Biomedicine & Pharmacotherapy 71 (2015) 79–83
Available online at
ScienceDirect www.sciencedirect.com
Original article
Up-regulation of BRAF activated non-coding RNA is associated with radiation therapy for lung cancer Jian-xiang Chen, Ming Chen, Yuan-da Zheng, Sheng-ye Wang, Zhu-ping Shen * Department of Radiation Oncology, Zhejiang Cancer Hospital, Hangzhou 310022, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 January 2015 Accepted 15 February 2015
Radiation therapy has become more effective in treating primary tumors, such as lung cancer. Recent evidence suggested that BRAF activated non-coding RNAs (BANCR) play a critical role in cellular processes and are found to be dysregulated in a variety of cancers. The clinical significance of BANCR in radiation therapy, and its molecular mechanisms controlling tumor growth are unclear. In the present study, C57BL/6 mice were inoculated Lewis lung cancer cells and exposed to radiation therapy, then BANCR expression was analyzed using qPCR. Chromatin immunoprecipitation and western blot were performed to calculate the enrichment of histone acetylation and HDAC3 protein levels in Lewis lung cancer cells, respectively. MTT assay was used to evaluate the effects of BANCR on Lewis lung cancer cell viability. Finally, we found that BANCR expression was significantly increased in C57BL/6 mice receiving radiation therapy (P < 0.05) compared with control group. Additionally, knockdown of BANCR expression was associated with larger tumor size in C57BL/6 mice inoculated Lewis lung cancer cells. Histone deacetylation was observed to involve in the regulation of BANCR in Lewis lung cancer cells. Moreover, over expression HDAC3 reversed the effect of rays on BANCR expression. MTT assay showed that knockdown of BANCR expression promoted cell viability surviving from radiation. In conclusion, these findings indicated that radiation therapy was an effective treatment for lung cancer, and it may exert function through up-regulation BANCR expression. ß 2015 Published by Elsevier Masson SAS.
Keywords: Radiation therapy Lung cancer BRAF activated non-coding RNA HDAC3 Histone acetylation Lewis lung cancer cell
1. Introduction Lung cancer, stemming from abnormal epithelial cells in the airways of the lung, becomes the second most commonly diagnosed cancer subjected to men and women in the United States. Unfortunately, lung cancer accounts for the majority of cancer-related deaths, especially in smoking prevalence region [1– 3]. The World Health Organization has divided lung cancer into two major classes: non–small cell lung cancer (NSCLC) and small cell lung cancer. With the development of chemotherapeutic agents and targeted therapies directed against the epidermal growth factor and vascular endothelial growth factor receptor pathways for NSCLC treatment, radiation therapy techniques has emerged as a prophylactically treatment of subclinical disease and provided effective relief from pain and obstructive symptoms [1,4]. Despite recent advances of radiation therapy in clinical
* Corresponding author at: Department of Radiation Oncology, Zhejiang Cancer Hospital, No. 38 Guangji Rd, Hangzhou 310022, Zhejiang Province, China. Tel.: +86 571 88128056; fax: +86 571 88128056. E-mail address: [email protected] (Z.-p. Shen). http://dx.doi.org/10.1016/j.biopha.2015.02.021 0753-3322/ß 2015 Published by Elsevier Masson SAS.
and experimental oncology, the ideal genetic marker in radiation therapy for lung cancer detection has far from being fully elucidated. Long non-coding RNAs (lncRNAs), important new members of the ncRNA family, recently have been found to participate in tumorigenesis and tumor progression [5,6]. Emerging studies reported that functional lncRNAs may be used for diagnosing cancer, determining prognosis and as a therapeutic target [7,8]. BRAF-activated lncRNA (BANCR) is a 693-bp lncRNA on chromosome 9, which is frequently over expressed in melanoma cells and plays crucial roles in melanoma cell migration [9,10]. Ming Sun reported that the expression of BANCR was significantly decreased in NSCLC tissues and could be a biomarker for poor prognosis of NSCLC. Furthermore, histone deacetylation was observed to involve in the downregulation of BANCR in NSCLC cells [11]. It has been appreciated for decades that histone deacetylases (HDACs), which catalyzed acetyl groups removal from the histone protein, associated with a variety of cellular oncogenes and tumorsuppressor genes and played an essential role in cancer development [12,13]. Based on the identification and cloning of HDACs, it was widely suggested that HDACs counteracted the activating
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effects of histone acetyltransferases by recruiting to promoters to repress transcription. In addition, HDAC inhibitors, such as trichostatin A (TSA), have attracted considerable attention as anticancer agents for their ability to trigger proteins leading cell cycle arrest, differentiation, or apoptosis in neoplastically transformed cell [14,15]. Therefore, detection of HDACs variation may contribute to understand the regulation of some factors in lung cancer, such as BANCR. In this study, we investigated the expression levels of BANCR in radiation therapy for treatment lung cancer and the function and molecular mechanisms of BANCR regulation in lung cancer. This study provides us a more detailed understanding of BANCR functioning as a regulator in lung cancer pathogenesis and facilitate the development of lncRNA-directed diagnostics and therapeutics. 2. Materials and methods 2.1. Animals and tumor model The procedures of animal experiments were performed by Guide for the Care and Use of Laboratory Animals and were approved by Nanjing Thoracic Hospital (Nanjing, China). Male C57B6/L mice were purchased from Better Biotechnology Co., Ltd. (Nanjing, China). Animals were kept in a temperature-controlled room with free access to standard chow and water. Lewis mice lung cancer cell line, cultured in vitro and existed in logarithmic phase, were prepared into the cell suspension, and then inoculated into the right hind leg of C57B6/L mice. Each C57B6/L was inoculated 1 106 cells. Tumor volumes were measured with calipers and calculated with the formula L W2 0.5. 2.2. Tumor irradiation After six days of Lewis mice lung cancer cell inoculation, C57B6/L mice were exposed to a high single-dose X-rays with 12 Gy/one fraction/day, and served as radiation therapy group; equal number C57B6/L mice without received X-rays were as control group. Lewis mice lung cancer cells were divided into 3 groups, which were exposed to X-rays with 0 Gy, 1 Gy and 3 Gy. Mice were anesthetized (Nembutal) during the radiation treatment, and were immobilized in a customized harness that allowed the right hind leg to be exposed, whereas the remainder of the body was shielded by 3.5 cm of lead. Mice were irradiated in a Gammacell Cesium 137 (Atomic Energy of Canada) source operating at a rate of 125 cGy/min. 2.3. Quantitative polymerase chain reaction (PCR) Total RNA was extracted from tissues and cells using RNAiso Plus (Takara, China), and reverse transcription (RT) reactions were performed using ImProm-IITM (Promega, USA) following the manufacturer’s instructions. Quantitative PCR reactions were prepared at a final volume of 20 ml using a standard protocol and the TransStartTM SYBR Green qPCR Supermix (TransGen Biotech, China), and the reactions were performed on the StepOnePlus Real-Time PCR System (Applied Biosystems, Inc., DDCT CA, USA). The 2 method was used to determine the relative BANCR expression levels, using b-actin as the endogenous control to normalize the data.
