University, Jinan, 250012 Shandong, PR China Department of Neurosurgery, Liaocheng People’s Hospital, Liaocheng 252000, Shandong, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 April 2015 Accepted 26 July 2015
It has been well documented that aberrant expression of microRNAs is associated with carcinogenesis of glioblastoma (GBM), however the underlying mechanisms are not clear. In this present study, we aimed to clarify the biological function of miR-454 in GBM. MiR-454 was identified to be significantly downregulated in GBM primary tumors and cell lines. Overexpression of miR-454 in GBM cells resulted in arresting cells at G0/G1 phase and thus inhibiting cell proliferation. Bioinformatic analysis predicted 3-phosphoinositide-dependent protein kinase-1 (PDK1) as a target of miR-454 which acted as a tumor promoter gene. Increased miR-454 significantly repressed PDK1 expression, and then regulating cell proliferation and cell cycle regulators, down-regulation of Cyclin D1 and p-pRb and p21 was up-regulated. Taken together, our study has revealed miR-454 as a tumor suppressor in GBM. ã 2015 Elsevier Masson SAS. All rights reserved.
Keywords: MiR-454 Glioblastoma PDK1 Cell proliferation Cell cycle
1. Introduction Glioblastoma (GBM) is the most common and aggressive Central nervous system malignancy in adults worldwide [1]. However, the causes of GBM carcinogenesis and progression are largely unknown. In recent years, numerous studies have revealed aberrant expression of microRNAs(miRNAs) played critical roles in cancer cell proliferation, metastasis, and response to cancer therapy [2–6]. MiRNAs are a novel class of small noncoding RNAs (19– 22 nucleotides) that play essential roles in multiple biological processes of cancer, such as cell proliferation, invasion, migration and cell cycle, by typically suppressing the translation of messenger RNAs (mRNAs) by binding to the 30 -untranslated regions (30 -UTR) of their target mRNAs [7–11]. However, the biological function of miR-454 in GBM remains largely unknown. In the current study, our results revealed that miR-454 was markedly downregulated in GBM primary tumors and cell lines. Using Bioinformatic analysis, MTT assays, colony formation and cell cycle assay, we found that miR-454 acted as a tumor suppressor in GBM, suppressing GBM cell proliferation was due to inhibit 3-phosphoinositide-dependent protein kinase-1
* Corresponding author at: Department of Neurosurgery, Qilu Hospital of Shandong University, 107 Wenhua West Road, Lixia District, Jinan, Shandong Province, PR China. E-mail address: [email protected] (G. Li).
(PDK1), and then regulating cell proliferation and cell cycle regulators, down-regulation of Cyclin D1 and p-pRb and p21 was up-regulated. 2. Materials and methods 2.1. Clinical specimens Eight paired human GBM tissues and their matched adjacent normal brain tissues (ANT) were obtained from GBM patients at Department of Neurosurgery, Qilu Hospital of Shandong University (Jinan, People’s Republic of China). The study was approved by the ethics committee of Qilu Hospital of Shandong University (Jinan, People’s Republic of China). All patients gave informed consent in written. Tissue samples were collected at surgery, immediately frozen in liquid nitrogen and stored until total RNAs or proteins were extracted. 2.2. Cell culture Human GBM cell lines U87, T98G, A-172, LN18 and LN-229 were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gbico, USA) supplemented with 10% fetal bovine serum (FBS, Sigma, USA), 100 units/ml of penicillin-streptomycin (Invitrogen, Carlsbad, CA), and NHAs were purchased from Lonza and cultured in the provided astrocyte growth media supplemented with rhEGF, insulin, ascorbic acid, GA-1000, L-glutamine and, 5%
http://dx.doi.org/10.1016/j.biopha.2015.07.029 0753-3322/ ã 2015 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: B. Fang, et al., MiR-454 inhibited cell proliferation of human glioblastoma cells by suppressing PDK1 expression, Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.07.029
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FBS. Cells were cultured in a humidified incubator in an atmosphere of 5% CO2 at 37 C.
luciferase activity using the Dual-Luciferase Reporter Assay system (Promega).
2.3. Plasmids and transfection
2.8. Western blotting
The PDK1 30 -UTR fragment containing putative binding sites for miR-454 and the mutated binding site of the PDK1 30 -UTR (as a negative control) were amplified using PCR and subcloned into the pGL3-luciferase reporter plasmid (Promega). The miR-454 mimics, miR-454 inhibitor and relative negative control were purchased from Shanghai GenePharma (Shanghai, China) and transfected into GBM cells using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer’s instructions.
