- Email: [email protected]
miR-1254 promotes lung cancer cell proliferation by targeting SFRP1
Biomedicine & Pharmacotherapy 92 (2017) 913–918
Available online at
ScienceDirect www.sciencedirect.com
miR-1254 promotes lung cancer cell proliferation by targeting SFRP1 Hong Li, Tian Yang, Dong Shang, Zhongmin Sun* Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, Shanxi, China
A R T I C L E I N F O
Article history: Received 12 March 2017 Received in revised form 19 May 2017 Accepted 24 May 2017 Keywords: miR-1254 SFRP1 Lung cancer Cell proliferation
A B S T R A C T
Lung cancer is the leading cause of cancer deaths worldwide, many miRNAs play critical role in lung cancer initiation and progression. Here, we demonstrated that miR-1254 was upregulated in lung cancer tissues and cells. miR-1254 overexpression promoted lung cancer cell proliferation determined by MTT assay, colony formation assay, soft agar growth ability assay and BrdU incorporation assay, miR-1254 knockdown suppressed lung cancer cell proliferation. Mechanism analysis revealed that Wnt/b-catenin pathway antagonist secreted frizzled related protein 1 (SFRP1) was its target, its expression was opposite to SFRP1 level, and directly bound to the 30 UTR of SFRP1. Double knockdown of miR-1254 and SFRP1 promoted lung cancer cell proliferation, suggesting miR-1254 promoted lung cancer cell proliferation by targeting SFRP1. © 2017 Published by Elsevier Masson SAS.
1. Introduction miRNA represents a new layer of post-transcriptional regulation, and plays a critical role in gene expression regulation, it binds to the 30 UTR of target mRNA by base pairing, and mediates the mRNA degradation, deadenylation or translational suppression. miRNA genes are transcribed into primary miRNA transcripts (primiRNA) by RNA polymerase II or III in nucleus, pri-miRNA can be edited by adenosine deaminases acting on RNA (ADARs). Then Drosha-DGCR8 microprocessor complex breaks pri-miRNA to form pre-miRNA. Pre-miRNA was transported to the cytoplasm by Exportin5 in a Ran-GTP-dependent manner. Finally, pre-miRNA interacts with RNase Dicer and TRBP to cleave pre-miRNA hairpin into its mature length. Mature miRNA is loaded with Argonaute (Ago2) to form RNA-induced silencing complex (RISC) and inhibits gene expression, the detailed biogenesis of miRNA is still to be discovered [1–5]. But the functions of miRNA have been extensively and deeply studied, especially in tumor biology. Many miRNAs regulate lung cancer initiation and progression, for example, epigenetic regulatory proteins EZH2 and JMJD1A promote lung cancer proliferation and tumorigenesis, let-7c directly binds to the 30 UTR of JMJD1A and EZH2, and inhibits their expression to suppress lung cancer proliferation and
* Corresponding author at: Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Xi’an Jiaotong University, 277 Yanta Road, Xi’an 710061, Shanxi, China. E-mail address: [email protected] (Z. Sun). http://dx.doi.org/10.1016/j.biopha.2017.05.116 0753-3322/© 2017 Published by Elsevier Masson SAS.
tumorigenesis [6]. miR-432 is a prognostic factor for lung cancer, it inhibits lung cancer cell proliferation and increases sensitivity to cisplatin by targeting E2F3 and AXL [7]. miR-1254 suppresses colorectal cancer migration by inhibiting PSMD10 [8], miR-1254 inhibits oral squamous cell carcinoma metastasis, tumor inhibition and resistance to chemotherapy by repressing MAP3K3 [9]. miR1254 level is significantly upregulated in the serum of lung cancer patients, and can function as a biomarker for early lung cancer diagnose [10,11], but the miR-1254’s role in the proliferation of lung cancer cell hasn’t been studied. Here, we studied the effect of miR-1254 on lung cancer cell proliferation, and found miR-1254 promoted lung cancer cell proliferation through directly inhibiting SFRP1. 2. Materials and methods 2.1. Patients specimens and cell culture Paired of lung cancer tissues and matched adjacent normal lung tissues were obtained from the First Affiliated Hospital of Xi’an Jiaotong University. For using of these clinical materials for research purpose, Informed consents were obtained from every patient, and the study was approved by the Institute Research Ethics Committee of institute. Lung cancer cells NCI-H1975, NCI-H460, 95D, NCI-H1650, A549, NCI-H358 and NCI-H1299 and immortalized normal lung epithelial cell BEAS–2 B were cultured using RPMI-1640 (Hyclone) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin. These cells were obtained from ATCC.
914
H. Li et al. / Biomedicine & Pharmacotherapy 92 (2017) 913–918
2.2. Transfection
2.4. Western blot
The sequence of miR-1254 including stem loop structure and 200 300 bp of up-stream and down-stream flanking regions were amplification by PCR for genomic DNA of BEAS-2B, and cloned into pMSCV-Pur vector, empty vector was used as the negative control. the vectors were transfected into lung cancer cells A549 and 95D using FuGENE HD (Promega) according to the instructions. Plasmids harboring mutations in the miR-1254 seed sites were prepared by the site-directed mutagenesis (QuickChang, Stratagene). miR-1254 antagomirs and negative control were synthesized by Guangzhou Ribobio, and transfected into lung cancer cells using Lipofectamine 2000 reagent (Invitrogen).
