Mol Cell Biochem (2014) 390:155–160 DOI 10.1007/s11010-014-1966-x
miR-96 downregulates RECK to promote growth and motility of non-small cell lung cancer cells Haizhou Guo • Qianping Li • Weihao Li • Tianliang Zheng • Song Zhao • Zhangsuo Liu
Received: 22 November 2013 / Accepted: 14 January 2014 / Published online: 29 January 2014 Ó Springer Science+Business Media New York 2014
Abstract MicroRNAs play critical roles in the development and progression of non-small cell lung cancer (NSCLC). miR-96 acts as an oncogene in some malignancies, while its role in NSCLC is unclear. Here, we validated that miR-96 was significantly increased in both human NSCLC tissues and cell lines. Inhibition of miR-96 expression remarkably reduced cell proliferation, colony formation, migration, and invasion of NSCLC cells. Reversion-inducing-cysteine-rich protein with kazal motifs (RECK) was identified as a target of miR-96 in NSCLC cells. In addition, the expression of RECK was found to be negatively correlated with the expression of miR-96 in NSCLC tissues. Our data suggest that miR-96 might promote the growth and motility of NSCLC cells partially by targeting RECK. Keywords miR-96 Non-small cell lung cancer Growth Migration Invasion RECK
Haizhou Guo and Qianping Li have contributed equally to this work. H. Guo W. Li T. Zheng S. Zhao Department of thoracic surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China Q. Li Department of Cardiothoracic Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China Z. Liu (&) Institute of Nephrology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China e-mail: [email protected]
Introduction Lung cancer is the leading cause of cancer-related mortality worldwide [1]. Non-small cell lung cancer (NSCLC) accounts for approximately 85 % of all lung cancer cases [2]. Despite recent advances in the diagnosis and treatment of NSCLC, the prognosis of NSCLC remains very poor, and the 5-year survival rate is a dismal 11 % [3]. Therefore, exploration of the potential mechanisms of the tumorigenesis of NSCLC is urgently needed. MicroRNAs (miRNAs) are a class of small non-coding RNA molecules (approximately 20–25 nucleotides), which negatively regulate the expression of target genes via either translational inhibition or mRNA degradation [4]. miRNAs can regulate the expression of many target genes involved in a number of biological processes including cell proliferation, differentiation, migration, and apoptosis [5]. Recently, mounting evidence suggests that aberrant expression of miRNAs correlates with a wide range of cancers, and they can act as oncogenes or tumor suppressors [6, 7]. In NSCLC, multiple miRNAs have been identified as tumor suppressors, such as miR-16, let-7c, and miR-143 [8–10]; on the other hand, miR-92b, miR-150, and miR-200a were found to promote NSCLC carcinogenesis [11–13]. miR-96 has been found to be upregulated and acts as oncogenes in several tumors, including bladder cancer, hepatoma, and prostate cancer [14–16]. Recently, miR-96 has been reported to be elevated in NSCLC [17]; however, the detailed role of miR96 in NSCLC remains poorly understood. Herein, we studied the effects of miR-96 on cell proliferation, colony formation, migration, and invasion of NSCLC cells. Reversion-inducing-cysteine-rich protein with kazal motifs (RECK) was confirmed to be a target of miR-96. Our results suggest that miR-96 might promote NSCLC cell growth and motility partially by suppressing RECK.
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Materials and methods
Colony formation assay
Tissue samples and cell lines
500 transfected cells were plated into a 6-well plate and cultured in DMEM containing 10 % FBS for 14 days. Colonies were fixed and stained with absolute methanol for 15 min, followed by 0.5 % crystal violet for 20 min. Colonies were quantified using Olympus INT-2 inverted microscope (Tokyo, Japan).
