Tumor Biol. DOI 10.1007/s13277-015-3550-8
RESEARCH ARTICLE
MiR-183 promotes growth of non-small cell lung cancer cells through FoxO1 inhibition Liqun Zhang 1 & Hongyu Quan 2 & Sihai Wang 1 & XueHui Li 1 & Xiaoyu Che 1
Received: 28 April 2015 / Accepted: 7 May 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Non-small cell lung cancer (NSCLC) is a prevalent cancer in lung of high incidence. NSCLCs often appear to be fast growing, which renders comprehension of the mechanisms underlying the growth of NSCLC extremely critical. Previous study has addressed a role of microRNA (miR) family member, miR-183, in the regulation of the invasiveness of NSCLC, whereas the role of miR-183 in the growth control of NSCLC is not clear. Here, we analyzed the regulation of FoxO1 by miR-183 in vitro using luciferase-reporter assay. We also analyzed the effects of miR-183 on NSCLC cell growth in vitro using a microculture tetrazolium (MTT) assay and in vivo by visualizing tumor growth using bioluminescence assay. We found that overexpression of miR-183 in NSCLC cells decreased FoxO1 protein levels, whereas inhibition of miR-183 increased FoxO1 protein levels without affecting FoxO1 transcripts. Moreover, miR-183 bound to FoxO1 mRNA to prevent its translation through its 3′untranslated region (UTR). Furthermore, administration of miR-183 suppressed FoxO1 levels in NSCLC, resulting in a significant increase in NSCLC growth in vitro and in vivo, while administration of antisense of miR-183 significantly increased FoxO1 levels in NSCLC resulting in a significant decrease in NSCLC growth. Taken together, our data demonstrate that miR-183/FoxO1 axis may be a novel therapeutic target for regulating the growth of NSCLC.
* Liqun Zhang [email protected] 1
Department of Respiratory Diseases, Beijing Military Region General Hospital of PLA, 5 Nanmencang, Dongcheng District, Beijing 100700, China
2
Battalion 19 of the Department of Biomedical Engineering, Third Military Medical University, Chongqing 400038, China
Keywords Non-small cell lung cancer (NSCLC) . FoxO1 . miR-183
Introduction Non-small cell lung cancer (NSCLC) is a prevalent cancer in lung of high incidence. NSCLC mainly consists three subtypes: squamous cell carcinoma, large cell carcinoma, and adenocarcinoma [1–4]. NSCLCs are often resistant to chemotherapy and radiation therapy and often appear to be fast growing [1–5]. Thus, a better comprehension of the mechanisms underlying the growth of NSCLC appears to be extremely critical for developing effective therapy. MicroRNAs (miRs) are a class of small, non-coding RNA that play important roles in various biological processes [6–8]. Of note, bioinformatics approaches have predicted that approximately one-third of all mammalian genes were targeted and regulated by miRs [6–8]. Interestingly, the expression of miRs has been detected in various tumors [6–8]. Previous studies have shown that aberrant miRNA expression could impact normal biological processes in NSCLC cells, resulting in tumor initiation and progression. Specifically, miR-183 has been highlighted as a major aberrantly expressed miRNA in some cancers [9–13], whereas the role of miR-183 in NSCLC has been rarely studied. A recent study has shown a role of miR-183 in the regulation of the invasiveness of NSCLC [14], whereas the role of miR-183 in the growth control of NSCLC is not clear. Forkhead box protein (Fox) proteins are a subgroup of the Forkhead family of transcription factors, characterized by a conserved DNA-binding domain. Fox proteins comprise more than 100 members in humans, classified from FoxA to FoxR on the basis of sequence similarity [15]. These proteins participate in very diverse functions. FoxO1 transcription factors
Tumor Biol.
orchestrate various cellular events like apoptosis and cellcycle control [15]. However, whether FoxO1 may be involved in the miR-183-mediated increases in NSCLC growth is unknown. Here we report that overexpression of miR-183 in NSCLC cells decreased FoxO1 protein levels, whereas inhibition of miR-183 increased FoxO1 protein levels, without affecting FoxO1 transcripts. Moreover, miR-183 bound to FoxO1 mRNA to prevent its translation through its 3′untranslated region (UTR). Furthermore, administration of miR-183 suppressed FoxO1 levels in NSCLC, resulting in a significant increase in NSCLC growth in vitro and in vivo, while administration of antisense of miR-183 significantly increased FoxO1 levels in NSCLC, resulting in a significant decrease in NSCLC growth.