penicillin and 100 mg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) at 37 8C/5% CO2. 2.5. Plasmid generation To detect the expression alteration of BANCR with HDAC3 over expression, the HDAC3 sequence was synthesized and sub cloned into the pCDNA3.1 (Invitrogen, Shanghai, China) vector. Over expression of HDAC3 was achieved through pCDNA-3.1-HDAC3 transfection, with an empty pCDNA3.1 vector used as a control. 2.6. Cell transfection The siRNAs si-BANCR or si-NC were transfected into Lewis lung cancer cells. Plasmid vectors pCDNA-3.1-HDAC3, pCDNA3.1 and BANCR RNAi Lentivirus expression vector (Shanghai R&S Biotechnology Co., Ltd., China; 10 MOI) for transfection were prepared using DNA Midi prep or Midi prep kits (Qiagen, Hilden, Germany), and transfected into Lewis lung cancer cells. Lewis lung cancer cells were grown on six-well plates to confluency and transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. At 48 h post-transfection, cells were harvested for qPCR or western blot analysis. Cells transfected with BANCR RNAi Lentivirus expression vector were explanted under the skin of C57BL/6 mice, and then the mice exposed to X-rays for tumor volume determination. 2.7. Chromatin immunoprecipitation Chromatin immunoprecipitations were performed as described previously [16]. Briefly, 700 mL chromatin prepared from Lewis lung cancer cells were incubated with 100 mL protein A-agarose (50% slurry in ChIP buffer) for 1 h at 4 8C. ChIP buffer contained 50 mM NaCl, 10 mM Tris–HCI pH 7.5, 10 mM Na butyrate, 1 mM EDTA, 0.1 mM PMSF and 10 mg/ml aprotinin. After separating protein A-agarose and chromatin, DNA at this point used as the ‘‘input’’ sample. Antibodies specific for acetylated histones (100 mL) [anti-acetylated-histone H3, anti-acetylated-histone H4 supplied by Biotechnology] were incubated with protein Aclarified chromatin for 2 h at 4 8C under constant agitation. Then 100 mL protein A-agarose were added to the chromatin antibody mix and incubated overnight at 4 8C. The protein A-agarose and unbound chromatin were separated using 0.45 mm Microfuge filter units. Bound chromatin was eluted by washing protein Aagarose five times with ChIP buffer. DNA from bound chromatin and unbound chromatin were purified by phenol/chloroform extraction and ethanol precipitation. DNA samples were quantified using picogreen fluorescence. 2.8. Western blotting analysis Equal amounts of protein were electrophoresed on a 12% or 15% sodium dodecyl sulfate-polyacrylamide gel and subsequently transferred to a nitrocellulose membrane (Bio-Rad, USA), which were blocked in Tris-buffered saline with 5% milk and 0.05% Tween 20 and probed with primary antibodies at 4 8C overnight according to the manufacturer’s recommendations. b-Actin was served as a control protein to quantify the expression of target protein. 2.9. Cell viability assays
2.4. Cell culture Lewis lung cancer cells, purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China), were cultured in DMEM (GIBCO-BRL) medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml
Cell viability was monitored using MTT assay (Sigma, USA). Lewis lung cancer cells transfected with si-BANCR and si-control (3000 cells/well) were grown in 96-well plates. Cell viability was assessed every 24 h following the manufacturer’s protocol. Briefly, 5 mg/mL MTT reagent was added to the medium (the final
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Fig. 1. BANCR is frequently up-regulated in lung cancer tissues exposed to radiation therapy. (A) Tumor volume of radiation therapy group and control group. (B) BANCR expression of radiation therapy group and control group. Mean SD, all results were repeated for three times; *VS control group, P < 0.05.
concentration MTT is 0.5 mg/mL) and incubated with cells for 4 h. After incubation, 150 mL dimethylsulfoxide was used to dissolved the crystal products and then the absorbance was detected at a wavelength of 570 nm by SpectraMax M5 Multimode Reader (Molecular Devices, USA). 3. Results 3.1. BANCR is frequently up-regulated in lung cancer tissues exposed to radiation therapy Given the effective roles of radiotherapy in tumor treatment, we were interested in examining its efficacy on lung cancer. Therefore, after six days of inoculating Lewis lung cancer cells into C57BL/6 mice, the tumor growth in C57BL/6 mice which exposed to radiation therapy was evaluated. As a result, tumors growth was found to be significantly repressed in radiation therapy group comparing to control group (Fig. 1A). This suggests that radiation therapy may be a functional way to inhibit tumor growth. As the report of BANCR may being a biomarker for poor prognosis of NSCLC [11], we investigated the levels of BANCR using qPCR in C57BL/6 mice, and found that BANCR expression was significantly increased in radiation therapy group compared with control group (Fig. 1B).