Cells were lysed using RIPA buffer (Beyotime Biotechnology) supplemented with protease inhibitors (Roche). Equal amounts of protein extracts were separated on 12% SDS-PAGE, transferred to PVDF membranes (Millipore) and incubated with primary antibodies in TBST containing 5% non-fat milk powder. Primary antibodies were detected by a peroxidase-coupled secondary antibody, ECL was used to determine the bands. The following primary antibodies were used: anti-SFRP1 (#4690), anti-Cyclin D1 (#2922), anti-c-Myc (#9402) and anti-a-Tubulin (#2144), these antibodies were purchased from Cell signaling Technology. 2.5. Cell proliferation assay
2.3. Quantitative real time-PCR Total RNA was isolated using RNAiso reagent (TaKaRa), to examine gene expression, cDNA was synthesized using PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa). Quantitative real time-PCR was performed using SYBR Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa) on a CFX-96 system (BioRad). To determine miR-1254 expression, specific stem-loop primers and TaqMan MicroRNA Assay (Applied Biosystems) was used. GAPDH and U6 were used to normalize mRNA and miR-1254 expression, the relative expression levels were calculated using the 2DDCt method.
MTT assay, colony formation assay, Soft agar growth ability assay and BrdU incorporation assay were used to analyze the effect of miR-1254 on lung cancer cell proliferation, and performed according the standard methods previously reported [12–14]. 2.6. Luciferase reporter assay 30 UTR reporter constructs were generated through PCR amplification of 30 UTR from genomic DNA, the fragments were subcloned into psiCHECK2 vector (Promega, named as Target30 UTR). Target-30 UTR were cotransfected with pMSCV vector with miR-1254 or mut-miR-1254 overexpression or miR-1254
Fig 1. miR-1254 is regulated in lung cancer tissues and cells. A. miR-1254 was significantly upregulated in lung cancer tissues (T) compared to the normal lung cancer tissues (N), data were downloaded from TCGA dataset. B. miR-1254 was significantly upregulated in lung cancer tissues compared to the adjacent normal lung tissues. C. miR-1254 was upregulated in lung cancer cells. D. miR-1254 was upregulated in lung cancer tissues (T) compared to tumor adjacent normal lung tissues. Error bars indicate SEM. **p < 0.01.
H. Li et al. / Biomedicine & Pharmacotherapy 92 (2017) 913–918
antagomirs using Lipofectamine 2000 reagent (Invitrogen). 48 h after transfection, luciferase activities were determined using the Dual-Luciferase Assay System (Promega) following manufacturer’s instruction. 2.7. Statistical analysis Results are presented as mean SD of at least three independent experiments. Student’s t test was used for comparisons. p values < 0.05 were considered to reflect statistical significance. 3. Results 3.1. miR-1254 was upregulated in lung cancer tissues and cells We used TCGA data to identify miRNA differentially expressed in normal lung tissues and lung cancer tissues, and found miR1254 was significantly upregulated lung cancer tissues (Fig. 1A). Meanwhile, miR-1254 was significantly upregulated in lung cancer tissues compared to matched adjacent normal lung tissues (Fig. 1B). We also found that miR-1254 was upregulated in lung cancer cells compared to normal lung cells (Fig. 1C). Eight pairs of
915
lung cancer tissues and tumor adjacent lung tissues were used to demonstrate above finding, and found miR-1254 was upregulated in lung cancer tissues (Fig. 1D). Together, miR-1254 was upregulated in lung cancer tissues and cells.
3.2. miR-1254 promoted lung cancer cell proliferation miR-1254 was overexpressed in lung cancer cells A549 and 95D to analyze its role in cell proliferation. miR-1254 was significantly upregulated in lung cancer cells after transfection miR-1254overpressing vector (Fig. 2A). We used MTT assay, colony formation assay, soft agar growth ability assay and BrdU incorporation assay to examine the effect of miR-1254 on lung cancer cell proliferation. MTT assay and colony formation assay suggested miR-1254 overexpression significantly increased lung cancer cell proliferation rate (Fig. 2B and C). Soft agar growth ability assay suggested miR-1254 overexpression significantly promoted the anchorageindependent cell growth (Fig. 2D). BrdU incorporation assay suggested miR-1254 overexpression significantly increased the BrdU positive cell number (Fig. 2E), demonstrating miR-1254 overexpression promoted cell proliferation of lung cancer.
Fig. 2. miR-1254 overexpression promotes lung cancer cells proliferation. A. miR-1254 was upregulated when miR-1254 overexpression vectors were transfected into lung cancer cells. B. MTT assay suggested miR-1254 overexpression promoted lung cancer cell proliferation. C. Colony formation assay suggested miR-1254 overexpression promoted lung cancer cells proliferation. D. Soft agar growth ability assay suggested miR-1254 overexpression promoted cell anchorage-independent growth. E. BrdU incorporation assay suggested miR-1254 overexpression promoted lung cancer cells proliferation. Error bars indicate SEM. *p < 0.05.
916
H. Li et al. / Biomedicine & Pharmacotherapy 92 (2017) 913–918
Fig. 3. miR-1254 knockdown suppresses lung cancer cells proliferation. A. miR-1254 was downregulated when miR-1254 antagomirs were transfected into lung cancer cells. B. MTT assay suggested miR-1254 knockdown suppressed lung cancer cell proliferation. C. Colony formation assay suggested miR-1254 knockdown suppressed lung cancer cells proliferation. D. Soft agar growth ability assay suggested miR-1254 knockdown suppressed cell anchorage-independent growth. E. BrdU incorporation assay suggested miR-1254 knockdown suppressed lung cancer cells proliferation. Error bars indicate SEM. *p < 0.05.