Materials and methods Culture of human NSCLC cell line A549 is a widely used human NSCLC line purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and was first developed in 1972 by Dr. Giard through the removal and culturing of cancerous lung tissue in the explanted tumor a of a 58-year-old Caucasian male [16]. A549 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 20 % fetal bovine serum (Invitrogen, Carlsbad, CA, USA). Animal manipulation All animal procedures in the current study have been approved and conducted by the Institutional Review Boards of Beijing Military Region General Hospital of PLA. Female nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were purchased from Charles River Laboratories (China), kept pathogen free, and used for study at 12 weeks of age. 106 Luciferase-carrying A549 cells (A549LUC-GFP) were directly injected subcutaneously to form tumor in NOD/SCID mice. Adeno-associated viruses (AAV) of 109 were injected in 100 μl solution directly into the implanted tumor. Transfection of A549 cells in vitro A549 cells were transfected with a miR-183 construct, or an antisense (as) of miR-183, or a scramble sequence (scr) as a control, and after which the transfected cells were purified based on green fluorescence protein (GFP) to generate A549-miR-183, or A549-scr, or A549-as-miR-183 cells, respectively. MiR-183 sequence: 5′-UAUGGCACUGGUAG AAUUCACU-3′, miR-183 antisense sequence: 5′-AGUG
AAUUCUACCAGUGCCAUC-3′, and control sequence: 5′UUGUACUACACAAAAGUAAUG-3′. Transfection was performed with Lipofectamine 2000 reagent (Invitrogen), according to the manufacturer’s instructions. Transduction of A549 with AAV A549 cells were also transduced with an AAV carrying luciferase and GFP construct, to allow in vivo tracing in mice and purification of transduced cells by flow cytometry, respectively. AAV (serotype 8) was prepared as has been previously reported. Briefly, human embryonic kidney 293 cell line (HEK293) was used. We used a pAAV-CMV-LUC-GFP plasmid (Clontech, Mountain View, CA, USA), a packaging plasmid carrying the serotype 8 rep and cap genes, and a helper plasmid carrying the adenovirus helper functions (Applied Viromics, LLC. Fremont, CA, USA) in this study. AAV was prepared by triple transfection of the newly prepared plasmids, R2C8 (containing AAV2 Rep and AAV8 capsid genes) and plAd5 (containing adenovirus helper genes) into HEK293 cells by Lipofectamine 2000 reagent (Invitrogen). The viruses were purified using CsCl density centrifugation and then titered by a quantitative densitometric dot-blot assay. Then, the A549 cells were incubated with AAV at a MOI of 100 for 12 h. Luciferase-reporter activity assay Luciferase-reporters were successfully constructed using molecular cloning technology. Target sequence was inserted into pGL3-Basic vector (Promega, Madison, WI, USA) to obtain pGL3-FoxO1-3′UTR, which contains the miR-183 binding sequence (FoxO1-3′UTR sequence). A549-miR-183, or A549-scr, or A549-antisense (as)-miR-183 cells were seeded in 24-well plates for 24 h, after which they were transfected with 1 μg of luciferase-reporter plasmids per well using a PEI transfection reagent. Then luciferase activities were measured using the dual-luciferase reporter gene assay kit (Promega), according to the manufacturer’s instructions. Imaging of the implanted tumor by bioluminescence The tumor growth in the living animals was monitored and quantified by luminescence levels. Bioluminescence was measured with the IVIS imaging system (Xenogen Corp., Alameda, CA, USA). All of the images were taken 10 min after intraperitoneal injection of luciferin (Sigma-Aldrich) of 150 mg/kg body weight, as a 60-s acquisition and 10 of binning. During image acquisition, mice were sedated continuously via inhalation of 3 % isoflurane. Image analysis and bioluminescent quantification was performed using a Living Image software (Xenogen Corp).
Tumor Biol.