Cells were lysed with RIPA lysis buffer on ice for 20 min, proteins (40 mg) were subjected to 10%SDS-PAGE gel, transferred onto PVDF membranes by electroblotting. After probed with 1:1000 diluted anti-PDK1, anti-CyclinD1, anti-P21, anti-p-pRb and anti-pRb (Abcam, MA, USA) at 4 C overnight, the blots were washed with TBST and incubation with HRP (horseradish peroxidase)-conjugated secondary antibody (1:5000) for 2 h at room temperature. b-actin (Abcam, MA, USA) served as the loading control. Immunocomplexes were visualized using the ECL detection reagent (Beyotime, China) according to the manufacturer’s protocol.
2.4. RNA extraction and real-time quantitative PCR TRIzol Reagent (Invitrogen, Carlsbad, CA) was used to extract total RNA from the GBM cell lines and the clinical tissue samples according to the manufacturer’s instructions. The expression levels of miR-454 were quantified using miRNA-specific TaqMan miRNA assay kit (Applied Biosystems) according to the manufacturer’s instructions. The relative miR-454 expression levels after normalization to U6 small nuclear RNA were calculated using 2[(CtofmiR454)(CtofU6)].
All data were expressed as mean standard deviation (SD) and performed using the SPSS 18.0 (SPSS, Chicago, IL). The Student’s t-test was used to evaluate the statistical significance of differences between two groups of data in all pertinent experiments. A P-value less than 0.05 was considered to be statistically significant.
2.5. MTT assays and Colony formation
3. Result
To determine the effect of miR-454 on cell proliferation, MTT assay was used according to the Manufacturer’s instructions. For colony formation assay, miR-454 or miR-454-in or the relative miR-NC infected cells were placed in a fresh six well plate and incubated for 14 days in medium containing 10% FBS. Cells were fixed with methanol and stained with 0.1% crystal violet. Visible colonies were manually counted.
3.1. MiR-454 was downexpressed in human GBM tissues and GBM cell lines
2.6. Cell cycle assays by flow cytometry Pro-pidium iodide (PI) was attributable to cell cycle. The distribution of cells was analyzed by cell flow cytometry.
2.9. Statistical analysis
To test the expression of miR-454 in GBM, we examined miR454 expression in 8 GBM cell lines. The results showed that decreased expression of miR-454 was found in 9 GBM cell lines compared with the normal human astrocytes (NHAs) (Fig. 1A). In addition, we examined miR-454 expression in 8 GBM tissues and matched adjacent normal tissue using quantitative RT-PCR analysis. MiR-454 was differentially downregulated at the mRNA levels in GBM tissues compared with paired adjacent noncancerous tissues. Together, these results indicate that miR454 is downregulated in GBM primary tumors and cell lines.
2.7. Luciferase assays 3.2. MiR-454 inhibited GBM cell proliferation Cells (5 104) in each groups were seeded in 6-well plates and transfected with plasmid when cells reached 70% confluence, luciferase reporters were performed according to manufacturer’s instruction of lipofectamine 2000 (Invitrogen, USA). Cells were harvested 48 h after transfection, being analyzed for
To investigate the biological significance of miR-454 in GBM, we transfected the GBM cell lines, LN18 and T98G cells, with miR-454 mimics, miR-454 inhibitor or the respective controls. Real-time PCR was performed to detect the transfection efficiency
Fig. 1. Downregulation of miR-454 in human GBM tissues and cell lines. (A) Real-time PCR analysis of miR-454 expression in NHA and GBM cell lines, including U87, T98G, A-172, LN18 and LN-229. (B) Eight paired human GBM tissues and their matched adjacent normal brain tissues (ANT) were obtained from GBM patient, by PCR analysis. Experiments were repeated at least three times (A and B). Each bar represents the mean of three independent experiments. *P < 0.05.
Please cite this article in press as: B. Fang, et al., MiR-454 inhibited cell proliferation of human glioblastoma cells by suppressing PDK1 expression, Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.07.029
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Fig. 2. MiR-454 upregulation inhibited GBM cell proliferation. (A) Validation of miR-454 expression levels after transfection by PCR analysis. (B)MTT assays revealed that upregulation of miR-454 inhibited growth of GBM cell line LN18 and T98G. (C) Quantification of crystal violet-stained cell colonies. (D) Flow cytometric analysis of the indicated GBM cell line LN18 and T98G cells transfected with NC or miR-454. Each bar represents the mean of three independent experiments. *P < 0.05.