We downregulated miR-1254 in the same lung cancer cells to identify above results by transfection miR-1254 inhibitor (Fig. 3A). MTT assay and colony formation assay suggested miR-1254 knockdown significantly reduced lung cancer cell proliferation rate (Fig. 3B and C). Soft agar growth ability assay suggested miR1254 knockdown significantly inhibited anchorage-independent growth (Fig. 3D). BrdU incorporation assay suggested miR-1254 knockdown significantly reduced BrdU positive cell number (Fig. 3E). These results revealed miR-1254 knockdown inhibited cell proliferation. 3.3. miR-1254 promoted lung cancer cell proliferation by targeting SFRP1 To analyze the regulatory mechanism of miR-1254, we looked for the targets of miR-1254 through the online algorithms (TargetScan, Pictar and miRanda), and found Wnt/b-catenin pathway inhibitor SFRP1 was its target [15,16] (Fig. 4A). Western blot assay suggested miR-1254 inhibited SFRP1 expression
(Fig. 4B), suggesting miR-1253 might bound to the 30 UTR of SFRP1. Luciferase reporter assay suggested that miR-1254 overexpression inhibited luciferase activity, miR-1254 knockdown increased luciferase activity, but mutational miR-1254 overexpression hadn’t effect on luciferase activity (Fig. 4C). Wnt pathway plays central role in many biological process, and regulates many genes, such as Cyclin D1 and c-Myc. These two genes regulate cell proliferation, so we analyzed the regulation of miR-1254 on c-Myc and Cyclin D1. Cyclin D1 and c-Myc were significantly reduced after miR-1254 knockdown, they were increased after miR-1254 overexpression determined by qRT-PCR and western blot (Fig. 4D and E), suggesting miR-1254 might activate Wnt/b-catenin pathway to promote lung cancer cell proliferation. To examine whether miR-1254 promoted lung cancer proliferation by targeting SFRP1, miR-1254 inhibitor and SFPR1 small interference RNA were cotranfected into lung cancer cells, western blot assay suggested SFRP1 small interference RNA inhibited SFRP1 expression effectively in protein level (Fig. 5A). Functional analysis suggested double knockdown of miR-1254 and SFRP1 significantly
H. Li et al. / Biomedicine & Pharmacotherapy 92 (2017) 913–918
917
Fig. 4. SFRP1 is the target of miR-1254. A. Alignment between miR-1254 and its binding sequence on 30 UTR of SFRP1. B. Western blot suggested miR-1254 inhibited SFRP1 expression. C. Luciferase reporter assay suggested miR-1254 directly bound to the 30 UTR of SFRP1. D and E. Quantitative real time-PCR and western blot assay suggested miR1254 promoted CylinD1 and C-MYC expression. Error bars indicate SEM. *p < 0.05.
promoted lung cancer cell proliferation determined by colony formation assay, soft agar growth assay and BrdU incorporation assay (Fig. 5B–D). This phenotype was as the same as the miR-1254 overexpression, and suggested miR-1254 promoted lung cancer cell proliferation by inhibiting SFRP1.
4. Discussion In present study, we analyzed the role of miR-1254 in lung cancer cell proliferation, and found miR-1254 was upregulated in lung cancer tissues and cells, overexpression of miR-1254
Fig. 5. miR-1254 promotes lung cancer cells proliferation through inhibiting SFRP1. A. Western blot assay suggested SFRP1 siRNA inhibited SFRP1 expression in lung cancer cells with miR-1254 knockdown. B. Knockdown of miR-1254 and SFRP1 promoted cell proliferation determined by colony formation assay. C. Knockdown of miR-1254 and SFRP1 promoted cell proliferation determined by cell anchorage-independent growth determined by soft agar growth ability assay. D. Knockdown of miR-1254 and SFRP1 promoted cell proliferation determined BrdU incorporation assay. Error bars indicate SEM. *p < 0.05.
918
H. Li et al. / Biomedicine & Pharmacotherapy 92 (2017) 913–918
promoted cell proliferation, knockdown of miR-1254 inhibited cell proliferation. miRNA could regulate gene expression through basebased pairs, SFRP1 was the target of miR-1254, miR-1254 bound to the 30 UTR of SFRP1, knockdown of miR-1254 and SFRP1 reversed the effects of miR-1254 knockdown, suggesting miR-1254 promoted lung cancer cells proliferation by targeting SFRP1. SFRP1 is a secreted antagonist of Wnt/b-catenin pathway, it binds to Wnt/b-catenin family proteins and blocks the interaction with frizzled proteins, preventing activation of Wnt/b-catenin pathway [17,18]. Previous reports suggest SFRP1 inhibits lung cancer progression, for example, it inhibits epithelial-mesenchymal transition in A549 cell [19], SFRP1’s promoter is hypermethylation in non-small cell lung cancer (NSCLC) samples, it may be a potential biomarker for NSCLC. DNA methyltransferase inhibitor 5-aza-20 -deoxycytidine could enhance SFRP1 expression [20–24], suggesting SFRP1 was a potential target for NSCLC therapy, miR-1254 could suppress SFRP1 expression, miR-154 antagomirs combine with 5-aza-20 -deoxycytidine might effectively control NSCLC progression. In summary, we found miR-1254 promoted lung cancer cell proliferation by targeting Wnt/b-catenin pathway antagonist SFRP1, and might be a target for lung cancer therapy. Competing financial interests The authors declare no conflict interests. Ethical approval Informed consent was obtained from every patient, and the study was approved by the Institute Research Ethics Committee of institute. Role of the funding source None. References [1] B. Czech, G.J. Hannon, Small RNA sorting: matchmaking for Argonautes, Nat. Rev. Genet. 12 (1) (2011) 19–31. [2] W. Filipowicz, S.N. Bhattacharyya, N. Sonenberg, Mechanisms of posttranscriptional regulation by microRNAs: are the answers in sight? Nat. Rev. Genet. 9 (2) (2008) 102–114. [3] J. Winter, S. Jung, S. Keller, R.I. Gregory, S. Diederichs, Many roads to maturity: microRNA biogenesis pathways and their regulation, Nat. Cell Biol. 11 (3) (2009) 228–234. [4] V.N. Kim, MicroRNA biogenesis: coordinated cropping and dicing, Nat. Rev. Mol. Cell Biol. 6 (5) (2005) 376–385. [5] D.P. Bartel, MicroRNAs: genomics, biogenesis, mechanism, and function, Cell 116 (2) (2004) 281–297.