A total of 28 NSCLC tissues and matched normal tissues were obtained from our department. This work was approved by the Ethics Committee of The First Affiliated Hospital of Zhengzhou University, and informed consent was taken from all subjects. Three NSCLC cell lines, A549, SK-MES-1, and H1299, and a normal lung bronchus epithelial cell line BEAS-2B were obtained from the Chinese Academy of Sciences (Shanghai, China), cultured in DMEM media (Invitrogen, Carlsbad, CA, USA) supplemented 10 % FBS at 37 °C with 5 % CO2. RNA extraction and quantitative real-time PCR (qRTPCR) Total RNAs were extracted by Trizol reagent (Invitrogen, Carlsbad, CA, USA), and 1 lg of RNA was reversely transcripted. qRT-PCR was performed on ABI Stepone (ABI, Foster City, CA, USA) using SYBR Green Ò Premix Ex Taq (Takara, Tokyo, Japan). GAPDH was used as internal control. The primers for RECK: sense 50 -CCT CAGTGAGCACAGTTCAGA-30 and antisense 50 -GCAG CACACACACTGCTGTA-30 . miRNAs were extracted using mirVana miRNA isolation kit (Ambion, Carlsbad, CA, USA). Expression of miR-96 was quantified using TaqMan MicroRNA assay kit (ABI, Carlsbad, CA, USA), and U6 was used as internal control. Plasmids miR-96 mimic/inhibitor and the controls were obtained from RiboBio (Guangzhou, China). The 30 -UTR of RECK mRNA was amplified using the following primers: sense 50 -CCCTCGAGGCTGGGAAATGAGATGAC-30 and antisense 50 -TTGCGGCCGCTATGGCTATTCACCTTCTTC-30 . The PCR product was inserted into psiCHECK2 within XhoI and NotI restriction sites (Promega, Madison, WI, USA). Mutation experiment was performed using a fast mutation kit (NEB, Ipswich, Canada). MTT cell proliferation assay Approximately 3 9 103 cells were seeded in each well of a 96-well plate after 24-h incubation at the end of transfection. At various times following treatment, MTT (20 ll of 5 mg/ml) was added to each well and incubated at 37 °C for 4 h. Then, the cells were harvested and centrifuged, and 150 ll of DMSO was added to the formazan precipitates. The absorbance values at 490 nm were detected using a MRX II absorbance reader (DYNEX Technologies, Chantilly, VA, USA).
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Cell migration and invasion assays Cell migration and invasion were measured using a transwell chamber (Millipore, Billerica, MA, USA) with/without Matrigel (BD, San Jose, CA, USA). For the invasion assay, 1 9 105 transfected cells were plated into the upper chamber of transwells coated with Matrigel. In both assays, cells were cultured in medium with DMEM, while 600 ll of 10 % FBS-DMEM was added to the lower chamber. After 24 h incubation at 37 °C, migrated cells were fixed with methanol for 30 min. Non-migrated cells on the upper chamber were removed by cotton swabs. Cells on the bottom surface of the membrane were then stained by 0.5 % crystal violet for 20 min. Cell images were obtained using an inverted microscope (Olympus, Japan). Luciferase reporter assays HEK293 cells were cultured in 24-well plate for 24 h and co-transfected with 150 nM of miR-96 or control mimics and WT or Mut 30 -UTR of RECK using Lipofectamine 2000. 48 h after transfection, HEK293 cells were collected, and the relative luciferase activity was assayed using DualLuciferase Reporter Assay System (Promega, Wisconsin, WI, USA). Western blotting Cells were lysed and quantified by a BCA kit (Thermo, Waltham, MA, USA). Proteins were separated by 10 % SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membranes. Then the membranes were blocked with 3 % non-fat milk and incubated with antibodies against RECK (Abcam, Cambridge, UK) or GAPDH (Sangon Biotech, Shanghai, China) overnight. The membranes were incubated with HRP-conjugated secondary antibody for 1 h and detected by ECL system (Pierce, Rockford, IL, USA). Statistical analysis All data are presented as mean ± SD and analyzed by SPSS 16.0. One-way analysis of variance (ANOVA) or two-tailed Student’s t test was used to determine the
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Fig. 1 miR-96 was elevated in NSCLC tissues and cell lines. a miR96 was significantly increased in 28 NSCLC tissues compared to that in the non-tumoral tissues (NC). b miR-96 was remarkably elevated
in three NSCLC cell lines compared to that in the normal bronchial epithelial cell line BEAS-2B. U6 was used as an internal control. *P \ 0.05, **P \ 0.01 versus the control group
Fig. 2 Inhibition of miR-96 suppressed NSCLC cell growth in vitro. a At 24, 48, 36, 72, or 96 h after transfection, MTT assay was used to determine the proliferation of A549 cells. Anti-miR-96 transfection significantly suppressed A549 cell proliferation. b Anti-miR-96
transfection significantly suppressed A549 cells colony formation. c The expression of miR-96 was substantially decreased after miR-96 inhibitor transfection. Data were drawn from four independent experiments. *P \ 0.05, **P \ 0.01 versus the control group
statistical significance of differences. P \ 0.05 was considered to be statistically significant.