Quantitative PCR (RT-qPCR)
Results
MiRNA and total RNA were extracted from cultured cells with miRNeasy mini kit or RNeasy kit (Qiagen, Hilden, Germany), respectively, and used for cDNA synthesis. Quantitative PCR was performed in duplicates with QuantiTect SYBR Green PCR Kit (Qiagen). All primers were purchased from Qiagen. Values of genes were normalized against α-tubulin, and then compared to controls.
MiR-183 decreases FoxO1 protein but does not affect its transcription In the current study, we evaluated a role of miR-183 in the regulation of the growth of NSCLC. We used a human NSCLC cell line, A549, in our study. We transfected A549 cells with either miR-183 or antisense for miR-183 (as-miR-183). A549 cells were also transfected with a scramble scr. First, modulation of miR-183 levels in A549 cells was confirmed by RT-qPCR (Fig. 1a). Since FoxO1 has been shown to play a critical role in cell-cycle control and specifically express in NSCLC cells, we were thus prompted to evaluate whether FoxO1 may be regulated by miR-183 in NSCLC cell. We found that although the FoxO1 transcripts did not change by miR-183 levels (Fig. 1b), the protein levels of FoxO1 in miR-183overexpressing A549 cells was significantly decreased, while the protein levels of FoxO1 in miR-183-depleted A549 cells was significantly increased (Fig. 1c). These data
Statistical analysis All statistical analyses were carried out using the SPSS 17.0 statistical software package. All values are depicted as mean ± standard deviation and are considered significant if p < 0.05. All data were statistically analyzed using one-way ANOVA with a Bonferoni correction, followed by Fisher’s exact test to compare two groups.
10
B
*
5
*
1.0
0.5
0
0
A5
C
1.5 NS
Fold change of FoxO1 transcripts
Fold change of miR-183 levels
A
Fold change of FoxO1 protein
Fig. 1 MiR-183 decreases FoxO1 levels in NSCLC cells. We overexpressed or inhibited miR-183 expression in a human NSCLC cell line, A549, to obtain A549-miR-183 and A549-asmiR-183 cells, respectively. The cells transduced with a control plasmid carrying a scramble sequence (A549-scr). a, b RTqPCR on miR-183 (a) and FoxO1 (b). c Western blot images and quantification for FoxO1. *p
RESEARCH ARTICLE
MiR-183 promotes growth of non-small cell lung cancer cells through FoxO1 inhibition Liqun Zhang 1 & Hongyu Quan 2 & Sihai Wang 1 & XueHui Li 1 & Xiaoyu Che 1
Received: 28 April 2015 / Accepted: 7 May 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Non-small cell lung cancer (NSCLC) is a prevalent cancer in lung of high incidence. NSCLCs often appear to be fast growing, which renders comprehension of the mechanisms underlying the growth of NSCLC extremely critical. Previous study has addressed a role of microRNA (miR) family member, miR-183, in the regulation of the invasiveness of NSCLC, whereas the role of miR-183 in the growth control of NSCLC is not clear. Here, we analyzed the regulation of FoxO1 by miR-183 in vitro using luciferase-reporter assay. We also analyzed the effects of miR-183 on NSCLC cell growth in vitro using a microculture tetrazolium (MTT) assay and in vivo by visualizing tumor growth using bioluminescence assay. We found that overexpression of miR-183 in NSCLC cells decreased FoxO1 protein levels, whereas inhibition of miR-183 increased FoxO1 protein levels without affecting FoxO1 transcripts. Moreover, miR-183 bound to FoxO1 mRNA to prevent its translation through its 3′untranslated region (UTR). Furthermore, administration of miR-183 suppressed FoxO1 levels in NSCLC, resulting in a significant increase in NSCLC growth in vitro and in vivo, while administration of antisense of miR-183 significantly increased FoxO1 levels in NSCLC resulting in a significant decrease in NSCLC growth. Taken together, our data demonstrate that miR-183/FoxO1 axis may be a novel therapeutic target for regulating the growth of NSCLC.