[6] M. Zhan, F. Wen, L. Liu, Z. Chen, H. Wei, H. Zhou, JMJD1A promotes tumorigenesis and forms a feedback loop with EZH2/let-7c in NSCLC cells, Tumour Biol.: J. Int. Soc. Oncodev. Biol. Med. 37 (8) (2016) 11237–11247. [7] L. Chen, G. Kong, C. Zhang, H. Dong, C. Yang, G. Song, C. Guo, L. Wang, H. Yu, MicroRNA-432 functions as a tumor suppressor gene through targeting E2F3 and AXL in lung adenocarcinoma, Oncotarget 7 (15) (2016) 20041–20053. [8] Y.M. Chu, H.X. Peng, Y. Xu, D.M. Yang, F.L. Zhou, J. Li, R. Kuai, Y. Lin, MicroRNA1254 inhibits the migration of colon adenocarcinoma cells by targeting PSMD10, J. Dig. Dis. 18 (3) (2017) 169–178. [9] M. Lu, W.H. Chen, C.Y. Wang, C.Q. Mao, J. Wang, Reciprocal regulation of miR1254 and c-Myc in oral squamous cell carcinoma suppresses EMT-mediated metastasis and tumor-initiating properties through MAPK signaling, Biochem. Biophys. Res. Commun. 484 (4) (2017) 801–807. [10] K.M. Foss, C. Sima, D. Ugolini, M. Neri, K.E. Allen, G.J. Weiss, miR-1254 and miR574-5p: serum-based microRNA biomarkers for early-stage non-small cell lung cancer, J. Thoracic Oncol.: Off. Publ. Int. Assoc. Study Lung Cancer 6 (3) (2011) 482–488. [11] H. Peng, J. Wang, J. Li, M. Zhao, S.K. Huang, Y.Y. Gu, Y. Li, X.J. Sun, L. Yang, Q. Luo, C.Z. Huang, A circulating non-coding RNA panel as an early detection predictor of non-small cell lung cancer, Life Sci. 151 (2016) 235–242. [12] L. Zhao, Y. Zhang, miR-342-3p affects hepatocellular carcinoma cell proliferation via regulating NF-kappaB pathway, Biochem. Biophys. Res. Commun. 457 (3) (2015) 370–377. [13] L. Liu, J. Wang, X. Li, J. Ma, C. Shi, H. Zhu, Q. Xi, J. Zhang, X. Zhao, M. Gu, MiR-2045p suppresses cell proliferation by inhibiting IGFBP5 in papillary thyroid carcinoma, Biochem. Biophys. Res. Commun. 457 (4) (2015) 621–626. [14] M.S. Pang, X. Chen, B. Lu, J. Zhao, B.H. Li, Y.Q. Wei, Y.J. Guo, Lentiviral vectormediated doxycycline-inducible iASPP gene targeted RNA interference in hepatocellular carcinoma, Chin. J. Cancer 29 (9) (2010) 796–801. [15] L. Zheng, H. Jiang, Z.W. Zhang, K.N. Wang, Q.F. Wang, Q.L. Li, T. Jiang, Arsenic trioxide inhibits viability and induces apoptosis through reactivating the Wnt inhibitor secreted frizzled related protein-1 in prostate cancer cells, OncoTargets Therapy 9 (2016) 885–894. [16] Q. Xu, P.A. D'Amore, S.Y. Sokol, Functional and biochemical interactions of Wnts with FrzA, a secreted Wnt antagonist, Development 125 (23) (1998) 4767–4776. [17] J. Ezan, L. Leroux, L. Barandon, P. Dufourcq, B. Jaspard, C. Moreau, C. Allieres, D. Daret, T. Couffinhal, C. Duplaa, FrzA/sFRP-1, a secreted antagonist of the WntFrizzled pathway, controls vascular cell proliferation in vitro and in vivo, Cardiovasc. Res. 63 (4) (2004) 731–738. [18] S. Dennis, M. Aikawa, W. Szeto, P.A. d'Amore, J. Papkoff, A secreted frizzled related protein, FrzA, selectively associates with Wnt-1 protein and regulates wnt-1 signaling, J. Cell Sci. 112 (21) (1999) 3815–3820. [19] J. Ren, R. Wang, G. Huang, H. Song, Y. Chen, L. Chen, sFRP1 inhibits epithelialmesenchymal transition in A549 human lung adenocarcinoma cell line, Cancer Biother. Radiopharm. 28 (7) (2013) 565–571. [20] Y.H. Taguchi, M. Iwadate, H. Umeyama, SFRP1 is a possible candidate for epigenetic therapy in non-small cell lung cancer, BMC Med. Genomics 9 (Suppl 1) (2016) 28. [21] Y.W. Zhang, Y.F. Miao, J. Yi, J. Geng, R. Wang, L.B. Chen, Transcriptional inactivation of secreted frizzled-related protein 1 by promoter hypermethylation as a potential biomarker for non-small cell lung cancer, Neoplasma 57 (3) (2010) 228–233. [22] Y. Zhang, R. Wang, H. Song, G. Huang, J. Yi, Y. Zheng, J. Wang, L. Chen, Methylation of multiple genes as a candidate biomarker in non-small cell lung cancer, Cancer Lett. 303 (1) (2011) 21–28. [23] W.K. De Jong, G.F. Verpooten, H. Kramer, J. Louwagie, H.J. Groen, Promoter methylation primarily occurs in tumor cells of patients with non-small cell lung cancer, Anticancer Res. 29 (1) (2009) 363–369. [24] T. Fukui, M. Kondo, G. Ito, O. Maeda, N. Sato, H. Yoshioka, K. Yokoi, Y. Ueda, K. Shimokata, Y. Sekido, Transcriptional silencing of secreted frizzled related protein 1 (SFRP 1) by promoter hypermethylation in non-small-cell lung cancer, Oncogene 24 (41) (2005) 6323–6327.