(Fig. 2a). Similarly, suppression of miR-96 significantly inhibited the colony formation of A549 (Fig. 2b). The effect of miR-96 inhibitor was confirmed by qRT-PCR (Fig. 2c).
Results
Inhibition of miR-96 suppressed NSCLC cell migration and invasion
miR-96 was elevated in NSCLC tissues and cell lines Expression of miR-96 in 28 NSCLC and matched nontumoral normal tissue samples was measured by qRT-PCR. miR-96 was significantly increased in NSCLC tissues compared to that in the non-tumor normal tissues (Fig. 1a). Expression of miR-96 in three NSCLC cell lines, A549, SK-MES-1, and H1299, was significantly increased compared to that in the BEAS-2B cells (Fig. 1b). Inhibition of miR-96 suppressed NSCLC cell growth in vitro To explore the function of miR-96 in the regulation of NSCLC cell growth, A549 cells were transfected with miR96 or control inhibitors. Suppression of miR-96 by miR-96 inhibitor remarkably inhibited the growth of A549 cells
To study the effect of miR-96 on motility of NSCLC cells, miR-96 or control inhibitors were transfected into A549 cells, and migration and invasion assays were performed. Inhibition of miR-96 substantially suppressed the migration and invasion capabilities of NSCLC cells (Fig. 3a, b). RECK was a direct target of miR-96 in NSCLC cells To identify the target of miR-96 in NSCLC, TargetScan 6.2 was used. RECK was predicted to be a potential target of miR-96 (Fig. 4a). Relative luciferase activity assay found that miR-96 significantly inhibited the luciferase activity of the WT but not the Mut 30 -UTR of RECK in HEK293 cells (Fig. 4b). Moreover, overexpression of miR-96 significantly inhibited RECK’s expression, while inhibition of miR-96 significantly elevated RECK’s expression (Fig. 4c).
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Fig. 3 Inhibition of miR-96 suppressed NSCLC cell migration and invasion. a Migration assay of A549 cells transfected with miR-96 or control inhibitor. Inhibition of miR-96 significantly suppressed the migration of A549 cells. b Invasion assay of A549 cells transfected
Mol Cell Biochem (2014) 390:155–160
with miR-96 or control inhibitor. Inhibition of miR-96 significantly suppressed the invasion of A549 cells. Data were drawn from four independent experiments. *P \ 0.05, **P \ 0.01 versus the control group
Fig. 4 RECK was a direct target of miR-96 in NSCLC cells. a WT or Mut 30 -UTR of RECK sequence. b HEK293 cells were co-transfected with miR-96 or miR-NC with WT or Mut 30 -UTR of RECK. Relative luciferase activity was assayed. c A549 cells transfected with miR-96/miR-NC, or anti-miR96/anti-miR-NC, and Western blotting was used to detect the protein levels of RECK. GAPDH was used as control. Data were drawn from four independent experiments. *P \ 0.05, **P \ 0.01 versus the control group
miR-96 was negatively correlated with RECK in NSCLC tissues Expression of RECK in 28 NSCLC and non-tumor tissues was measured. Results showed that RECK mRNA level was significantly decreased in NSCLC tissues compared to that in the non-tumor tissues (Fig. 5a). Moreover, RECK was found to be inversely correlated with miR-96 level in NSCLC tissues (Fig. 5b).
Discussion Recently, miRNAs have been widely studied in a number of tumors, and the previous studies showed that miR-96
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was commonly increased in several human cancers. miR96 was found to play oncogenic roles in both prostate cancer and bladder cancer by inhibiting the expression of Forkhead box protein O1 (FOXO1), and blocking the binding site reversed the growth enhancement conveyed by miR-96 [14, 15]. Moreover, in hepatocellular carcinoma, miR-96 was found to suppress the expression of FOXO1 and FOXO3 to promote the cell proliferation and clonogenicity, while suppression of miR-96 expression inhibited the invasion of hepatocellular carcinoma cells [16, 18]. On the other hand, in some cancers, miR-96 was shown to act as tumor suppressor. For instance, in pancreatic cancer, miR-96 functioned as tumor suppressor by targeting GTPase Kras (KRAS) [19]. However, the function of miR96 in lung cancer remains poorly understood.