increasing (Fig. 3A). It has been reported that histone acetylation is a key factor in controlling BANCR expression. Thus we examined the levels of acetyl-histones. Additionally, it has been well known that all core histones are acetylated in vivo, while histones H3 and H4 modifications are much more extensively characterized than those of H2A and H2B [17]. Thus, chromatin immunoprecipitation was performed to examined the levels of histone H3 and H4 acetylation in BANCR promoter region. The result showed that they were both increased and positively correlated to ray dosage (Fig. 3B). Following, western blot was performed to determine HDAC3 and HDAC1 protein levels. Fig. 3C showed that HDAC3 protein levels was decreased with the increasing ray dosage, while HDAC1 protein levels remained constant. 3.4. Over expression HDAC3 reverses the effect of rays on BANCR expression As has been known that HDAC3 involved in the regulation of BANCR expression, we further explored the effect of over expression HDAC3 on BANCR expression. Fig. 4 showed that the expression of BANCR was significantly increased in Lewis lung cancer cell received 3 Gy rays, while over expression HDAC3 reversed this effect and significantly decreased the expression of BANCR.
3.2. Knockdown of BANCR promotes tumor growth in lung cancer tissues exposed to radiation therapy In order to further study the roles of BANCR on tumors growth, we examined the tumors volume in C57BL/6 mice, which was transplanted Lewis lung cancer cell transfected si-BANCR under the skin. Six days later, we observed that the emerging tumor was insensitive to X-rays, and tumors growth was significantly exceeded the control group (Fig. 2). Taken together, these data indicated that BANCR played important roles in tumor development. 3.3. HDAC involves in the regulation of BANCR expression in Lewis lung cancer cells exposed to X-rays To investigate the influence of X-rays on BANCR expression in Lewis lung cancer cell, it was received different X-ray dosage of 0.1 Gy and 3 Gy for 24 h. Then, qPCR showed that BANCR expression was significantly increased with the ray dosage
Fig. 2. Tumor volume of C57BL/6 mice transplanted Lewis lung cancer cell, which was transfected si-BANCR and NC, respectively. Mean SD, all results were repeated for three times; *VS control group, P < 0.05.
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Fig. 3. HDAC involves in the regulation of BANCR expression in Lewis lung cancer cells. Lewis lung cancer cell was received ray dosage with 0.1 Gy and 3 Gy. (A) BANCR expression of each group. (B) The levels of histone H3 and H4 acetylation of each group. (C) The HDAC3 and HDAC1 protein levels of each group. Mean SD, all results were repeated for three times; *VS control group, P < 0.05.
Lung cancer is one of the most mortal malignant tumors, and is becoming one of most lethal threat to human health and life [18]. Nowadays several urgent or emergent symptoms due to metastatic lung cancer are often effectively treated with radiation therapy. Radiation therapy is frequently used as an adjunct to surgery or an alternative for the patient when considered
inoperable for medical or technical reasons [19,20]. Therefore, identification of gene regulation associated in radiation therapy for lung cancer treatment and investigation of their clinical significance and functions may provide an insight into the well-known oncogenic and tumor suppressor network. It has been reported that lncRNA dysregulation resulted in a range of biological functions and provided a cellular growth advantage for progressive and uncontrolled tumor growth [21,22]. Therefore, effective control cell proliferation is pivotal to the prevention of oncogenesis and successful cancer therapy. Recently, Ming Sun et al. have suggested that BANCR may actively functions as a regulator of epithelial-mesenchymal transition during NSCLC metastasis, which indicated that BANCR could be a biomarker for poor prognosis of NSCLC [11]. However, the roles of BANCR in radiation therapy for lung cancer treatment and the carcinogenesis of LC are far from being fully illuminated. In this study, we found that radiation therapy was effective against repressing tumor growth in C57BL/6 mice transfected
Fig. 4. HDAC3 reverses the effect of X-rays on BANCR expression. Mean SD, all results were repeated for three times; *VS control group, P < 0.05.
Fig. 5. The effect of rays on Lewis lung cancer cell viability. Mean SD, all results were repeated for three times; *VS control group, P < 0.05.
3.5. The effect of X-rays on Lewis lung cancer cells viability Following Lewis lung cancer cells subjected to 3 Gy X-rays for 72 h, cell viability was examined using MTT assay. As was showed in Fig. 5, we found that cell viability was significantly decreased after exposed to 3 Gy X-rays (Fig. 5). In addition, when Lewis lung cancer cell were transfected si-BANCR, the cell viability was reversed and significantly increased compared to control group (Fig. 5). 4. Discussion
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Lewis lung cancer cells, which suggested that radiation therapy indeed inhibited tumor growth impactfully. Additionally, we also found that the expression of BANCR was significantly upregulated in lung cancer tissues which were exposed to radiation therapy. Furthermore, in vivo examination of the potential role of BANCR in C57BL/6 mice transfected Lewis lung cancer cells demonstrated that the knockdown of BANCR in Lewis lung cancer cells was associated with the promotion of tumor growth in spite of radiation therapy. This observation was consistent with previous studies regarding the expression of BANCR in NSCLC tissues [11]. Moreover, cell viability of Lewis lung cancer cells also verified this result in vitro, as MTT assays showed that Lewis lung cancer cells been exposed to X-rays possessed significantly lower cell viability, while knockdown of BANCR in Lewis lung cancer cells reversed this