(Figs. 2 and 3A). To further investigate the functional role of miR454 in cell growth of GBM, MTT and colony formation assays were used. MTT and colony formation assays showed that miR454 significantly decreased cell growth and the ability of colony formation in LN18 and T98G cells (Fig. 2B and C). In contrary, miR454-in was used to investigate the role of miR-454 depletion in GBM cell lines. MiR-454-in increased the ability of cell proliferation and colony formation in LN18 and T98G cells (Fig. 3B and C). To further determine if cell growth rate of GBM cells was due to perturbation of cell cycles progression, cell cycle assays were used. Results of cell cycle assays revealed that overexpression of miR454 resulted in the arrest of cell cycle in G0/G1 phase and prevention of cells from entering into the S phase(Fig. 2D). On the contrary, miR-454-in caused more cells entering to S phase and thus promoted cell growth (Fig. 3D). Taken together, our results suggested that overexpression of miR-454 in GBM cells resulted in arresting cells at G0/G1 phase and thus inhibiting tumor cell growth.
To further investigate the direct effect of miR-454 on PDK1 expression, luciferase report assay was used. LN18 and T98G cells were cotransfected with miR-454 and PDK1 30 -UTR wild-type or mut-type. As shown in Fig. 4C, in miR-454 overexpressed cells, the expression of luciferase was dramatically inhibited. Furthermore, miR-454 had no effect on the luciferase activity of mut-type PDK1 30 -UTR. To evaluate whether PDK1 is regulated by miR-454, expressions of PDK1 were detected in the GBM cell lines LN18 and T98G cells, which were transfected with miR-454 mimics, miR-454-in or the respective controls. Expression of PDK1 was decreased in miR-454overexpression both GBM cell lines, LN18 and T98G cells, while miR-454 showed the opposite effect. Consistently in these cells, the downstream effectors of PDK1, Cyclin D1 and p-pRb were down-regulated, while p21 was up-regulated by miR-454 (Fig. 4b). All these findings suggested that miR-454 probably regulates cell growth through PDK1. 4. Discussion
3.3. MiR-454 directly targets PDK1 by binding to its 30 -UTR To further detect how miR-454 inhibits growth of GBM cells, we used a bioinformatic analysis (TargetScan Huaman 6.2) to find PDK1 acting as the target gene of miR-454. The wild-type or mut-type PDK1 30 -UTR were cloned into cloned into the 3’end of luciferase gene (Fig. 4A).
In the current study, we found that miR-454 is markedly downregulated in GBM primary tumors and cell lines. Furthermore, ectopic expression of miR-454 arrested GBM cells at G0/G1 phase and thus decreased cell proliferation, whereas miR-454-in had the opposite effect. More specifically, we showed that the molecular mechanism by which miR-454 inhibited GBM cell
Please cite this article in press as: B. Fang, et al., MiR-454 inhibited cell proliferation of human glioblastoma cells by suppressing PDK1 expression, Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.07.029
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Fig. 3. Inhibition of miR-454 promoted GBM cell proliferation. (A) Validation of miR-454 expression levels after transfection by PCR analysis. (B)MTT assays revealed that inhibition of miR-454 promoted growth of GBM cell line LN18 and T98G. (C) Quantification (right) of crystal violet-stained cell colonies. (D) Flow cytometric analysis of the indicated GBM cell line LN18 and T98G cells transfected with NC or miR-454-in. Each bar represents the mean of three independent experiments. *P < 0.05.
Fig. 4. MiR-454 suppresses PDK1 expression by directly targeting the PDK1 30 -UTR and altered levels of proteins related to cell proliferation and cell cycle in GBM cell line LN18 and T98G. (A) Predicted miR-454 target sequence in the 30 -UTR of PDK1 (PDK1-30 -UTR) and positions of three mutated nucleotides (red) in the 30 -UTR of PDK1 (PDK1-30 -UTR mut). (B) Western blotting analysis of protein expression of PDK1, cyclin D1, p21, p-pRb and pRb in indicated LN18 and T98G cells. b-actin served as the loading control. (C) Luciferase reporter assay of the indicated LN18 and T98G cells transfected with the pGL3-PDK1-30 -UTR reporter and miR-454 or miR-454 inhibitor oligonucleotides. *P < 0.05.