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Fig. 5 miR-96 was negatively correlated with RECK in NSCLC tissues. a RECK mRNA level was significantly decreased in NSCLC tissues compared to that in the non-tumoral tissues. b RECK mRNA
level was inversely correlated with miR-96 level in NSCLC tissues (Spearman’s correlation analysis, r = -0.759). **P \ 0.01 versus the control group
Here we expanded the function of miR-96 in the progression of NSCLC. We confirmed that the expression of miR-96 was robustly increased in NSCLC tissues and cell lines compared to that in the normal non-tumor controls. We also demonstrated that the inhibition of miR-96 substantially suppressed the proliferation and colony formation of NSCLC cells. Furthermore, inhibition of miR-96 dramatically inhibited the migration and invasion abilities of NSCLC cells. Our data together suggest that miR-96 might contribute to the development of NSCLC. Moreover, we validated that RECK, which was a tumor suppressor and decreased in some cancers [6, 12, 20, 21], was a target of miR-96. RECK was first identified as a matrix metalloproteinase (MMP) inhibitor, and it suppressed the expression of several MMPs which played important roles in cancer invasion and metastasis, including MMP-2, MMP-9, and MMP14 [22, 23]. Decreased expression of RECK was correlated with poor survival in a wide range of cancers [6, 12]. RECK also played critical roles in the regulation of embryogenesis and vasculogenesis [24, 25]. RECK could be regulated by several miRNAs in many cancers, including miR-15a, miR-21, miR-25, miR-92b, miR182, and miR-222 [6, 12, 21, 26, 27]. In the present study, we found that miR-96 negatively regulated RECK expression, and RECK was inversely correlated with miR-96 level in NSCLC tissues. In conclusion, miR-96 was overexpressed in NSCLC, and increased miR-96 could affect various biological processes of NSCLC, including proliferation, colony formation, migration, and invasion, partially by inhibiting RECK expression. miR-96 might function as a biomarker for NSCLC, providing novel strategy for the treatment of NSCLC.
References
Conflict of interest
We declare that we have no conflict of interest.
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miR-96 downregulates RECK to promote growth and motility of non-small cell lung cancer cells Haizhou Guo • Qianping Li • Weihao Li • Tianliang Zheng • Song Zhao • Zhangsuo Liu
Received: 22 November 2013 / Accepted: 14 January 2014 / Published online: 29 January 2014 Ó Springer Science+Business Media New York 2014
Abstract MicroRNAs play critical roles in the development and progression of non-small cell lung cancer (NSCLC). miR-96 acts as an oncogene in some malignancies, while its role in NSCLC is unclear. Here, we validated that miR-96 was significantly increased in both human NSCLC tissues and cell lines. Inhibition of miR-96 expression remarkably reduced cell proliferation, colony formation, migration, and invasion of NSCLC cells. Reversion-inducing-cysteine-rich protein with kazal motifs (RECK) was identified as a target of miR-96 in NSCLC cells. In addition, the expression of RECK was found to be negatively correlated with the expression of miR-96 in NSCLC tissues. Our data suggest that miR-96 might promote the growth and motility of NSCLC cells partially by targeting RECK. Keywords miR-96 Non-small cell lung cancer Growth Migration Invasion RECK
Haizhou Guo and Qianping Li have contributed equally to this work. H. Guo W. Li T. Zheng S. Zhao Department of thoracic surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China Q. Li Department of Cardiothoracic Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China Z. Liu (&) Institute of Nephrology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China e-mail: [email protected]
Introduction Lung cancer is the leading cause of cancer-related mortality worldwide [1]. Non-small cell lung cancer (NSCLC) accounts for approximately 85 % of all lung cancer cases [2]. Despite recent advances in the diagnosis and treatment of NSCLC, the prognosis of NSCLC remains very poor, and the 5-year survival rate is a dismal 11 % [3]. Therefore, exploration of the potential mechanisms of the tumorigenesis of NSCLC is urgently needed. MicroRNAs (miRNAs) are a class of small non-coding RNA molecules (approximately 20–25 nucleotides), which negatively regulate the expression of target genes via either translational inhibition or mRNA degradation [4]. miRNAs can regulate the expression of many target genes involved in a number of biological processes including cell proliferation, differentiation, migration, and apoptosis [5]. Recently, mounting evidence suggests that aberrant expression of miRNAs correlates with a wide range of cancers, and they can act as oncogenes or tumor suppressors [6, 7]. In NSCLC, multiple miRNAs have been identified as tumor suppressors, such as miR-16, let-7c, and miR-143 [8–10]; on the other hand, miR-92b, miR-150, and miR-200a were found to promote NSCLC carcinogenesis [11–13]. miR-96 has been found to be upregulated and acts as oncogenes in several tumors, including bladder cancer, hepatoma, and prostate cancer [14–16]. Recently, miR-96 has been reported to be elevated in NSCLC [17]; however, the detailed role of miR96 in NSCLC remains poorly understood. Herein, we studied the effects of miR-96 on cell proliferation, colony formation, migration, and invasion of NSCLC cells. Reversion-inducing-cysteine-rich protein with kazal motifs (RECK) was confirmed to be a target of miR-96. Our results suggest that miR-96 might promote NSCLC cell growth and motility partially by suppressing RECK.