effect. However, Yong Wang et al. reported that BANCR was highly expressed in papillary thyroid carcinoma, as well as in colorectal cancer reported by Qinhao Guo et al. [23,24]. These observations suggested that the function of BANCR could be tissue-specific. Taken together, these findings suggest that BANCR plays a direct role in the course of radiation therapy for treatment lung cancer. Fu Yang et al. determined that a hypoxic microenvironment suppressed lncRNA-LET through HDAC3 activation, which resulted in reduced histone H3 and H4 acetylation levels in the lncRNA-LET promoter region [25]. Similarly, our study showed that with the increasing expression of BANCR in Lewis lung cancer cells exposed to X-rays, histone H3 and H4 acetylation was also increased in BANCR promoter region. Western blot further suggested that HDAC3 protein levels were decreased, while HDAC1 protein levels was no change. Accordingly, we addressed whether the upregulation of BANCR under radiation therapy was mediated by HDAC3. Over expression of HDAC3 showed that the expression of BANCR was significantly decreased in Lewis lung cancer cells exposed to X-rays. These results support that the upregulation of BANCR expression under radiation therapy is mediated by HDAC3. In summary, the expression of BANCR was increased in LC tissues exposed to radiation, suggesting that BANCR may be a positive prognostic factor for LC. This study showed that radiation may regulate the proliferation of LC cells partly through upregulation of BANCR, and HDAC3 protein was involved in the regulation. These findings further the understanding on the mechanisms of treating LC by radiation, and facilitate the development of BANCR-directed diagnostics and therapeutics against this deadly disease.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
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References [1] Haas ML. Advances in radiation therapy for lung cancer. Semin Oncol Nurs 2008;24:34–40. [2] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin 2013;63:11–30. [3] Salim EI, Jazieh AR, Moore MA. Lung cancer incidence in the Arab league countries: risk factors and control. Asian Pac J Cancer Prev 2011;12:17–34. [4] Shibuya K, Hiraoka M. Radiation therapy for lung cancer. Gan Kagaku Ryoho/ Cancer Chemother 2007;34:544–9. [5] Nakagawa T, Endo H, Yokoyama M, Abe J, Tamai K, Tanaka N, et al. Large noncoding RNA HOTAIR enhances aggressive biological behavior and is associated with short disease-free survival in human non-small cell lung cancer. Biochem Biophys Res Commun 2013;436:319–24. [6] Mercer TR, Dinger ME, Mattick JS. Long non-coding RNAs: insights into functions. Nat Rev Genet 2009;10:155–9. [7] Qi P, Du X. The long non-coding RNAs, a new cancer diagnostic and therapeutic gold mine. Mod Pathol 2012;26:155–65. [8] Spizzo R, Almeida MI, Colombatti A, Calin GA. Long non-coding RNAs and cancer: a new frontier of translational research. Oncogene 2012;31:4577–87. [9] Flockhart RJ, Webster DE, Qu K, Mascarenhas N, Kovalski J, Kretz M, et al. BRAFV600E remodels the melanocyte transcriptome and induces BANCR to regulate melanoma cell migration. Genome Res 2012;22:1006–14. [10] McCarthy N. Epigenetics: going places with BANCR. Nat Rev Cancer 2012;12:451. [11] Sun M, Liu X-H, Wang K-M, Nie F-Q, Kong R, Yang J-S, et al. Downregulation of BRAF activated non-coding RNA is associated with poor prognosis for nonsmall cell lung cancer and promotes metastasis by affecting epithelial-mesenchymal transition. Mol Cancer 2014;13:68–80. [12] De Ruijter A, Van Gennip A, Caron H, Kemp S, van Kuilenburg A. Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochem J 2003;370:737–49. [13] Glozak M, Seto E. Histone deacetylases and cancer. Oncogene 2007;26:5420–32. [14] Myzak MC, Karplus PA, Chung F-L, Dashwood RH. A novel mechanism of chemoprotection by sulforaphane inhibition of histone deacetylase. Cancer Res 2004;64:5767–74. [15] Shi Q-Q, Zuo G-W, Feng Z-Q, Zhao L-C, Luo L, You Z, et al. Effect of trichostatin A on anti HepG2 liver carcinoma cells: inhibition of HDAC activity and activation of Wnt/b-catenin signaling. Asian Pac J Cancer Prev 2014;15:7849–55. [16] Hebbes T, Clayton A, Thorne A, Crane-Robinson C. Core histone hyperacetylation co-maps with generalized DNase I sensitivity in the chicken beta-globin chromosomal domain. EMBO J 1994;13:1823–30. [17] Wade PA. Transcriptional control at regulatory checkpoints by histone deacetylases: molecular connections between cancer and chromatin. Hum Mol Genet 2001;10:693–8. [18] Zheng D-J, Yu G-H, Gao J-F, Gu J-D. Concomitant EGFR inhibitors combined with radiation for treatment of non-small cell lung carcinoma. Asian Pac J Cancer Prev 2013;14:4485–94. [19] Van Houtte P. Postoperative radiotherapy for lung cancer. Lung Cancer 1991;7:57–64. [20] Graham MV, Purdy JA, Emami B, Matthews JW, Harms WB. Preliminary results of a prospective trial using three dimensional radiotherapy for lung cancer. Int J Radiat Oncol Biol Phys 1995;33:993–1000. [21] Gupta RA, Shah N, Wang KC, Kim J, Horlings HM, Wong DJ, et al. Long noncoding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature 2010;464:1071–6. [22] Kotake Y, Nakagawa T, Kitagawa K, Suzuki S, Liu N, Kitagawa M, et al. Long non-coding RNA ANRIL is required for the PRC2 recruitment to and silencing of p15INK4B tumor suppressor gene. Oncogene 2010;30:1956–62. [23] Wang Y, Guo Q, Zhao Y, Chen J, Wang S, Hu J, et al. BRAF-activated long noncoding RNA contributes to cell proliferation and activates autophagy in papillary thyroid carcinoma. Oncol Lett 2014;8:1947–52. [24] Guo Q, Zhao Y, Chen J, Hu J, Wang S, Zhang D, et al. BRAF-activated long noncoding RNA contributes to colorectal cancer migration by inducing epithelialmesenchymal transition. Oncol Lett 2014;8:869–75. [25] Yang F, Huo X-S, Yuan S-X, Zhang L, Zhou W-P, Wang F, et al. Repression of the long noncoding RNA-LET by histone deacetylase 3 contributes to hypoxiamediated metastasis. Mol Cell 2013;49:1083–96.