Please cite this article in press as: B. Fang, et al., MiR-454 inhibited cell proliferation of human glioblastoma cells by suppressing PDK1 expression, Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.07.029
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proliferation was due to suppress PDK1, and then regulating cell proliferation regulators, down-regulation of Cyclin D1 and p-pRb and p21 was up-regulated. Taken together, we propose that miR454 is a candidate tumor suppressor miRNA in GBM. Lots of reports have shown that that miRNAs are 19-25nucleotides regulatory non-protein coding RNA molecules that play essential roles in multiple biological processes of cancer [9,12,13]. MiRNAs usually function as endogenous repressors of target genes resulting in mRNA degradation [14–16]. Findings by SG Liu et al. indicated that expression of miR-454 was downregulated in esophageal cancer [17]. However, the molecular mechanism of miR-454-involved GBM progression has not been clarified. Our data expanded the role of miR-454 in GBM. In this study, qRT-PCR analysis indicated that miR-454 is significantly downregulated in GBM primary tumors and cell lines. Furthermore, the effect of miR-454 mimics and antisense transfection in LN18 and T98G cells showed that miR-454 decreased cell growth, colony formation via arresting GBM cells at G0/G1 phase. Bioinformatic algorithms predicted that PDK1, which was bona fide target genes of miR-454. Result of luciferase experiments showed that miR-454 could bind to the PDK1 30 UTR but not the mutant PDK1 30 UTR. Previous research showed that PDK1 played a pivotal role in the cell growth of angiosarcoma and inhibition of PDK1 may be a more appropriate strategy for the treatment of angiosarcoma [18]. Yang Z et al. reported that increased PDK1 expression associates with poor prognosis in esophageal squamous cell carcinoma with facilitating tumorigenicity in vitro [19]. Western blotting showed that downstream effectors of PDK1, Cyclin D1 and p-pRb were down-regulated, while p21 was upregulated in miR-454 transfected GBM cells. Taken together, this study clearly illustrated that miR-454 inhibited GBM cell proliferation was due to arresting GBM cells at G0/G1 phase, upregulation of p21 and downregulation of Cyclin D1 and p-pRb. In conclusion, the current study revealed that an essential link between miR-454-mediated tumor growth of GBM. Therefore, our results indicated that ectopic expression of miR-454 led to downregulation of Cyclin D1 and p-pRb and upregulation of p21, and thus to promote cell cycle and cell proliferation in GBM. Hence, miR-454 could be considered as a novel therapeutic target for GBM patients. Conflict of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments This work was supported by Department of Neurosurgery, Qilu Hospital of Shandong University. All authors designed the study together, performed the experiment together; all authors analyzed
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the data and wrote the paper; all authors approved the final manuscript. References [1] P.R. Sullivan, Brain tumors, N. Engl. J. Med. 344 (2001) 1478–1479 author reply 1479. [2] R. Wang, Y. Ma, D. Yu, J. Zhao, P. Ma, Mir-377 functions as a tumor suppressor in human clear cell renal cell carcinoma by targeting ets1, Biomed. Pharmacother. 70 (2015) 64–71. [3] P.M. Costa, A.L. Cardoso, C. Custodia, P. Cunha, A. de, L. lmeida, L. de, M.C. ima, Mirna-21 silencing mediated by tumor-targeted nanoparticles combined with sunitinib: A new multimodal gene therapy approach for glioblastoma, J. Control. Release 207 (2015) 31–39. [4] H. Chen, X. Li, W. Li, H. Zheng, Mir-130a can predict response to temozolomide in patients with glioblastoma multiforme, independently of o6methylguanine-DNA methyltransferase, J. Transl. Med. 13 (2015) 69. [5] H. Wang, T. Xu, Y. Jiang, Y. Yan, R. Qin, J. Chen, Micrornas in human glioblastoma: From bench to beside, Front. Biosci. (Landmark ed.) 20 (2015) 105–118. [6] C. Qin, R.Y. Huang, Z.X. Wang, Potential role of mir-100 in cancer diagnosis, prognosis, and therapy, Tumour Biol. 36 (2015) 1403–1409. [7] S.L. Romero-Cordoba, I. Salido-Guadarrama, M. Rodriguez-Dorantes, A. Hidalgo-Miranda, Mirna biogenesis: Biological impact in the development of cancer, Cancer Biol. Ther. 15 (2014) 1444–1455. [8] X.M. Zhao, K.Q. Liu, G. Zhu, F. He, B. Duval, J.M. Richer, D.S. Huang, C.J. Jiang, J.K. Hao, L. Chen, Identifying cancer-related micrornas based on gene expression data, Bioinformatics (Oxf. Engl.) 31 (2015) 1226–1234. [9] F. Shen, W.S. Cai, Z. Feng, J.L. Li, J.W. Chen, J. Cao, B. Xu, Mir-492 contributes to cell proliferation and cell cycle of human breast cancer cells by suppressing sox7 expression, Tumour Biol. 36 (2015) 1913–1921. [10] D. Jeansonne, M. DeLuca, L. Marrero, A. Lassak, M. Pacifici, D. Wyczechowska, A. Wilk, K. Reiss, F. Peruzzi, Anti-tumoral effects of mir-3189-3p in glioblastoma, J. Biol. Chem. 290 (2015) 8067–8080. [11] Z. Wang, X. Ma, Q. Cai, X. Wang, B. Yu, Q. Cai, B. liu, Z. Zhu, C. Li, Mir-199a-3p promotes gastric cancer progression by targeting zhx1, FEBS Lett. 588 (2014) 4504–4512. [12] T. Rosenberg, M. Thomassen, S.S. Jensen, M.J. Larsen, K.P. Sorensen, S.K. Hermansen, T.A. Kruse, B.W. Kristensen, Acute hypoxia induces upregulation of microrna-210 expression in glioblastoma spheroids, CNS Oncol. 