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Materials and methods
Colony formation assay
Tissue samples and cell lines
500 transfected cells were plated into a 6-well plate and cultured in DMEM containing 10 % FBS for 14 days. Colonies were fixed and stained with absolute methanol for 15 min, followed by 0.5 % crystal violet for 20 min. Colonies were quantified using Olympus INT-2 inverted microscope (Tokyo, Japan).
A total of 28 NSCLC tissues and matched normal tissues were obtained from our department. This work was approved by the Ethics Committee of The First Affiliated Hospital of Zhengzhou University, and informed consent was taken from all subjects. Three NSCLC cell lines, A549, SK-MES-1, and H1299, and a normal lung bronchus epithelial cell line BEAS-2B were obtained from the Chinese Academy of Sciences (Shanghai, China), cultured in DMEM media (Invitrogen, Carlsbad, CA, USA) supplemented 10 % FBS at 37 °C with 5 % CO2. RNA extraction and quantitative real-time PCR (qRTPCR) Total RNAs were extracted by Trizol reagent (Invitrogen, Carlsbad, CA, USA), and 1 lg of RNA was reversely transcripted. qRT-PCR was performed on ABI Stepone (ABI, Foster City, CA, USA) using SYBR Green Ò Premix Ex Taq (Takara, Tokyo, Japan). GAPDH was used as internal control. The primers for RECK: sense 50 -CCT CAGTGAGCACAGTTCAGA-30 and antisense 50 -GCAG CACACACACTGCTGTA-30 . miRNAs were extracted using mirVana miRNA isolation kit (Ambion, Carlsbad, CA, USA). Expression of miR-96 was quantified using TaqMan MicroRNA assay kit (ABI, Carlsbad, CA, USA), and U6 was used as internal control. Plasmids miR-96 mimic/inhibitor and the controls were obtained from RiboBio (Guangzhou, China). The 30 -UTR of RECK mRNA was amplified using the following primers: sense 50 -CCCTCGAGGCTGGGAAATGAGATGAC-30 and antisense 50 -TTGCGGCCGCTATGGCTATTCACCTTCTTC-30 . The PCR product was inserted into psiCHECK2 within XhoI and NotI restriction sites (Promega, Madison, WI, USA). Mutation experiment was performed using a fast mutation kit (NEB, Ipswich, Canada). MTT cell proliferation assay Approximately 3 9 103 cells were seeded in each well of a 96-well plate after 24-h incubation at the end of transfection. At various times following treatment, MTT (20 ll of 5 mg/ml) was added to each well and incubated at 37 °C for 4 h. Then, the cells were harvested and centrifuged, and 150 ll of DMSO was added to the formazan precipitates. The absorbance values at 490 nm were detected using a MRX II absorbance reader (DYNEX Technologies, Chantilly, VA, USA).