Available online at
ScienceDirect www.sciencedirect.com
miR-1254 promotes lung cancer cell proliferation by targeting SFRP1 Hong Li, Tian Yang, Dong Shang, Zhongmin Sun* Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, Shanxi, China
A R T I C L E I N F O
Article history: Received 12 March 2017 Received in revised form 19 May 2017 Accepted 24 May 2017 Keywords: miR-1254 SFRP1 Lung cancer Cell proliferation
A B S T R A C T
Lung cancer is the leading cause of cancer deaths worldwide, many miRNAs play critical role in lung cancer initiation and progression. Here, we demonstrated that miR-1254 was upregulated in lung cancer tissues and cells. miR-1254 overexpression promoted lung cancer cell proliferation determined by MTT assay, colony formation assay, soft agar growth ability assay and BrdU incorporation assay, miR-1254 knockdown suppressed lung cancer cell proliferation. Mechanism analysis revealed that Wnt/b-catenin pathway antagonist secreted frizzled related protein 1 (SFRP1) was its target, its expression was opposite to SFRP1 level, and directly bound to the 30 UTR of SFRP1. Double knockdown of miR-1254 and SFRP1 promoted lung cancer cell proliferation, suggesting miR-1254 promoted lung cancer cell proliferation by targeting SFRP1. © 2017 Published by Elsevier Masson SAS.
1. Introduction miRNA represents a new layer of post-transcriptional regulation, and plays a critical role in gene expression regulation, it binds to the 30 UTR of target mRNA by base pairing, and mediates the mRNA degradation, deadenylation or translational suppression. miRNA genes are transcribed into primary miRNA transcripts (primiRNA) by RNA polymerase II or III in nucleus, pri-miRNA can be edited by adenosine deaminases acting on RNA (ADARs). Then Drosha-DGCR8 microprocessor complex breaks pri-miRNA to form pre-miRNA. Pre-miRNA was transported to the cytoplasm by Exportin5 in a Ran-GTP-dependent manner. Finally, pre-miRNA interacts with RNase Dicer and TRBP to cleave pre-miRNA hairpin into its mature length. Mature miRNA is loaded with Argonaute (Ago2) to form RNA-induced silencing complex (RISC) and inhibits gene expression, the detailed biogenesis of miRNA is still to be discovered [1–5]. But the functions of miRNA have been extensively and deeply studied, especially in tumor biology. Many miRNAs regulate lung cancer initiation and progression, for example, epigenetic regulatory proteins EZH2 and JMJD1A promote lung cancer proliferation and tumorigenesis, let-7c directly binds to the 30 UTR of JMJD1A and EZH2, and inhibits their expression to suppress lung cancer proliferation and
* Corresponding author at: Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Xi’an Jiaotong University, 277 Yanta Road, Xi’an 710061, Shanxi, China. E-mail address: [email protected] (Z. Sun). http://dx.doi.org/10.1016/j.biopha.2017.05.116 0753-3322/© 2017 Published by Elsevier Masson SAS.
tumorigenesis [6]. miR-432 is a prognostic factor for lung cancer, it inhibits lung cancer cell proliferation and increases sensitivity to cisplatin by targeting E2F3 and AXL [7]. miR-1254 suppresses colorectal cancer migration by inhibiting PSMD10 [8], miR-1254 inhibits oral squamous cell carcinoma metastasis, tumor inhibition and resistance to chemotherapy by repressing MAP3K3 [9]. miR1254 level is significantly upregulated in the serum of lung cancer patients, and can function as a biomarker for early lung cancer diagnose [10,11], but the miR-1254’s role in the proliferation of lung cancer cell hasn’t been studied. Here, we studied the effect of miR-1254 on lung cancer cell proliferation, and found miR-1254 promoted lung cancer cell proliferation through directly inhibiting SFRP1. 2. Materials and methods 2.1. Patients specimens and cell culture Paired of lung cancer tissues and matched adjacent normal lung tissues were obtained from the First Affiliated Hospital of Xi’an Jiaotong University. For using of these clinical materials for research purpose, Informed consents were obtained from every patient, and the study was approved by the Institute Research Ethics Committee of institute. Lung cancer cells NCI-H1975, NCI-H460, 95D, NCI-H1650, A549, NCI-H358 and NCI-H1299 and immortalized normal lung epithelial cell BEAS–2 B were cultured using RPMI-1640 (Hyclone) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin. These cells were obtained from ATCC.
914
H. Li et al. / Biomedicine & Pharmacotherapy 92 (2017) 913–918
2.2. Transfection
2.4. Western blot
The sequence of miR-1254 including stem loop structure and 200 300 bp of up-stream and down-stream flanking regions were amplification by PCR for genomic DNA of BEAS-2B, and cloned into pMSCV-Pur vector, empty vector was used as the negative control. the vectors were transfected into lung cancer cells A549 and 95D using FuGENE HD (Promega) according to the instructions. Plasmids harboring mutations in the miR-1254 seed sites were prepared by the site-directed mutagenesis (QuickChang, Stratagene). miR-1254 antagomirs and negative control were synthesized by Guangzhou Ribobio, and transfected into lung cancer cells using Lipofectamine 2000 reagent (Invitrogen).