Available online at
ScienceDirect www.sciencedirect.com
Original article
Up-regulation of BRAF activated non-coding RNA is associated with radiation therapy for lung cancer Jian-xiang Chen, Ming Chen, Yuan-da Zheng, Sheng-ye Wang, Zhu-ping Shen * Department of Radiation Oncology, Zhejiang Cancer Hospital, Hangzhou 310022, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 January 2015 Accepted 15 February 2015
Radiation therapy has become more effective in treating primary tumors, such as lung cancer. Recent evidence suggested that BRAF activated non-coding RNAs (BANCR) play a critical role in cellular processes and are found to be dysregulated in a variety of cancers. The clinical significance of BANCR in radiation therapy, and its molecular mechanisms controlling tumor growth are unclear. In the present study, C57BL/6 mice were inoculated Lewis lung cancer cells and exposed to radiation therapy, then BANCR expression was analyzed using qPCR. Chromatin immunoprecipitation and western blot were performed to calculate the enrichment of histone acetylation and HDAC3 protein levels in Lewis lung cancer cells, respectively. MTT assay was used to evaluate the effects of BANCR on Lewis lung cancer cell viability. Finally, we found that BANCR expression was significantly increased in C57BL/6 mice receiving radiation therapy (P < 0.05) compared with control group. Additionally, knockdown of BANCR expression was associated with larger tumor size in C57BL/6 mice inoculated Lewis lung cancer cells. Histone deacetylation was observed to involve in the regulation of BANCR in Lewis lung cancer cells. Moreover, over expression HDAC3 reversed the effect of rays on BANCR expression. MTT assay showed that knockdown of BANCR expression promoted cell viability surviving from radiation. In conclusion, these findings indicated that radiation therapy was an effective treatment for lung cancer, and it may exert function through up-regulation BANCR expression. ß 2015 Published by Elsevier Masson SAS.
Keywords: Radiation therapy Lung cancer BRAF activated non-coding RNA HDAC3 Histone acetylation Lewis lung cancer cell
1. Introduction Lung cancer, stemming from abnormal epithelial cells in the airways of the lung, becomes the second most commonly diagnosed cancer subjected to men and women in the United States. Unfortunately, lung cancer accounts for the majority of cancer-related deaths, especially in smoking prevalence region [1– 3]. The World Health Organization has divided lung cancer into two major classes: non–small cell lung cancer (NSCLC) and small cell lung cancer. With the development of chemotherapeutic agents and targeted therapies directed against the epidermal growth factor and vascular endothelial growth factor receptor pathways for NSCLC treatment, radiation therapy techniques has emerged as a prophylactically treatment of subclinical disease and provided effective relief from pain and obstructive symptoms [1,4]. Despite recent advances of radiation therapy in clinical
* Corresponding author at: Department of Radiation Oncology, Zhejiang Cancer Hospital, No. 38 Guangji Rd, Hangzhou 310022, Zhejiang Province, China. Tel.: +86 571 88128056; fax: +86 571 88128056. E-mail address: [email protected] (Z.-p. Shen). http://dx.doi.org/10.1016/j.biopha.2015.02.021 0753-3322/ß 2015 Published by Elsevier Masson SAS.
and experimental oncology, the ideal genetic marker in radiation therapy for lung cancer detection has far from being fully elucidated. Long non-coding RNAs (lncRNAs), important new members of the ncRNA family, recently have been found to participate in tumorigenesis and tumor progression [5,6]. Emerging studies reported that functional lncRNAs may be used for diagnosing cancer, determining prognosis and as a therapeutic target [7,8]. BRAF-activated lncRNA (BANCR) is a 693-bp lncRNA on chromosome 9, which is frequently over expressed in melanoma cells and plays crucial roles in melanoma cell migration [9,10]. Ming Sun reported that the expression of BANCR was significantly decreased in NSCLC tissues and could be a biomarker for poor prognosis of NSCLC. Furthermore, histone deacetylation was observed to involve in the downregulation of BANCR in NSCLC cells [11]. It has been appreciated for decades that histone deacetylases (HDACs), which catalyzed acetyl groups removal from the histone protein, associated with a variety of cellular oncogenes and tumorsuppressor genes and played an essential role in cancer development [12,13]. Based on the identification and cloning of HDACs, it was widely suggested that HDACs counteracted the activating
80
J.- Chen et al. / Biomedicine & Pharmacotherapy 71 (2015) 79–83
effects of histone acetyltransferases by recruiting to promoters to repress transcription. In addition, HDAC inhibitors, such as trichostatin A (TSA), have attracted considerable attention as anticancer agents for their ability to trigger proteins leading cell cycle arrest, differentiation, or apoptosis in neoplastically transformed cell [14,15]. Therefore, detection of HDACs variation may contribute to understand the regulation of some factors in lung cancer, such as BANCR. In this study, we investigated the expression levels of BANCR in radiation therapy for treatment lung cancer and the function and molecular mechanisms of BANCR regulation in lung cancer. This study provides us a more detailed understanding of BANCR functioning as a regulator in lung cancer pathogenesis and facilitate the development of lncRNA-directed diagnostics and therapeutics. 2. Materials and methods 2.1. Animals and tumor model The procedures of animal experiments were performed by Guide for the Care and Use of Laboratory Animals and were approved by Nanjing Thoracic Hospital (Nanjing, China). Male C57B6/L mice were purchased from Better Biotechnology Co., Ltd. (Nanjing, China). Animals were kept in a temperature-controlled room with free access to standard chow and water. Lewis mice lung cancer cell line, cultured in vitro and existed in logarithmic phase, were prepared into the cell suspension, and then inoculated into the right hind leg of C57B6/L mice. Each C57B6/L was inoculated 1 106 cells. Tumor volumes were measured with calipers and calculated with the formula L W2 0.5. 2.2. Tumor irradiation After six days of Lewis mice lung cancer cell inoculation, C57B6/L mice were exposed to a high single-dose X-rays with 12 Gy/one fraction/day, and served as radiation therapy group; equal number C57B6/L mice without received X-rays were as control group. Lewis mice lung cancer cells were divided into 3 groups, which were exposed to X-rays with 0 Gy, 1 Gy and 3 Gy. Mice were anesthetized (Nembutal) during the radiation treatment, and were immobilized in a customized harness that allowed the right hind leg to be exposed, whereas the remainder of the body was shielded by 3.5 cm of lead. Mice were irradiated in a Gammacell Cesium 137 (Atomic Energy of Canada) source operating at a rate of 125 cGy/min. 2.3. Quantitative polymerase chain reaction (PCR) Total RNA was extracted from tissues and cells using RNAiso Plus (Takara, China), and reverse transcription (RT) reactions were performed using ImProm-IITM (Promega, USA) following the manufacturer’s instructions. Quantitative PCR reactions were prepared at a final volume of 20 ml using a standard protocol and the TransStartTM SYBR Green qPCR Supermix (TransGen Biotech, China), and the reactions were performed on the StepOnePlus Real-Time PCR System (Applied Biosystems, Inc., DDCT CA, USA). The 2 method was used to determine the relative BANCR expression levels, using b-actin as the endogenous control to normalize the data.