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Cell migration and invasion assays Cell migration and invasion were measured using a transwell chamber (Millipore, Billerica, MA, USA) with/without Matrigel (BD, San Jose, CA, USA). For the invasion assay, 1 9 105 transfected cells were plated into the upper chamber of transwells coated with Matrigel. In both assays, cells were cultured in medium with DMEM, while 600 ll of 10 % FBS-DMEM was added to the lower chamber. After 24 h incubation at 37 °C, migrated cells were fixed with methanol for 30 min. Non-migrated cells on the upper chamber were removed by cotton swabs. Cells on the bottom surface of the membrane were then stained by 0.5 % crystal violet for 20 min. Cell images were obtained using an inverted microscope (Olympus, Japan). Luciferase reporter assays HEK293 cells were cultured in 24-well plate for 24 h and co-transfected with 150 nM of miR-96 or control mimics and WT or Mut 30 -UTR of RECK using Lipofectamine 2000. 48 h after transfection, HEK293 cells were collected, and the relative luciferase activity was assayed using DualLuciferase Reporter Assay System (Promega, Wisconsin, WI, USA). Western blotting Cells were lysed and quantified by a BCA kit (Thermo, Waltham, MA, USA). Proteins were separated by 10 % SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membranes. Then the membranes were blocked with 3 % non-fat milk and incubated with antibodies against RECK (Abcam, Cambridge, UK) or GAPDH (Sangon Biotech, Shanghai, China) overnight. The membranes were incubated with HRP-conjugated secondary antibody for 1 h and detected by ECL system (Pierce, Rockford, IL, USA). Statistical analysis All data are presented as mean ± SD and analyzed by SPSS 16.0. One-way analysis of variance (ANOVA) or two-tailed Student’s t test was used to determine the
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Fig. 1 miR-96 was elevated in NSCLC tissues and cell lines. a miR96 was significantly increased in 28 NSCLC tissues compared to that in the non-tumoral tissues (NC). b miR-96 was remarkably elevated
in three NSCLC cell lines compared to that in the normal bronchial epithelial cell line BEAS-2B. U6 was used as an internal control. *P \ 0.05, **P \ 0.01 versus the control group
Fig. 2 Inhibition of miR-96 suppressed NSCLC cell growth in vitro. a At 24, 48, 36, 72, or 96 h after transfection, MTT assay was used to determine the proliferation of A549 cells. Anti-miR-96 transfection significantly suppressed A549 cell proliferation. b Anti-miR-96
transfection significantly suppressed A549 cells colony formation. c The expression of miR-96 was substantially decreased after miR-96 inhibitor transfection. Data were drawn from four independent experiments. *P \ 0.05, **P \ 0.01 versus the control group
statistical significance of differences. P \ 0.05 was considered to be statistically significant.
(Fig. 2a). Similarly, suppression of miR-96 significantly inhibited the colony formation of A549 (Fig. 2b). The effect of miR-96 inhibitor was confirmed by qRT-PCR (Fig. 2c).
Results
Inhibition of miR-96 suppressed NSCLC cell migration and invasion
miR-96 was elevated in NSCLC tissues and cell lines Expression of miR-96 in 28 NSCLC and matched nontumoral normal tissue samples was measured by qRT-PCR. miR-96 was significantly increased in NSCLC tissues compared to that in the non-tumor normal tissues (Fig. 1a). Expression of miR-96 in three NSCLC cell lines, A549, SK-MES-1, and H1299, was significantly increased compared to that in the BEAS-2B cells (Fig. 1b). Inhibition of miR-96 suppressed NSCLC cell growth in vitro To explore the function of miR-96 in the regulation of NSCLC cell growth, A549 cells were transfected with miR96 or control inhibitors. Suppression of miR-96 by miR-96 inhibitor remarkably inhibited the growth of A549 cells
To study the effect of miR-96 on motility of NSCLC cells, miR-96 or control inhibitors were transfected into A549 cells, and migration and invasion assays were performed. Inhibition of miR-96 substantially suppressed the migration and invasion capabilities of NSCLC cells (Fig. 3a, b). RECK was a direct target of miR-96 in NSCLC cells To identify the target of miR-96 in NSCLC, TargetScan 6.2 was used. RECK was predicted to be a potential target of miR-96 (Fig. 4a). Relative luciferase activity assay found that miR-96 significantly inhibited the luciferase activity of the WT but not the Mut 30 -UTR of RECK in HEK293 cells (Fig. 4b). Moreover, overexpression of miR-96 significantly inhibited RECK’s expression, while inhibition of miR-96 significantly elevated RECK’s expression (Fig. 4c).