4 (2015) 25–35. [13] X. Yang, C. Zhang, T. Guo, Y. Feng, Q. Liu, Y. Chen, Q. Zhang, Reduced expression of microrna206 regulates cell proliferation via cyclind2 in gliomas, Mol. Med. Rep. 11 (2015) 3295–3300. [14] B.D. Clarke, J.A. Roby, A. Slonchak, A.A. Khromykh, Functional non-coding rnas derived from the flavivirus 30 untranslated region, Virus Res. 206 (2015) 53–61. [15] Y. Kim, J. Yeo, J.H. Lee, J. Cho, D. Seo, J.S. Kim, V.N. Kim, Deletion of human tarbp2 reveals cellular microrna targets and cell-cycle function of trbp, Cell Rep. 9 (2014) 1061–1074. [16] D.R. Morais, S.T. Reis, N. Viana, C.B. Piantino, C. Massoco, C. Moura, N. Dip, I.A. Silva, M. Srougi, K.R. Leite, The involvement of mir-100 in bladder urothelial carcinogenesis changing the expression levels of mrna and proteins of genes related to cell proliferation, survival, apoptosis and chromosomal stability, Cancer cell Int. 14 (2014) 119. [17] S.G. Liu, X.G. Qin, B.S. Zhao, B. Qi, W.J. Yao, T.Y. Wang, H.C. Li, X.N. Wu, Differential expression of mirnas in esophageal cancer tissue, Oncol. Lett. 5 (2013) 1639–1642. [18] M. Wada, M. Horinaka, S. Yasuda, M. Masuzawa, T. Sakai, N. Katoh, Pdk1 is a potential therapeutic target against angiosarcoma cells, J. Dermatol. Sci. 78 (2015) 44–50. [19] Z. Yang, Z. Wu, T. Liu, L. Han, C. Wang, B. Yang, F. Zheng, Upregulation of pdk1 associates with poor prognosis in esophageal squamous cell carcinoma with facilitating tumorigenicity in vitro, Med. Oncol. (Northwood Lond. Engl.) 31 (2014) 337.
Please cite this article in press as: B. Fang, et al., MiR-454 inhibited cell proliferation of human glioblastoma cells by suppressing PDK1 expression, Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.07.029
Biomedicine & Pharmacotherapy xxx (2015) xxx–xxx
Available online at
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Original article
MiR-454 inhibited cell proliferation of human glioblastoma cells by suppressing PDK1 expression Baojun Fanga,b , Jianxin Zhua,b , Yunhua Wangb , Fengyang Gengb , Gang Lia,* a b
Department of Neurosurgery, Qilu Hospital of Shandong University, Jinan, 250012 Shandong, PR China Department of Neurosurgery, Liaocheng People’s Hospital, Liaocheng 252000, Shandong, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 April 2015 Accepted 26 July 2015
It has been well documented that aberrant expression of microRNAs is associated with carcinogenesis of glioblastoma (GBM), however the underlying mechanisms are not clear. In this present study, we aimed to clarify the biological function of miR-454 in GBM. MiR-454 was identified to be significantly downregulated in GBM primary tumors and cell lines. Overexpression of miR-454 in GBM cells resulted in arresting cells at G0/G1 phase and thus inhibiting cell proliferation. Bioinformatic analysis predicted 3-phosphoinositide-dependent protein kinase-1 (PDK1) as a target of miR-454 which acted as a tumor promoter gene. Increased miR-454 significantly repressed PDK1 expression, and then regulating cell proliferation and cell cycle regulators, down-regulation of Cyclin D1 and p-pRb and p21 was up-regulated. Taken together, our study has revealed miR-454 as a tumor suppressor in GBM. ã 2015 Elsevier Masson SAS. All rights reserved.
Keywords: MiR-454 Glioblastoma PDK1 Cell proliferation Cell cycle
1. Introduction Glioblastoma (GBM) is the most common and aggressive Central nervous system malignancy in adults worldwide [1]. However, the causes of GBM carcinogenesis and progression are largely unknown. In recent years, numerous studies have revealed aberrant expression of microRNAs(miRNAs) played critical roles in cancer cell proliferation, metastasis, and response to cancer therapy [2–6]. MiRNAs are a novel class of small noncoding RNAs (19– 22 nucleotides) that play essential roles in multiple biological processes of cancer, such as cell proliferation, invasion, migration and cell cycle, by typically suppressing the translation of messenger RNAs (mRNAs) by binding to the 30 -untranslated regions (30 -UTR) of their target mRNAs [7–11]. However, the biological function of miR-454 in GBM remains largely unknown. In the current study, our results revealed that miR-454 was markedly downregulated in GBM primary tumors and cell lines. Using Bioinformatic analysis, MTT assays, colony formation and cell cycle assay, we found that miR-454 acted as a tumor suppressor in GBM, suppressing GBM cell proliferation was due to inhibit 3-phosphoinositide-dependent protein kinase-1
* Corresponding author at: Department of Neurosurgery, Qilu Hospital of Shandong University, 107 Wenhua West Road, Lixia District, Jinan, Shandong Province, PR China. E-mail address: [email protected] (G. Li).