Cells were lysed using RIPA buffer (Beyotime Biotechnology) supplemented with protease inhibitors (Roche). Equal amounts of protein extracts were separated on 12% SDS-PAGE, transferred to PVDF membranes (Millipore) and incubated with primary antibodies in TBST containing 5% non-fat milk powder. Primary antibodies were detected by a peroxidase-coupled secondary antibody, ECL was used to determine the bands. The following primary antibodies were used: anti-SFRP1 (#4690), anti-Cyclin D1 (#2922), anti-c-Myc (#9402) and anti-a-Tubulin (#2144), these antibodies were purchased from Cell signaling Technology. 2.5. Cell proliferation assay
2.3. Quantitative real time-PCR Total RNA was isolated using RNAiso reagent (TaKaRa), to examine gene expression, cDNA was synthesized using PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa). Quantitative real time-PCR was performed using SYBR Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa) on a CFX-96 system (BioRad). To determine miR-1254 expression, specific stem-loop primers and TaqMan MicroRNA Assay (Applied Biosystems) was used. GAPDH and U6 were used to normalize mRNA and miR-1254 expression, the relative expression levels were calculated using the 2DDCt method.
MTT assay, colony formation assay, Soft agar growth ability assay and BrdU incorporation assay were used to analyze the effect of miR-1254 on lung cancer cell proliferation, and performed according the standard methods previously reported [12–14]. 2.6. Luciferase reporter assay 30 UTR reporter constructs were generated through PCR amplification of 30 UTR from genomic DNA, the fragments were subcloned into psiCHECK2 vector (Promega, named as Target30 UTR). Target-30 UTR were cotransfected with pMSCV vector with miR-1254 or mut-miR-1254 overexpression or miR-1254
Fig 1. miR-1254 is regulated in lung cancer tissues and cells. A. miR-1254 was significantly upregulated in lung cancer tissues (T) compared to the normal lung cancer tissues (N), data were downloaded from TCGA dataset. B. miR-1254 was significantly upregulated in lung cancer tissues compared to the adjacent normal lung tissues. C. miR-1254 was upregulated in lung cancer cells. D. miR-1254 was upregulated in lung cancer tissues (T) compared to tumor adjacent normal lung tissues. Error bars indicate SEM. **p < 0.01.
H. Li et al. / Biomedicine & Pharmacotherapy 92 (2017) 913–918
antagomirs using Lipofectamine 2000 reagent (Invitrogen). 48 h after transfection, luciferase activities were determined using the Dual-Luciferase Assay System (Promega) following manufacturer’s instruction. 2.7. Statistical analysis Results are presented as mean SD of at least three independent experiments. Student’s t test was used for comparisons. p values < 0.05 were considered to reflect statistical significance. 3. Results 3.1. miR-1254 was upregulated in lung cancer tissues and cells We used TCGA data to identify miRNA differentially expressed in normal lung tissues and lung cancer tissues, and found miR1254 was significantly upregulated lung cancer tissues (Fig. 1A). Meanwhile, miR-1254 was significantly upregulated in lung cancer tissues compared to matched adjacent normal lung tissues (Fig. 1B). We also found that miR-1254 was upregulated in lung cancer cells compared to normal lung cells (Fig. 1C). Eight pairs of
915
lung cancer tissues and tumor adjacent lung tissues were used to demonstrate above finding, and found miR-1254 was upregulated in lung cancer tissues (Fig. 1D). Together, miR-1254 was upregulated in lung cancer tissues and cells.
3.2. miR-1254 promoted lung cancer cell proliferation miR-1254 was overexpressed in lung cancer cells A549 and 95D to analyze its role in cell proliferation. miR-1254 was significantly upregulated in lung cancer cells after transfection miR-1254overpressing vector (Fig. 2A). We used MTT assay, colony formation assay, soft agar growth ability assay and BrdU incorporation assay to examine the effect of miR-1254 on lung cancer cell proliferation. MTT assay and colony formation assay suggested miR-1254 overexpression significantly increased lung cancer cell proliferation rate (Fig. 2B and C). Soft agar growth ability assay suggested miR-1254 overexpression significantly promoted the anchorageindependent cell growth (Fig. 2D). BrdU incorporation assay suggested miR-1254 overexpression significantly increased the BrdU positive cell number (Fig. 2E), demonstrating miR-1254 overexpression promoted cell proliferation of lung cancer.
Fig. 2. miR-1254 overexpression promotes lung cancer cells proliferation. A. miR-1254 was upregulated when miR-1254 overexpression vectors were transfected into lung cancer cells. B. MTT assay suggested miR-1254 overexpression promoted lung cancer cell proliferation. C. Colony formation assay suggested miR-1254 overexpression promoted lung cancer cells proliferation. D. Soft agar growth ability assay suggested miR-1254 overexpression promoted cell anchorage-independent growth. E. BrdU incorporation assay suggested miR-1254 overexpression promoted lung cancer cells proliferation. Error bars indicate SEM. *p < 0.05.
916
H. Li et al. / Biomedicine & Pharmacotherapy 92 (2017) 913–918
Fig. 3. miR-1254 knockdown suppresses lung cancer cells proliferation. A. miR-1254 was downregulated when miR-1254 antagomirs were transfected into lung cancer cells. B. MTT assay suggested miR-1254 knockdown suppressed lung cancer cell proliferation. C. Colony formation assay suggested miR-1254 knockdown suppressed lung cancer cells proliferation. D. Soft agar growth ability assay suggested miR-1254 knockdown suppressed cell anchorage-independent growth. E. BrdU incorporation assay suggested miR-1254 knockdown suppressed lung cancer cells proliferation. Error bars indicate SEM. *p < 0.05.