penicillin and 100 mg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) at 37 8C/5% CO2. 2.5. Plasmid generation To detect the expression alteration of BANCR with HDAC3 over expression, the HDAC3 sequence was synthesized and sub cloned into the pCDNA3.1 (Invitrogen, Shanghai, China) vector. Over expression of HDAC3 was achieved through pCDNA-3.1-HDAC3 transfection, with an empty pCDNA3.1 vector used as a control. 2.6. Cell transfection The siRNAs si-BANCR or si-NC were transfected into Lewis lung cancer cells. Plasmid vectors pCDNA-3.1-HDAC3, pCDNA3.1 and BANCR RNAi Lentivirus expression vector (Shanghai R&S Biotechnology Co., Ltd., China; 10 MOI) for transfection were prepared using DNA Midi prep or Midi prep kits (Qiagen, Hilden, Germany), and transfected into Lewis lung cancer cells. Lewis lung cancer cells were grown on six-well plates to confluency and transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. At 48 h post-transfection, cells were harvested for qPCR or western blot analysis. Cells transfected with BANCR RNAi Lentivirus expression vector were explanted under the skin of C57BL/6 mice, and then the mice exposed to X-rays for tumor volume determination. 2.7. Chromatin immunoprecipitation Chromatin immunoprecipitations were performed as described previously [16]. Briefly, 700 mL chromatin prepared from Lewis lung cancer cells were incubated with 100 mL protein A-agarose (50% slurry in ChIP buffer) for 1 h at 4 8C. ChIP buffer contained 50 mM NaCl, 10 mM Tris–HCI pH 7.5, 10 mM Na butyrate, 1 mM EDTA, 0.1 mM PMSF and 10 mg/ml aprotinin. After separating protein A-agarose and chromatin, DNA at this point used as the ‘‘input’’ sample. Antibodies specific for acetylated histones (100 mL) [anti-acetylated-histone H3, anti-acetylated-histone H4 supplied by Biotechnology] were incubated with protein Aclarified chromatin for 2 h at 4 8C under constant agitation. Then 100 mL protein A-agarose were added to the chromatin antibody mix and incubated overnight at 4 8C. The protein A-agarose and unbound chromatin were separated using 0.45 mm Microfuge filter units. Bound chromatin was eluted by washing protein Aagarose five times with ChIP buffer. DNA from bound chromatin and unbound chromatin were purified by phenol/chloroform extraction and ethanol precipitation. DNA samples were quantified using picogreen fluorescence. 2.8. Western blotting analysis Equal amounts of protein were electrophoresed on a 12% or 15% sodium dodecyl sulfate-polyacrylamide gel and subsequently transferred to a nitrocellulose membrane (Bio-Rad, USA), which were blocked in Tris-buffered saline with 5% milk and 0.05% Tween 20 and probed with primary antibodies at 4 8C overnight according to the manufacturer’s recommendations. b-Actin was served as a control protein to quantify the expression of target protein. 2.9. Cell viability assays
2.4. Cell culture Lewis lung cancer cells, purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China), were cultured in DMEM (GIBCO-BRL) medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml
Cell viability was monitored using MTT assay (Sigma, USA). Lewis lung cancer cells transfected with si-BANCR and si-control (3000 cells/well) were grown in 96-well plates. Cell viability was assessed every 24 h following the manufacturer’s protocol. Briefly, 5 mg/mL MTT reagent was added to the medium (the final
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Fig. 1. BANCR is frequently up-regulated in lung cancer tissues exposed to radiation therapy. (A) Tumor volume of radiation therapy group and control group. (B) BANCR expression of radiation therapy group and control group. Mean SD, all results were repeated for three times; *VS control group, P < 0.05.
concentration MTT is 0.5 mg/mL) and incubated with cells for 4 h. After incubation, 150 mL dimethylsulfoxide was used to dissolved the crystal products and then the absorbance was detected at a wavelength of 570 nm by SpectraMax M5 Multimode Reader (Molecular Devices, USA). 3. Results 3.1. BANCR is frequently up-regulated in lung cancer tissues exposed to radiation therapy Given the effective roles of radiotherapy in tumor treatment, we were interested in examining its efficacy on lung cancer. Therefore, after six days of inoculating Lewis lung cancer cells into C57BL/6 mice, the tumor growth in C57BL/6 mice which exposed to radiation therapy was evaluated. As a result, tumors growth was found to be significantly repressed in radiation therapy group comparing to control group (Fig. 1A). This suggests that radiation therapy may be a functional way to inhibit tumor growth. As the report of BANCR may being a biomarker for poor prognosis of NSCLC [11], we investigated the levels of BANCR using qPCR in C57BL/6 mice, and found that BANCR expression was significantly increased in radiation therapy group compared with control group (Fig. 1B).