(PDK1), and then regulating cell proliferation and cell cycle regulators, down-regulation of Cyclin D1 and p-pRb and p21 was up-regulated. 2. Materials and methods 2.1. Clinical specimens Eight paired human GBM tissues and their matched adjacent normal brain tissues (ANT) were obtained from GBM patients at Department of Neurosurgery, Qilu Hospital of Shandong University (Jinan, People’s Republic of China). The study was approved by the ethics committee of Qilu Hospital of Shandong University (Jinan, People’s Republic of China). All patients gave informed consent in written. Tissue samples were collected at surgery, immediately frozen in liquid nitrogen and stored until total RNAs or proteins were extracted. 2.2. Cell culture Human GBM cell lines U87, T98G, A-172, LN18 and LN-229 were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gbico, USA) supplemented with 10% fetal bovine serum (FBS, Sigma, USA), 100 units/ml of penicillin-streptomycin (Invitrogen, Carlsbad, CA), and NHAs were purchased from Lonza and cultured in the provided astrocyte growth media supplemented with rhEGF, insulin, ascorbic acid, GA-1000, L-glutamine and, 5%
http://dx.doi.org/10.1016/j.biopha.2015.07.029 0753-3322/ ã 2015 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: B. Fang, et al., MiR-454 inhibited cell proliferation of human glioblastoma cells by suppressing PDK1 expression, Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.07.029
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B. Fang et al. / Biomedicine & Pharmacotherapy xxx (2015) xxx–xxx
FBS. Cells were cultured in a humidified incubator in an atmosphere of 5% CO2 at 37 C.
luciferase activity using the Dual-Luciferase Reporter Assay system (Promega).
2.3. Plasmids and transfection
2.8. Western blotting
The PDK1 30 -UTR fragment containing putative binding sites for miR-454 and the mutated binding site of the PDK1 30 -UTR (as a negative control) were amplified using PCR and subcloned into the pGL3-luciferase reporter plasmid (Promega). The miR-454 mimics, miR-454 inhibitor and relative negative control were purchased from Shanghai GenePharma (Shanghai, China) and transfected into GBM cells using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer’s instructions.
Cells were lysed with RIPA lysis buffer on ice for 20 min, proteins (40 mg) were subjected to 10%SDS-PAGE gel, transferred onto PVDF membranes by electroblotting. After probed with 1:1000 diluted anti-PDK1, anti-CyclinD1, anti-P21, anti-p-pRb and anti-pRb (Abcam, MA, USA) at 4 C overnight, the blots were washed with TBST and incubation with HRP (horseradish peroxidase)-conjugated secondary antibody (1:5000) for 2 h at room temperature. b-actin (Abcam, MA, USA) served as the loading control. Immunocomplexes were visualized using the ECL detection reagent (Beyotime, China) according to the manufacturer’s protocol.
2.4. RNA extraction and real-time quantitative PCR TRIzol Reagent (Invitrogen, Carlsbad, CA) was used to extract total RNA from the GBM cell lines and the clinical tissue samples according to the manufacturer’s instructions. The expression levels of miR-454 were quantified using miRNA-specific TaqMan miRNA assay kit (Applied Biosystems) according to the manufacturer’s instructions. The relative miR-454 expression levels after normalization to U6 small nuclear RNA were calculated using 2[(CtofmiR454)(CtofU6)].
All data were expressed as mean standard deviation (SD) and performed using the SPSS 18.0 (SPSS, Chicago, IL). The Student’s t-test was used to evaluate the statistical significance of differences between two groups of data in all pertinent experiments. A P-value less than 0.05 was considered to be statistically significant.
2.5. MTT assays and Colony formation
3. Result
To determine the effect of miR-454 on cell proliferation, MTT assay was used according to the Manufacturer’s instructions. For colony formation assay, miR-454 or miR-454-in or the relative miR-NC infected cells were placed in a fresh six well plate and incubated for 14 days in medium containing 10% FBS. Cells were fixed with methanol and stained with 0.1% crystal violet. Visible colonies were manually counted.
3.1. MiR-454 was downexpressed in human GBM tissues and GBM cell lines
2.6. Cell cycle assays by flow cytometry Pro-pidium iodide (PI) was attributable to cell cycle. The distribution of cells was analyzed by cell flow cytometry.