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Fig. 3 Inhibition of miR-96 suppressed NSCLC cell migration and invasion. a Migration assay of A549 cells transfected with miR-96 or control inhibitor. Inhibition of miR-96 significantly suppressed the migration of A549 cells. b Invasion assay of A549 cells transfected
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with miR-96 or control inhibitor. Inhibition of miR-96 significantly suppressed the invasion of A549 cells. Data were drawn from four independent experiments. *P \ 0.05, **P \ 0.01 versus the control group
Fig. 4 RECK was a direct target of miR-96 in NSCLC cells. a WT or Mut 30 -UTR of RECK sequence. b HEK293 cells were co-transfected with miR-96 or miR-NC with WT or Mut 30 -UTR of RECK. Relative luciferase activity was assayed. c A549 cells transfected with miR-96/miR-NC, or anti-miR96/anti-miR-NC, and Western blotting was used to detect the protein levels of RECK. GAPDH was used as control. Data were drawn from four independent experiments. *P \ 0.05, **P \ 0.01 versus the control group
miR-96 was negatively correlated with RECK in NSCLC tissues Expression of RECK in 28 NSCLC and non-tumor tissues was measured. Results showed that RECK mRNA level was significantly decreased in NSCLC tissues compared to that in the non-tumor tissues (Fig. 5a). Moreover, RECK was found to be inversely correlated with miR-96 level in NSCLC tissues (Fig. 5b).
Discussion Recently, miRNAs have been widely studied in a number of tumors, and the previous studies showed that miR-96
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was commonly increased in several human cancers. miR96 was found to play oncogenic roles in both prostate cancer and bladder cancer by inhibiting the expression of Forkhead box protein O1 (FOXO1), and blocking the binding site reversed the growth enhancement conveyed by miR-96 [14, 15]. Moreover, in hepatocellular carcinoma, miR-96 was found to suppress the expression of FOXO1 and FOXO3 to promote the cell proliferation and clonogenicity, while suppression of miR-96 expression inhibited the invasion of hepatocellular carcinoma cells [16, 18]. On the other hand, in some cancers, miR-96 was shown to act as tumor suppressor. For instance, in pancreatic cancer, miR-96 functioned as tumor suppressor by targeting GTPase Kras (KRAS) [19]. However, the function of miR96 in lung cancer remains poorly understood.
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Fig. 5 miR-96 was negatively correlated with RECK in NSCLC tissues. a RECK mRNA level was significantly decreased in NSCLC tissues compared to that in the non-tumoral tissues. b RECK mRNA
level was inversely correlated with miR-96 level in NSCLC tissues (Spearman’s correlation analysis, r = -0.759). **P \ 0.01 versus the control group
Here we expanded the function of miR-96 in the progression of NSCLC. We confirmed that the expression of miR-96 was robustly increased in NSCLC tissues and cell lines compared to that in the normal non-tumor controls. We also demonstrated that the inhibition of miR-96 substantially suppressed the proliferation and colony formation of NSCLC cells. Furthermore, inhibition of miR-96 dramatically inhibited the migration and invasion abilities of NSCLC cells. Our data together suggest that miR-96 might contribute to the development of NSCLC. Moreover, we validated that RECK, which was a tumor suppressor and decreased in some cancers [6, 12, 20, 21], was a target of miR-96. RECK was first identified as a matrix metalloproteinase (MMP) inhibitor, and it suppressed the expression of several MMPs which played important roles in cancer invasion and metastasis, including MMP-2, MMP-9, and MMP14 [22, 23]. Decreased expression of RECK was correlated with poor survival in a wide range of cancers [6, 12]. RECK also played critical roles in the regulation of embryogenesis and vasculogenesis [24, 25]. RECK could be regulated by several miRNAs in many cancers, including miR-15a, miR-21, miR-25, miR-92b, miR182, and miR-222 [6, 12, 21, 26, 27]. In the present study, we found that miR-96 negatively regulated RECK expression, and RECK was inversely correlated with miR-96 level in NSCLC tissues. In conclusion, miR-96 was overexpressed in NSCLC, and increased miR-96 could affect various biological processes of NSCLC, including proliferation, colony formation, migration, and invasion, partially by inhibiting RECK expression. miR-96 might function as a biomarker for NSCLC, providing novel strategy for the treatment of NSCLC.
References
Conflict of interest
We declare that we have no conflict of interest.
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