increasing (Fig. 3A). It has been reported that histone acetylation is a key factor in controlling BANCR expression. Thus we examined the levels of acetyl-histones. Additionally, it has been well known that all core histones are acetylated in vivo, while histones H3 and H4 modifications are much more extensively characterized than those of H2A and H2B [17]. Thus, chromatin immunoprecipitation was performed to examined the levels of histone H3 and H4 acetylation in BANCR promoter region. The result showed that they were both increased and positively correlated to ray dosage (Fig. 3B). Following, western blot was performed to determine HDAC3 and HDAC1 protein levels. Fig. 3C showed that HDAC3 protein levels was decreased with the increasing ray dosage, while HDAC1 protein levels remained constant. 3.4. Over expression HDAC3 reverses the effect of rays on BANCR expression As has been known that HDAC3 involved in the regulation of BANCR expression, we further explored the effect of over expression HDAC3 on BANCR expression. Fig. 4 showed that the expression of BANCR was significantly increased in Lewis lung cancer cell received 3 Gy rays, while over expression HDAC3 reversed this effect and significantly decreased the expression of BANCR.
3.2. Knockdown of BANCR promotes tumor growth in lung cancer tissues exposed to radiation therapy In order to further study the roles of BANCR on tumors growth, we examined the tumors volume in C57BL/6 mice, which was transplanted Lewis lung cancer cell transfected si-BANCR under the skin. Six days later, we observed that the emerging tumor was insensitive to X-rays, and tumors growth was significantly exceeded the control group (Fig. 2). Taken together, these data indicated that BANCR played important roles in tumor development. 3.3. HDAC involves in the regulation of BANCR expression in Lewis lung cancer cells exposed to X-rays To investigate the influence of X-rays on BANCR expression in Lewis lung cancer cell, it was received different X-ray dosage of 0.1 Gy and 3 Gy for 24 h. Then, qPCR showed that BANCR expression was significantly increased with the ray dosage
Fig. 2. Tumor volume of C57BL/6 mice transplanted Lewis lung cancer cell, which was transfected si-BANCR and NC, respectively. Mean SD, all results were repeated for three times; *VS control group, P < 0.05.
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Fig. 3. HDAC involves in the regulation of BANCR expression in Lewis lung cancer cells. Lewis lung cancer cell was received ray dosage with 0.1 Gy and 3 Gy. (A) BANCR expression of each group. (B) The levels of histone H3 and H4 acetylation of each group. (C) The HDAC3 and HDAC1 protein levels of each group. Mean SD, all results were repeated for three times; *VS control group, P < 0.05.
Lung cancer is one of the most mortal malignant tumors, and is becoming one of most lethal threat to human health and life [18]. Nowadays several urgent or emergent symptoms due to metastatic lung cancer are often effectively treated with radiation therapy. Radiation therapy is frequently used as an adjunct to surgery or an alternative for the patient when considered
inoperable for medical or technical reasons [19,20]. Therefore, identification of gene regulation associated in radiation therapy for lung cancer treatment and investigation of their clinical significance and functions may provide an insight into the well-known oncogenic and tumor suppressor network. It has been reported that lncRNA dysregulation resulted in a range of biological functions and provided a cellular growth advantage for progressive and uncontrolled tumor growth [21,22]. Therefore, effective control cell proliferation is pivotal to the prevention of oncogenesis and successful cancer therapy. Recently, Ming Sun et al. have suggested that BANCR may actively functions as a regulator of epithelial-mesenchymal transition during NSCLC metastasis, which indicated that BANCR could be a biomarker for poor prognosis of NSCLC [11]. However, the roles of BANCR in radiation therapy for lung cancer treatment and the carcinogenesis of LC are far from being fully illuminated. In this study, we found that radiation therapy was effective against repressing tumor growth in C57BL/6 mice transfected
Fig. 4. HDAC3 reverses the effect of X-rays on BANCR expression. Mean SD, all results were repeated for three times; *VS control group, P < 0.05.
Fig. 5. The effect of rays on Lewis lung cancer cell viability. Mean SD, all results were repeated for three times; *VS control group, P < 0.05.
3.5. The effect of X-rays on Lewis lung cancer cells viability Following Lewis lung cancer cells subjected to 3 Gy X-rays for 72 h, cell viability was examined using MTT assay. As was showed in Fig. 5, we found that cell viability was significantly decreased after exposed to 3 Gy X-rays (Fig. 5). In addition, when Lewis lung cancer cell were transfected si-BANCR, the cell viability was reversed and significantly increased compared to control group (Fig. 5). 4. Discussion
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Lewis lung cancer cells, which suggested that radiation therapy indeed inhibited tumor growth impactfully. Additionally, we also found that the expression of BANCR was significantly upregulated in lung cancer tissues which were exposed to radiation therapy. Furthermore, in vivo examination of the potential role of BANCR in C57BL/6 mice transfected Lewis lung cancer cells demonstrated that the knockdown of BANCR in Lewis lung cancer cells was associated with the promotion of tumor growth in spite of radiation therapy. This observation was consistent with previous studies regarding the expression of BANCR in NSCLC tissues [11]. Moreover, cell viability of Lewis lung cancer cells also verified this result in vitro, as MTT assays showed that Lewis lung cancer cells been exposed to X-rays possessed significantly lower cell viability, while knockdown of BANCR in Lewis lung cancer cells reversed this