-
肺癌是全球范围内影响人类健康的第一大恶性肿瘤[1]。肺癌的组织学亚型主要包括非小细胞肺癌(non-small cell lung cacer, NSCLC)和小细胞肺癌,分别占肺癌的85%和15%,其中NSCLC包括鳞癌、腺癌和大细胞癌[2]。放疗是利用一种或多种高能量电离辐射对恶性肿瘤患者进行的治疗,通过能量的释放阻止细胞生长和破坏癌细胞的正常功能,达到使肿瘤缩小甚至消失的目的[3-4]。放疗是肺癌综合治疗中的一个主要手段,在国际上已被广泛研究和应用[4]。但是,由于肺癌细胞的放射抵抗作用,需要提高照射剂量,这将给患者带来严重的不良反应和并发症,严重影响肺癌患者的放疗效果和生存质量。因此,有效地提高肺癌细胞的放射敏感性,从而提升患者的放疗效果和治愈率是近年来肺癌治疗研究的热点之一。
姜黄素是从姜科植物姜黄(Curcuma longa L.)中提取的一种相对分子质量较小的多酚类物质,通常认为它是姜黄中的最有效成分。研究结果证明,姜黄素具有抗氧化、抗炎、抗癌、清除自由基、抗微生物的作用,对心血管和消化系统等也具有药理作用[5]。研究结果显示,姜黄素单独使用或与其他药剂联合使用可对多种肿瘤发挥预防或治疗作用,包括结肠癌、胰腺癌、乳腺癌、前列腺癌、多发性骨髓瘤和口腔癌等[6]。在肺癌中,姜黄素可通过抑制Wnt/β-catenin、Sonic Hedgehog通路和靶向表皮生长因子受体等对肺癌的发展起到阻抑作用[7-8]。然而,关于姜黄素通过调控长链非编码RNA(long non-coding RNA,lncRNA)提高NSCLC放射敏感性的研究尚未见报道。
lncRNA是一类长度为200~100 000个核苷酸、缺少特异且完整的开放阅读框、无蛋白质编码功能的RNA分子[9-10]。同源异型盒基因转录的反义基因间RNA(homeotic complex gene anti-sense intergenic RNA,HOTAIR)是一个长度为2158个核苷酸的反义非编码RNA,该lncRNA由同源异型盒基因C位点转录,作为“脚手架”分子可以通过其5′端募集染色质重构复合体2,并将其定位到同源异型盒基因D位点,从而抑制该位点的转录[11-12]。HOTAIR在乳腺癌、结肠癌、肝癌、胃癌和肺癌等多种肿瘤中发挥调控功能,对肿瘤的发生发展及患者的预后和生存质量等具有重要影响[13-14]。然而,HOTAIR在介导姜黄素调控肺癌放射敏感性中的作用尚未见报道。
本研究选用NSCLC细胞A549和H460为模型,研究姜黄素对NSCLC放射敏感性的影响,并探索lncRNA在其中的调节机制,为姜黄素作为NSCLC放射增敏剂的应用并阐明其中的分子机制提供新的实验依据,并为探索肺癌的放射增敏新途径提供理论基础。
姜黄素对非小细胞肺癌细胞A549和H460放射敏感性的影响
Effects of curcumin on the radiosensitivity of non-small cell lung cancer A549 and H460 cells
-
摘要:
目的 探讨姜黄素对非小细胞肺癌(NSCLC)细胞A549和H460放射敏感性的影响,并探索长链非编码RNA(lncRNA)同源异型盒基因转录的反义基因间RNA(HOTAIR)在其中的调节机制。 方法 将培养的NSCLC细胞A549和H460根据处理方法的不同,采用4种方式进行分组。(1)分别将NSCLC细胞A549和H460分为6组:0、15、30、60、120和240 μmol/L姜黄素组,采用细胞计数试剂盒8(CCK-8)实验检测各组细胞的增殖活力;(2)分别将NSCLC细胞A549和H460分为8组:0、2、4、6 Gy γ射线照射组及其与30 μmol/L姜黄素联合组,采用CCK-8实验和平板克隆形成实验检测各组细胞的增殖活力;(3)将NSCLC细胞A549分为4组:空白对照组、30 μmol/L姜黄素组、4 Gy γ射线照射组和30 μmol/L姜黄素+4 Gy γ射线照射联合组,采用实时定量聚合酶链反应(qRT-PCR)实验检测各组细胞中5种促癌lncRNA和5种抑癌lncRNA的表达情况;(4)将NSCLC细胞A549分为6组:空白对照组、30 μmol/L姜黄素组、30 μmol/L姜黄素+lncRNA HOTAIR过表达组、4 Gy γ射线照射组、4 Gy γ射线照射+30 μmol/L姜黄素联合组和4 Gy γ射线照射+30 μmol/L姜黄素+lncRNA HOTAIR过表达组,采用CCK-8实验和平板克隆形成实验检测各组细胞的增殖活力。采用学生t检验进行统计学分析。 结果 (1)CCK-8实验结果显示,不同浓度的姜黄素处理可以剂量和时间依赖的方式降低NSCLC细胞A549和H460的增殖活力,且除了15 μmol/L姜黄素作用24 h之外,其余浓度和时间的姜黄素组与对照组相比,差异有统计学意义(t=3.884~5.731,P=0.000~0.043)。(2)CCK-8实验和克隆形成实验结果显示,30 μmol/L姜黄素+不同剂量的γ射线照射联合处理可使γ射线照射单独处理下的NSCLC细胞A549和H460的增殖活力和克隆形成能力进一步降低,差异有统计学意义(t=2.503~12.418,P=0.000~0.044)。(3)qRT-PCR实验结果显示,与空白对照组相比,lncRNA HOTAIR在γ射线单独处理后表达升高(t=3.317,P=0.040),而30 μmol/L姜黄素单独处理和30 μmol/L姜黄素+4 Gy γ射线联合处理均可下调A549细胞中促癌lncRNA HOTAIR的表达(t=3.205、5.916,P=0.038、0.000)。(4)对HOTAIR进行过表达处理,可消除姜黄素对NSCLC细胞A549增殖的抑制并增强其放射敏感性(t=3.584~5.802,P=0.000~0.004)。 结论 姜黄素可以通过下调促癌lncRNA HOTAIR的表达从而抑制NSCLC细胞的增殖活力,并增强其放射敏感性。 -
关键词:
- 姜黄素 /
- RNA,长链非编码 /
- 癌,非小细胞肺 /
- 辐射耐受性 /
- 同源异型盒基因转录的反义基因间RNA
Abstract:Objective To explore the effects of curcumin on the radiosensitivity of non-small cell lung cancer (NSCLC) A549 and H460 cells and determine the underlying mechanism as mediated by long non-coding RNA (lncRNA) homeotic complex gene anti-sense intergenic RNA (HOTAIR). Methods NSCLC were divided into different groups according to the experimental scheme. (1) A549 and H460 cells were divided into six groups each treated by 0, 15, 30, 60, and 240 μmol/L curcumin. Cell-counting kit 8 (CCK-8) assay was performed to measure the cell viability after each treatment. (2) A549 and H460 cells were divided into eight groups of 0, 2, 4, and 6 Gy γ-irradiation treatment groups and their corresponding combinations with 30 μmol/L curcumin. CCK-8 and colony formation assays were conducted to determine the cell proliferative abilities of each group. (3) A549 cells were divided into four groups: control, 30 μmol/L curcumin treatment, 4 Gy γ-irradiation treatment, and combinational treatment of 30 μmol/L curcumin and 4 Gy γ-irradiation groups. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed to monitor the expression levels of five oncogenic lncRNAs and five tumor-suppressive lncRNAs in A549 cells from each group. (4) A549 cells were divided into six groups: control, 30 μmol/L curcumin treatment, 30 μmol/L curcumin+overexpression of lncRNA HOTAIR, 4 Gy γ-irradiation treatment, combinational treatment of 30 μmol/L curcumin and 4 Gy γ-irradiation, and combinational treatment of 30 μmol/L curcumin and 4 Gy γ-irradiation+overexpression of lncRNA HOTAIR groups. The cell proliferative capacities after each treatment were detected by CCK-8 and colony formation assays. Statistical significance was determined by SPSS statistical software and analyzed by Student t-test. Results (1) CCK-8 assay results showed that curcumin treatment decreased the cell viability of A549 and H460 cells in a dose- and time-dependent manner, and the difference between each treatment group and the control group was statistically significant (t=3.884 –5.731, P=0.000–0.043). (2) CCK-8 and colony formation assay revealed that the combinational treatment of 30 μmol/L curcumin and different γ-irradiation doses significantly promoted the further reduction of cell proliferation and colony formation abilities compared with γ-irradiation alone (t=2.503–12.418, P=0.000–0.044). (3) qRT-PCR detection revealed that the expression level of lncRNA HOTAIR elevated after γ-irradiation treatment (t=3.317, P =0.040), but the sole treatment of 30 μmol/L curcumin and the combinational treatment of 30 μmol/L curcumin with γ-irradiation could down-regulate the expression of tumor-promoting lncRNA HOTAIR in A549 cells compared with the control group (t=3.205, 5.916, P=0.038, 0.000). (4) HOTAIR overexpression could reverse the inhibitory effects of curcumin on the cell proliferation capacity and radiation resistance of A549 cells (t=3.584–5.802, P=0.000–0.004). Conclusion Curcumin can suppress the cell proliferation capacity and enhance the radiation sensitivity of NSCLC by down-regulating lncRNA HOTAIR. -
-
[1] Smith RA, Andrews KS, Brooks D, et al. Cancer Screening in the United States, 2019: A Review of Current American Cancer Society Guidelines and Current Issues in Cancer Screening[J]. CA Cancer J Clin, 2019, 69(3): 184−210. DOI: 10.3322/caac.21557. [2] Ramalingam SS, Owonikoko TK, Khuri FR. Lung cancer: New biological insights and recent therapeutic advances[J]. CA Cancer J Clin, 2011, 61(2): 91−112. DOI: 10.3322/caac.20102. [3] Sargos P, Stoeckle E, De Figueiredo BH, et al. Radiotherapy for retroperitoneal sarcomas[J]. Cancer, 2016, 20(6/7): 677−684. DOI: 10.1016/j.canrad.2016.07.040. [4] 郎锦义, 吴大可. 我国放射治疗发展现状与展望[J]. 四川医学, 2004, 25(9): 1035−1038. DOI: 10.3969/j.issn.1004−0501.2004.09.050.