2.9. Statistical analysis
To test the expression of miR-454 in GBM, we examined miR454 expression in 8 GBM cell lines. The results showed that decreased expression of miR-454 was found in 9 GBM cell lines compared with the normal human astrocytes (NHAs) (Fig. 1A). In addition, we examined miR-454 expression in 8 GBM tissues and matched adjacent normal tissue using quantitative RT-PCR analysis. MiR-454 was differentially downregulated at the mRNA levels in GBM tissues compared with paired adjacent noncancerous tissues. Together, these results indicate that miR454 is downregulated in GBM primary tumors and cell lines.
2.7. Luciferase assays 3.2. MiR-454 inhibited GBM cell proliferation Cells (5 104) in each groups were seeded in 6-well plates and transfected with plasmid when cells reached 70% confluence, luciferase reporters were performed according to manufacturer’s instruction of lipofectamine 2000 (Invitrogen, USA). Cells were harvested 48 h after transfection, being analyzed for
To investigate the biological significance of miR-454 in GBM, we transfected the GBM cell lines, LN18 and T98G cells, with miR-454 mimics, miR-454 inhibitor or the respective controls. Real-time PCR was performed to detect the transfection efficiency
Fig. 1. Downregulation of miR-454 in human GBM tissues and cell lines. (A) Real-time PCR analysis of miR-454 expression in NHA and GBM cell lines, including U87, T98G, A-172, LN18 and LN-229. (B) Eight paired human GBM tissues and their matched adjacent normal brain tissues (ANT) were obtained from GBM patient, by PCR analysis. Experiments were repeated at least three times (A and B). Each bar represents the mean of three independent experiments. *P < 0.05.
Please cite this article in press as: B. Fang, et al., MiR-454 inhibited cell proliferation of human glioblastoma cells by suppressing PDK1 expression, Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.07.029
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Fig. 2. MiR-454 upregulation inhibited GBM cell proliferation. (A) Validation of miR-454 expression levels after transfection by PCR analysis. (B)MTT assays revealed that upregulation of miR-454 inhibited growth of GBM cell line LN18 and T98G. (C) Quantification of crystal violet-stained cell colonies. (D) Flow cytometric analysis of the indicated GBM cell line LN18 and T98G cells transfected with NC or miR-454. Each bar represents the mean of three independent experiments. *P < 0.05.
(Figs. 2 and 3A). To further investigate the functional role of miR454 in cell growth of GBM, MTT and colony formation assays were used. MTT and colony formation assays showed that miR454 significantly decreased cell growth and the ability of colony formation in LN18 and T98G cells (Fig. 2B and C). In contrary, miR454-in was used to investigate the role of miR-454 depletion in GBM cell lines. MiR-454-in increased the ability of cell proliferation and colony formation in LN18 and T98G cells (Fig. 3B and C). To further determine if cell growth rate of GBM cells was due to perturbation of cell cycles progression, cell cycle assays were used. Results of cell cycle assays revealed that overexpression of miR454 resulted in the arrest of cell cycle in G0/G1 phase and prevention of cells from entering into the S phase(Fig. 2D). On the contrary, miR-454-in caused more cells entering to S phase and thus promoted cell growth (Fig. 3D). Taken together, our results suggested that overexpression of miR-454 in GBM cells resulted in arresting cells at G0/G1 phase and thus inhibiting tumor cell growth.
To further investigate the direct effect of miR-454 on PDK1 expression, luciferase report assay was used. LN18 and T98G cells were cotransfected with miR-454 and PDK1 30 -UTR wild-type or mut-type. As shown in Fig. 4C, in miR-454 overexpressed cells, the expression of luciferase was dramatically inhibited. Furthermore, miR-454 had no effect on the luciferase activity of mut-type PDK1 30 -UTR. To evaluate whether PDK1 is regulated by miR-454, expressions of PDK1 were detected in the GBM cell lines LN18 and T98G cells, which were transfected with miR-454 mimics, miR-454-in or the respective controls. Expression of PDK1 was decreased in miR-454overexpression both GBM cell lines, LN18 and T98G cells, while miR-454 showed the opposite effect. Consistently in these cells, the downstream effectors of PDK1, Cyclin D1 and p-pRb were down-regulated, while p21 was up-regulated by miR-454 (Fig. 4b). All these findings suggested that miR-454 probably regulates cell growth through PDK1. 4. Discussion
3.3. MiR-454 directly targets PDK1 by binding to its 30 -UTR To further detect how miR-454 inhibits growth of GBM cells, we used a bioinformatic analysis (TargetScan Huaman 6.2) to find PDK1 acting as the target gene of miR-454. The wild-type or mut-type PDK1 30 -UTR were cloned into cloned into the 3’end of luciferase gene (Fig. 4A).