effect. However, Yong Wang et al. reported that BANCR was highly expressed in papillary thyroid carcinoma, as well as in colorectal cancer reported by Qinhao Guo et al. [23,24]. These observations suggested that the function of BANCR could be tissue-specific. Taken together, these findings suggest that BANCR plays a direct role in the course of radiation therapy for treatment lung cancer. Fu Yang et al. determined that a hypoxic microenvironment suppressed lncRNA-LET through HDAC3 activation, which resulted in reduced histone H3 and H4 acetylation levels in the lncRNA-LET promoter region [25]. Similarly, our study showed that with the increasing expression of BANCR in Lewis lung cancer cells exposed to X-rays, histone H3 and H4 acetylation was also increased in BANCR promoter region. Western blot further suggested that HDAC3 protein levels were decreased, while HDAC1 protein levels was no change. Accordingly, we addressed whether the upregulation of BANCR under radiation therapy was mediated by HDAC3. Over expression of HDAC3 showed that the expression of BANCR was significantly decreased in Lewis lung cancer cells exposed to X-rays. These results support that the upregulation of BANCR expression under radiation therapy is mediated by HDAC3. In summary, the expression of BANCR was increased in LC tissues exposed to radiation, suggesting that BANCR may be a positive prognostic factor for LC. This study showed that radiation may regulate the proliferation of LC cells partly through upregulation of BANCR, and HDAC3 protein was involved in the regulation. These findings further the understanding on the mechanisms of treating LC by radiation, and facilitate the development of BANCR-directed diagnostics and therapeutics against this deadly disease.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
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References [1] Haas ML. Advances in radiation therapy for lung cancer. Semin Oncol Nurs 2008;24:34–40. [2] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin 2013;63:11–30. [3] Salim EI, Jazieh AR, Moore MA. Lung cancer incidence in the Arab league countries: risk factors and control. Asian Pac J Cancer Prev 2011;12:17–34. [4] Shibuya K, Hiraoka M. Radiation therapy for lung cancer. Gan Kagaku Ryoho/ Cancer Chemother 2007;34:544–9. [5] Nakagawa T, Endo H, Yokoyama M, Abe J, Tamai K, Tanaka N, et al. Large noncoding RNA HOTAIR enhances aggressive biological behavior and is associated with short disease-free survival in human non-small cell lung cancer. Biochem Biophys Res Commun 2013;436:319–24. [6] Mercer TR, Dinger ME, Mattick JS. Long non-coding RNAs: insights into functions. Nat Rev Genet 2009;10:155–9. [7] Qi P, Du X. The long non-coding RNAs, a new cancer diagnostic and therapeutic gold mine. Mod Pathol 2012;26:155–65. [8] Spizzo R, Almeida MI, Colombatti A, Calin GA. Long non-coding RNAs and cancer: a new frontier of translational research. Oncogene 2012;31:4577–87. [9] Flockhart RJ, Webster DE, Qu K, Mascarenhas N, Kovalski J, Kretz M, et al. BRAFV600E remodels the melanocyte transcriptome and induces BANCR to regulate melanoma cell migration. Genome Res 2012;22:1006–14. [10] McCarthy N. Epigenetics: going places with BANCR. Nat Rev Cancer 2012;12:451. [11] Sun M, Liu X-H, Wang K-M, Nie F-Q, Kong R, Yang J-S, et al. Downregulation of BRAF activated non-coding RNA is associated with poor prognosis for nonsmall cell lung cancer and promotes metastasis by affecting epithelial-mesenchymal transition. Mol Cancer 2014;13:68–80. [12] De Ruijter A, Van Gennip A, Caron H, Kemp S, van Kuilenburg A. Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochem J 2003;370:737–49. [13] Glozak M, Seto E. Histone deacetylases and cancer. Oncogene 2007;26:5420–32. [14] Myzak MC, Karplus PA, Chung F-L, Dashwood RH. A novel mechanism of chemoprotection by sulforaphane inhibition of histone deacetylase. Cancer Res 2004;64:5767–74. [15] Shi Q-Q, Zuo G-W, Feng Z-Q, Zhao L-C, Luo L, You Z, et al. Effect of trichostatin A on anti HepG2 liver carcinoma cells: inhibition of HDAC activity and activation of Wnt/b-catenin signaling. Asian Pac J Cancer Prev 2014;15:7849–55. [16] Hebbes T, Clayton A, Thorne A, Crane-Robinson C. Core histone hyperacetylation co-maps with generalized DNase I sensitivity in the chicken beta-globin chromosomal domain. EMBO J 1994;13:1823–30. [17] Wade PA. Transcriptional control at regulatory checkpoints by histone deacetylases: molecular connections between cancer and chromatin. Hum Mol Genet 2001;10:693–8. [18] Zheng D-J, Yu G-H, Gao J-F, Gu J-D. Concomitant EGFR inhibitors combined with radiation for treatment of non-small cell lung carcinoma. Asian Pac J Cancer Prev 2013;14:4485–94. [19] Van Houtte P. Postoperative radiotherapy for lung cancer. Lung Cancer 1991;7:57–64. [20] Graham MV, Purdy JA, Emami B, Matthews JW, Harms WB. Preliminary results of a prospective trial using three dimensional radiotherapy for lung cancer. Int J Radiat Oncol Biol Phys 1995;33:993–1000. [21] Gupta RA, Shah N, Wang KC, Kim J, Horlings HM, Wong DJ, et al. Long noncoding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature 2010;464:1071–6. [22] Kotake Y, Nakagawa T, Kitagawa K, Suzuki S, Liu N, Kitagawa M, et al. Long non-coding RNA ANRIL is required for the PRC2 recruitment to and silencing of p15INK4B tumor suppressor gene. Oncogene 2010;30:1956–62. [23] Wang Y, Guo Q, Zhao Y, Chen J, Wang S, Hu J, et al. BRAF-activated long noncoding RNA contributes to cell proliferation and activates autophagy in papillary thyroid carcinoma. Oncol Lett 2014;8:1947–52. [24] Guo Q, Zhao Y, Chen J, Hu J, Wang S, Zhang D, et al. BRAF-activated long noncoding RNA contributes to colorectal cancer migration by inducing epithelialmesenchymal transition. Oncol Lett 2014;8:869–75. [25] Yang F, Huo X-S, Yuan S-X, Zhang L, Zhou W-P, Wang F, et al. Repression of the long noncoding RNA-LET by histone deacetylase 3 contributes to hypoxiamediated metastasis. Mol Cell 2013;49:1083–96.