* Liqun Zhang [email protected] 1
Department of Respiratory Diseases, Beijing Military Region General Hospital of PLA, 5 Nanmencang, Dongcheng District, Beijing 100700, China
2
Battalion 19 of the Department of Biomedical Engineering, Third Military Medical University, Chongqing 400038, China
Keywords Non-small cell lung cancer (NSCLC) . FoxO1 . miR-183
Introduction Non-small cell lung cancer (NSCLC) is a prevalent cancer in lung of high incidence. NSCLC mainly consists three subtypes: squamous cell carcinoma, large cell carcinoma, and adenocarcinoma [1–4]. NSCLCs are often resistant to chemotherapy and radiation therapy and often appear to be fast growing [1–5]. Thus, a better comprehension of the mechanisms underlying the growth of NSCLC appears to be extremely critical for developing effective therapy. MicroRNAs (miRs) are a class of small, non-coding RNA that play important roles in various biological processes [6–8]. Of note, bioinformatics approaches have predicted that approximately one-third of all mammalian genes were targeted and regulated by miRs [6–8]. Interestingly, the expression of miRs has been detected in various tumors [6–8]. Previous studies have shown that aberrant miRNA expression could impact normal biological processes in NSCLC cells, resulting in tumor initiation and progression. Specifically, miR-183 has been highlighted as a major aberrantly expressed miRNA in some cancers [9–13], whereas the role of miR-183 in NSCLC has been rarely studied. A recent study has shown a role of miR-183 in the regulation of the invasiveness of NSCLC [14], whereas the role of miR-183 in the growth control of NSCLC is not clear. Forkhead box protein (Fox) proteins are a subgroup of the Forkhead family of transcription factors, characterized by a conserved DNA-binding domain. Fox proteins comprise more than 100 members in humans, classified from FoxA to FoxR on the basis of sequence similarity [15]. These proteins participate in very diverse functions. FoxO1 transcription factors
Tumor Biol.
orchestrate various cellular events like apoptosis and cellcycle control [15]. However, whether FoxO1 may be involved in the miR-183-mediated increases in NSCLC growth is unknown. Here we report that overexpression of miR-183 in NSCLC cells decreased FoxO1 protein levels, whereas inhibition of miR-183 increased FoxO1 protein levels, without affecting FoxO1 transcripts. Moreover, miR-183 bound to FoxO1 mRNA to prevent its translation through its 3′untranslated region (UTR). Furthermore, administration of miR-183 suppressed FoxO1 levels in NSCLC, resulting in a significant increase in NSCLC growth in vitro and in vivo, while administration of antisense of miR-183 significantly increased FoxO1 levels in NSCLC, resulting in a significant decrease in NSCLC growth.
Materials and methods Culture of human NSCLC cell line A549 is a widely used human NSCLC line purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and was first developed in 1972 by Dr. Giard through the removal and culturing of cancerous lung tissue in the explanted tumor a of a 58-year-old Caucasian male [16]. A549 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 20 % fetal bovine serum (Invitrogen, Carlsbad, CA, USA). Animal manipulation All animal procedures in the current study have been approved and conducted by the Institutional Review Boards of Beijing Military Region General Hospital of PLA. Female nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were purchased from Charles River Laboratories (China), kept pathogen free, and used for study at 12 weeks of age. 106 Luciferase-carrying A549 cells (A549LUC-GFP) were directly injected subcutaneously to form tumor in NOD/SCID mice. Adeno-associated viruses (AAV) of 109 were injected in 100 μl solution directly into the implanted tumor. Transfection of A549 cells in vitro A549 cells were transfected with a miR-183 construct, or an antisense (as) of miR-183, or a scramble sequence (scr) as a control, and after which the transfected cells were purified based on green fluorescence protein (GFP) to generate A549-miR-183, or A549-scr, or A549-as-miR-183 cells, respectively. MiR-183 sequence: 5′-UAUGGCACUGGUAG AAUUCACU-3′, miR-183 antisense sequence: 5′-AGUG