We downregulated miR-1254 in the same lung cancer cells to identify above results by transfection miR-1254 inhibitor (Fig. 3A). MTT assay and colony formation assay suggested miR-1254 knockdown significantly reduced lung cancer cell proliferation rate (Fig. 3B and C). Soft agar growth ability assay suggested miR1254 knockdown significantly inhibited anchorage-independent growth (Fig. 3D). BrdU incorporation assay suggested miR-1254 knockdown significantly reduced BrdU positive cell number (Fig. 3E). These results revealed miR-1254 knockdown inhibited cell proliferation. 3.3. miR-1254 promoted lung cancer cell proliferation by targeting SFRP1 To analyze the regulatory mechanism of miR-1254, we looked for the targets of miR-1254 through the online algorithms (TargetScan, Pictar and miRanda), and found Wnt/b-catenin pathway inhibitor SFRP1 was its target [15,16] (Fig. 4A). Western blot assay suggested miR-1254 inhibited SFRP1 expression
(Fig. 4B), suggesting miR-1253 might bound to the 30 UTR of SFRP1. Luciferase reporter assay suggested that miR-1254 overexpression inhibited luciferase activity, miR-1254 knockdown increased luciferase activity, but mutational miR-1254 overexpression hadn’t effect on luciferase activity (Fig. 4C). Wnt pathway plays central role in many biological process, and regulates many genes, such as Cyclin D1 and c-Myc. These two genes regulate cell proliferation, so we analyzed the regulation of miR-1254 on c-Myc and Cyclin D1. Cyclin D1 and c-Myc were significantly reduced after miR-1254 knockdown, they were increased after miR-1254 overexpression determined by qRT-PCR and western blot (Fig. 4D and E), suggesting miR-1254 might activate Wnt/b-catenin pathway to promote lung cancer cell proliferation. To examine whether miR-1254 promoted lung cancer proliferation by targeting SFRP1, miR-1254 inhibitor and SFPR1 small interference RNA were cotranfected into lung cancer cells, western blot assay suggested SFRP1 small interference RNA inhibited SFRP1 expression effectively in protein level (Fig. 5A). Functional analysis suggested double knockdown of miR-1254 and SFRP1 significantly
H. Li et al. / Biomedicine & Pharmacotherapy 92 (2017) 913–918
917
Fig. 4. SFRP1 is the target of miR-1254. A. Alignment between miR-1254 and its binding sequence on 30 UTR of SFRP1. B. Western blot suggested miR-1254 inhibited SFRP1 expression. C. Luciferase reporter assay suggested miR-1254 directly bound to the 30 UTR of SFRP1. D and E. Quantitative real time-PCR and western blot assay suggested miR1254 promoted CylinD1 and C-MYC expression. Error bars indicate SEM. *p < 0.05.
promoted lung cancer cell proliferation determined by colony formation assay, soft agar growth assay and BrdU incorporation assay (Fig. 5B–D). This phenotype was as the same as the miR-1254 overexpression, and suggested miR-1254 promoted lung cancer cell proliferation by inhibiting SFRP1.
4. Discussion In present study, we analyzed the role of miR-1254 in lung cancer cell proliferation, and found miR-1254 was upregulated in lung cancer tissues and cells, overexpression of miR-1254
Fig. 5. miR-1254 promotes lung cancer cells proliferation through inhibiting SFRP1. A. Western blot assay suggested SFRP1 siRNA inhibited SFRP1 expression in lung cancer cells with miR-1254 knockdown. B. Knockdown of miR-1254 and SFRP1 promoted cell proliferation determined by colony formation assay. C. Knockdown of miR-1254 and SFRP1 promoted cell proliferation determined by cell anchorage-independent growth determined by soft agar growth ability assay. D. Knockdown of miR-1254 and SFRP1 promoted cell proliferation determined BrdU incorporation assay. Error bars indicate SEM. *p < 0.05.
918
H. Li et al. / Biomedicine & Pharmacotherapy 92 (2017) 913–918
promoted cell proliferation, knockdown of miR-1254 inhibited cell proliferation. miRNA could regulate gene expression through basebased pairs, SFRP1 was the target of miR-1254, miR-1254 bound to the 30 UTR of SFRP1, knockdown of miR-1254 and SFRP1 reversed the effects of miR-1254 knockdown, suggesting miR-1254 promoted lung cancer cells proliferation by targeting SFRP1. SFRP1 is a secreted antagonist of Wnt/b-catenin pathway, it binds to Wnt/b-catenin family proteins and blocks the interaction with frizzled proteins, preventing activation of Wnt/b-catenin pathway [17,18]. Previous reports suggest SFRP1 inhibits lung cancer progression, for example, it inhibits epithelial-mesenchymal transition in A549 cell [19], SFRP1’s promoter is hypermethylation in non-small cell lung cancer (NSCLC) samples, it may be a potential biomarker for NSCLC. DNA methyltransferase inhibitor 5-aza-20 -deoxycytidine could enhance SFRP1 expression [20–24], suggesting SFRP1 was a potential target for NSCLC therapy, miR-1254 could suppress SFRP1 expression, miR-154 antagomirs combine with 5-aza-20 -deoxycytidine might effectively control NSCLC progression. In summary, we found miR-1254 promoted lung cancer cell proliferation by targeting Wnt/b-catenin pathway antagonist SFRP1, and might be a target for lung cancer therapy. Competing financial interests The authors declare no conflict interests. Ethical approval Informed consent was obtained from every patient, and the study was approved by the Institute Research Ethics Committee of institute. Role of the funding source None. References [1] B. Czech, G.J. Hannon, Small RNA sorting: matchmaking for Argonautes, Nat. Rev. Genet. 12 (1) (2011) 19–31. [2] W. Filipowicz, S.N. Bhattacharyya, N. Sonenberg, Mechanisms of posttranscriptional regulation by microRNAs: are the answers in sight? Nat. Rev. Genet. 9 (2) (2008) 102–114. [3] J. Winter, S. Jung, S. Keller, R.I. Gregory, S. Diederichs, Many roads to maturity: microRNA biogenesis pathways and their regulation, Nat. Cell Biol. 11 (3) (2009) 228–234. [4] V.N. Kim, MicroRNA biogenesis: coordinated cropping and dicing, Nat. Rev. Mol. Cell Biol. 6 (5) (2005) 376–385. [5] D.P. Bartel, MicroRNAs: genomics, biogenesis, mechanism, and function, Cell 116 (2) (2004) 281–297.