In the current study, we found that miR-454 is markedly downregulated in GBM primary tumors and cell lines. Furthermore, ectopic expression of miR-454 arrested GBM cells at G0/G1 phase and thus decreased cell proliferation, whereas miR-454-in had the opposite effect. More specifically, we showed that the molecular mechanism by which miR-454 inhibited GBM cell
Please cite this article in press as: B. Fang, et al., MiR-454 inhibited cell proliferation of human glioblastoma cells by suppressing PDK1 expression, Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.07.029
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Fig. 3. Inhibition of miR-454 promoted GBM cell proliferation. (A) Validation of miR-454 expression levels after transfection by PCR analysis. (B)MTT assays revealed that inhibition of miR-454 promoted growth of GBM cell line LN18 and T98G. (C) Quantification (right) of crystal violet-stained cell colonies. (D) Flow cytometric analysis of the indicated GBM cell line LN18 and T98G cells transfected with NC or miR-454-in. Each bar represents the mean of three independent experiments. *P < 0.05.
Fig. 4. MiR-454 suppresses PDK1 expression by directly targeting the PDK1 30 -UTR and altered levels of proteins related to cell proliferation and cell cycle in GBM cell line LN18 and T98G. (A) Predicted miR-454 target sequence in the 30 -UTR of PDK1 (PDK1-30 -UTR) and positions of three mutated nucleotides (red) in the 30 -UTR of PDK1 (PDK1-30 -UTR mut). (B) Western blotting analysis of protein expression of PDK1, cyclin D1, p21, p-pRb and pRb in indicated LN18 and T98G cells. b-actin served as the loading control. (C) Luciferase reporter assay of the indicated LN18 and T98G cells transfected with the pGL3-PDK1-30 -UTR reporter and miR-454 or miR-454 inhibitor oligonucleotides. *P < 0.05.
Please cite this article in press as: B. Fang, et al., MiR-454 inhibited cell proliferation of human glioblastoma cells by suppressing PDK1 expression, Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.07.029
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proliferation was due to suppress PDK1, and then regulating cell proliferation regulators, down-regulation of Cyclin D1 and p-pRb and p21 was up-regulated. Taken together, we propose that miR454 is a candidate tumor suppressor miRNA in GBM. Lots of reports have shown that that miRNAs are 19-25nucleotides regulatory non-protein coding RNA molecules that play essential roles in multiple biological processes of cancer [9,12,13]. MiRNAs usually function as endogenous repressors of target genes resulting in mRNA degradation [14–16]. Findings by SG Liu et al. indicated that expression of miR-454 was downregulated in esophageal cancer [17]. However, the molecular mechanism of miR-454-involved GBM progression has not been clarified. Our data expanded the role of miR-454 in GBM. In this study, qRT-PCR analysis indicated that miR-454 is significantly downregulated in GBM primary tumors and cell lines. Furthermore, the effect of miR-454 mimics and antisense transfection in LN18 and T98G cells showed that miR-454 decreased cell growth, colony formation via arresting GBM cells at G0/G1 phase. Bioinformatic algorithms predicted that PDK1, which was bona fide target genes of miR-454. Result of luciferase experiments showed that miR-454 could bind to the PDK1 30 UTR but not the mutant PDK1 30 UTR. Previous research showed that PDK1 played a pivotal role in the cell growth of angiosarcoma and inhibition of PDK1 may be a more appropriate strategy for the treatment of angiosarcoma [18]. Yang Z et al. reported that increased PDK1 expression associates with poor prognosis in esophageal squamous cell carcinoma with facilitating tumorigenicity in vitro [19]. Western blotting showed that downstream effectors of PDK1, Cyclin D1 and p-pRb were down-regulated, while p21 was upregulated in miR-454 transfected GBM cells. Taken together, this study clearly illustrated that miR-454 inhibited GBM cell proliferation was due to arresting GBM cells at G0/G1 phase, upregulation of p21 and downregulation of Cyclin D1 and p-pRb. In conclusion, the current study revealed that an essential link between miR-454-mediated tumor growth of GBM. Therefore, our results indicated that ectopic expression of miR-454 led to downregulation of Cyclin D1 and p-pRb and upregulation of p21, and thus to promote cell cycle and cell proliferation in GBM. Hence, miR-454 could be considered as a novel therapeutic target for GBM patients. Conflict of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments This work was supported by Department of Neurosurgery, Qilu Hospital of Shandong University. All authors designed the study together, performed the experiment together; all authors analyzed
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Please cite this article in press as: B. Fang, et al., MiR-454 inhibited cell proliferation of human glioblastoma cells by suppressing PDK1 expression, Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.07.029