AAUUCUACCAGUGCCAUC-3′, and control sequence: 5′UUGUACUACACAAAAGUAAUG-3′. Transfection was performed with Lipofectamine 2000 reagent (Invitrogen), according to the manufacturer’s instructions. Transduction of A549 with AAV A549 cells were also transduced with an AAV carrying luciferase and GFP construct, to allow in vivo tracing in mice and purification of transduced cells by flow cytometry, respectively. AAV (serotype 8) was prepared as has been previously reported. Briefly, human embryonic kidney 293 cell line (HEK293) was used. We used a pAAV-CMV-LUC-GFP plasmid (Clontech, Mountain View, CA, USA), a packaging plasmid carrying the serotype 8 rep and cap genes, and a helper plasmid carrying the adenovirus helper functions (Applied Viromics, LLC. Fremont, CA, USA) in this study. AAV was prepared by triple transfection of the newly prepared plasmids, R2C8 (containing AAV2 Rep and AAV8 capsid genes) and plAd5 (containing adenovirus helper genes) into HEK293 cells by Lipofectamine 2000 reagent (Invitrogen). The viruses were purified using CsCl density centrifugation and then titered by a quantitative densitometric dot-blot assay. Then, the A549 cells were incubated with AAV at a MOI of 100 for 12 h. Luciferase-reporter activity assay Luciferase-reporters were successfully constructed using molecular cloning technology. Target sequence was inserted into pGL3-Basic vector (Promega, Madison, WI, USA) to obtain pGL3-FoxO1-3′UTR, which contains the miR-183 binding sequence (FoxO1-3′UTR sequence). A549-miR-183, or A549-scr, or A549-antisense (as)-miR-183 cells were seeded in 24-well plates for 24 h, after which they were transfected with 1 μg of luciferase-reporter plasmids per well using a PEI transfection reagent. Then luciferase activities were measured using the dual-luciferase reporter gene assay kit (Promega), according to the manufacturer’s instructions. Imaging of the implanted tumor by bioluminescence The tumor growth in the living animals was monitored and quantified by luminescence levels. Bioluminescence was measured with the IVIS imaging system (Xenogen Corp., Alameda, CA, USA). All of the images were taken 10 min after intraperitoneal injection of luciferin (Sigma-Aldrich) of 150 mg/kg body weight, as a 60-s acquisition and 10 of binning. During image acquisition, mice were sedated continuously via inhalation of 3 % isoflurane. Image analysis and bioluminescent quantification was performed using a Living Image software (Xenogen Corp).
Tumor Biol.
Quantitative PCR (RT-qPCR)
Results
MiRNA and total RNA were extracted from cultured cells with miRNeasy mini kit or RNeasy kit (Qiagen, Hilden, Germany), respectively, and used for cDNA synthesis. Quantitative PCR was performed in duplicates with QuantiTect SYBR Green PCR Kit (Qiagen). All primers were purchased from Qiagen. Values of genes were normalized against α-tubulin, and then compared to controls.
MiR-183 decreases FoxO1 protein but does not affect its transcription In the current study, we evaluated a role of miR-183 in the regulation of the growth of NSCLC. We used a human NSCLC cell line, A549, in our study. We transfected A549 cells with either miR-183 or antisense for miR-183 (as-miR-183). A549 cells were also transfected with a scramble scr. First, modulation of miR-183 levels in A549 cells was confirmed by RT-qPCR (Fig. 1a). Since FoxO1 has been shown to play a critical role in cell-cycle control and specifically express in NSCLC cells, we were thus prompted to evaluate whether FoxO1 may be regulated by miR-183 in NSCLC cell. We found that although the FoxO1 transcripts did not change by miR-183 levels (Fig. 1b), the protein levels of FoxO1 in miR-183overexpressing A549 cells was significantly decreased, while the protein levels of FoxO1 in miR-183-depleted A549 cells was significantly increased (Fig. 1c). These data
Statistical analysis All statistical analyses were carried out using the SPSS 17.0 statistical software package. All values are depicted as mean ± standard deviation and are considered significant if p < 0.05. All data were statistically analyzed using one-way ANOVA with a Bonferoni correction, followed by Fisher’s exact test to compare two groups.
10
B
*
5
*
1.0
0.5
0
0
A5
C
1.5 NS
Fold change of FoxO1 transcripts
Fold change of miR-183 levels
A
Fold change of FoxO1 protein
Fig. 1 MiR-183 decreases FoxO1 levels in NSCLC cells. We overexpressed or inhibited miR-183 expression in a human NSCLC cell line, A549, to obtain A549-miR-183 and A549-asmiR-183 cells, respectively. The cells transduced with a control plasmid carrying a scramble sequence (A549-scr). a, b RTqPCR on miR-183 (a) and FoxO1 (b). c Western blot images and quantification for FoxO1. *p