[6] M. Zhan, F. Wen, L. Liu, Z. Chen, H. Wei, H. Zhou, JMJD1A promotes tumorigenesis and forms a feedback loop with EZH2/let-7c in NSCLC cells, Tumour Biol.: J. Int. Soc. Oncodev. Biol. Med. 37 (8) (2016) 11237–11247. [7] L. Chen, G. Kong, C. Zhang, H. Dong, C. Yang, G. Song, C. Guo, L. Wang, H. Yu, MicroRNA-432 functions as a tumor suppressor gene through targeting E2F3 and AXL in lung adenocarcinoma, Oncotarget 7 (15) (2016) 20041–20053. [8] Y.M. Chu, H.X. Peng, Y. Xu, D.M. Yang, F.L. Zhou, J. Li, R. Kuai, Y. Lin, MicroRNA1254 inhibits the migration of colon adenocarcinoma cells by targeting PSMD10, J. Dig. Dis. 18 (3) (2017) 169–178. [9] M. Lu, W.H. Chen, C.Y. Wang, C.Q. Mao, J. Wang, Reciprocal regulation of miR1254 and c-Myc in oral squamous cell carcinoma suppresses EMT-mediated metastasis and tumor-initiating properties through MAPK signaling, Biochem. Biophys. Res. Commun. 484 (4) (2017) 801–807. [10] K.M. Foss, C. Sima, D. Ugolini, M. Neri, K.E. Allen, G.J. Weiss, miR-1254 and miR574-5p: serum-based microRNA biomarkers for early-stage non-small cell lung cancer, J. Thoracic Oncol.: Off. Publ. Int. Assoc. Study Lung Cancer 6 (3) (2011) 482–488. [11] H. Peng, J. Wang, J. Li, M. Zhao, S.K. Huang, Y.Y. Gu, Y. Li, X.J. Sun, L. Yang, Q. Luo, C.Z. Huang, A circulating non-coding RNA panel as an early detection predictor of non-small cell lung cancer, Life Sci. 151 (2016) 235–242. [12] L. Zhao, Y. Zhang, miR-342-3p affects hepatocellular carcinoma cell proliferation via regulating NF-kappaB pathway, Biochem. Biophys. Res. Commun. 457 (3) (2015) 370–377. [13] L. Liu, J. Wang, X. Li, J. Ma, C. Shi, H. Zhu, Q. Xi, J. Zhang, X. Zhao, M. Gu, MiR-2045p suppresses cell proliferation by inhibiting IGFBP5 in papillary thyroid carcinoma, Biochem. Biophys. Res. Commun. 457 (4) (2015) 621–626. [14] M.S. Pang, X. Chen, B. Lu, J. Zhao, B.H. Li, Y.Q. Wei, Y.J. Guo, Lentiviral vectormediated doxycycline-inducible iASPP gene targeted RNA interference in hepatocellular carcinoma, Chin. J. Cancer 29 (9) (2010) 796–801. [15] L. Zheng, H. Jiang, Z.W. Zhang, K.N. Wang, Q.F. Wang, Q.L. Li, T. Jiang, Arsenic trioxide inhibits viability and induces apoptosis through reactivating the Wnt inhibitor secreted frizzled related protein-1 in prostate cancer cells, OncoTargets Therapy 9 (2016) 885–894. [16] Q. Xu, P.A. D'Amore, S.Y. Sokol, Functional and biochemical interactions of Wnts with FrzA, a secreted Wnt antagonist, Development 125 (23) (1998) 4767–4776. [17] J. Ezan, L. Leroux, L. Barandon, P. Dufourcq, B. Jaspard, C. Moreau, C. Allieres, D. Daret, T. Couffinhal, C. Duplaa, FrzA/sFRP-1, a secreted antagonist of the WntFrizzled pathway, controls vascular cell proliferation in vitro and in vivo, Cardiovasc. Res. 63 (4) (2004) 731–738. [18] S. Dennis, M. Aikawa, W. Szeto, P.A. d'Amore, J. Papkoff, A secreted frizzled related protein, FrzA, selectively associates with Wnt-1 protein and regulates wnt-1 signaling, J. Cell Sci. 112 (21) (1999) 3815–3820. [19] J. Ren, R. Wang, G. Huang, H. Song, Y. Chen, L. Chen, sFRP1 inhibits epithelialmesenchymal transition in A549 human lung adenocarcinoma cell line, Cancer Biother. Radiopharm. 28 (7) (2013) 565–571. [20] Y.H. Taguchi, M. Iwadate, H. Umeyama, SFRP1 is a possible candidate for epigenetic therapy in non-small cell lung cancer, BMC Med. Genomics 9 (Suppl 1) (2016) 28. [21] Y.W. Zhang, Y.F. Miao, J. Yi, J. Geng, R. Wang, L.B. Chen, Transcriptional inactivation of secreted frizzled-related protein 1 by promoter hypermethylation as a potential biomarker for non-small cell lung cancer, Neoplasma 57 (3) (2010) 228–233. [22] Y. Zhang, R. Wang, H. Song, G. Huang, J. Yi, Y. Zheng, J. Wang, L. Chen, Methylation of multiple genes as a candidate biomarker in non-small cell lung cancer, Cancer Lett. 303 (1) (2011) 21–28. [23] W.K. De Jong, G.F. Verpooten, H. Kramer, J. Louwagie, H.J. Groen, Promoter methylation primarily occurs in tumor cells of patients with non-small cell lung cancer, Anticancer Res. 29 (1) (2009) 363–369. [24] T. Fukui, M. Kondo, G. Ito, O. Maeda, N. Sato, H. Yoshioka, K. Yokoi, Y. Ueda, K. Shimokata, Y. Sekido, Transcriptional silencing of secreted frizzled related protein 1 (SFRP 1) by promoter hypermethylation in non-small-cell lung cancer, Oncogene 24 (41) (2005) 6323–6327.