Lang JY, Wu DK. Current situation and prospect of radiotherapy in China[J]. Sichuan Med J, 2004, 25(9): 1035−1038. DOI: 10.3969/j.issn.1004−0501.2004.09.050.[5] 国大量, 朱晓薇, 张艳军. 姜黄素自微乳颗粒的研究[J]. 齐鲁药事, 2012, 31(6): 316−318. DOI: 10.3969/j.issn.1672−7738.2012.06.002.
Guo DL, Zhu XW, Zhang YJ. Study on self-microemulsifying granules of curcumin[J]. Qilu Pharma Affa, 2012, 31(6): 316−318. DOI: 10.3969/j.issn.1672−7738.2012.06.002.[6] Devassy JG, Nwachukwu ID, Jones PJH. Curcumin and cancer: barriers to obtaining a health claim[J]. Nutr Rev, 2015, 73(3): 155−165. DOI: 10.1093/nutrit/nuu064. [7] Zhu JY, Yang X, Chen Y, et al. Curcumin Suppresses Lung Cancer Stem Cells via Inhibiting Wnt/β-catenin and Sonic Hedgehog Pathways[J]. Phytother Res, 2017, 31(4): 680−688. DOI: 10.1002/ptr.5791. [8] Shafiee M, Mohamadzade E, Shahidsales S, et al. Current Status and Perspectives Regarding the Therapeutic Potential of Targeting EGFR Pathway by Curcumin in Lung Cancer[J]. Curr Pharm Des, 2017, 23(13): 2002−2008. DOI: 10.2174/1381612823666170123143648. [9] Cui M, Xiao ZL, Wang Y, et al. Long Noncoding RNA HULC Modulates Abnormal Lipid Metabolism in Hepatoma Cells Through an miR-9-Mediated RXRA Signaling Pathway[J]. Cancer Res, 2015, 75(5): 846−857. DOI: 10.1158/0008−5472.CAN−14−1192. [10] Tay Y, Rinn J, Pandolfi PP. The multilayered complexity of ceRNA crosstalk and competition[J]. Nature, 2014, 505(7483): 344−352. DOI: 10.1038/nature12986. [11] 韩聪, 胡建宏, 胡姗, 等. 长链非编码RNA研究进展[J]. 生物技术通讯, 2018, 29(1): 123−130. DOI: 10.3969/j.issn.1009−0002.2018.01.024.
Han C, Hu JH, Hu S, et al. Research progress of long non-coding RNA[J]. Lett in Biotech, 2018, 29(1): 123−130. DOI: 10.3969/j.issn.1009−0002.2018.01.024.[12] 李伟松, 肖学文, 苏惠, 等. 长链非编码RNA研究进展[J]. 赣南医学院学报, 2017, 37(3): 433−437. DOI: 10.3969/j.issn.1001−5779.2017.03.036.
Li WS, Xiao XW, Su H, et al. The research progress of lncRNA[J]. J GN Med Univ, 2017, 37(3): 433−437. DOI: 10.3969/j.issn.1001−5779.2017.03.036.[13] Qu XH, Alsager S, Zhuo Y, et al. HOX transcript antisense RNA (HOTAIR) in cancer[J]. Cancer Lett, 2019, 454: 90−97. DOI: 10.1016/j.canlet.2019.04.016. [14] Woo CJ, Kingston RE. HOTAIR Lifts Noncoding RNAs to New Levels[J]. Cell, 2007, 129(7): 1257−1259. DOI: 10.1016/j.cell.2007.06.014. [15] 孙晓辉, 孔阳阳, 路倩颖, 等. 肺癌细胞A549和H460对137Cs γ射线辐射敏感性差异的研究[J]. 国际放射医学核医学杂志, 2018, 42(4): 346−351. DOI: 10.3760/cma.j.issn.1673−4114.2018.04.011.
Sun XH, Kong YY, Lu QY, et al. Difference of radiosensitivity for 137Cs γ-radiation between A549 and H460 lung cancer cell lines[J]. Int J Radiat Med Nucl Med, 2018, 42(4): 346−351. DOI: 10.3760/cma.j.issn.1673−4114.2018.04.011.[16] 廖晓宁, 罗彪, 张倬彬. 非小细胞肺癌放射治疗研究进展[J]. 医药前沿, 2017, 7(6): 6−7. DOI: 10.3969/j.issn.2095−1752.2017.06.012.
Liao XN, Luo B, Zhang ZB. Non-small cell lung cancer radiotherapy were reviewed[J]. J Front Med, 2017, 7(6): 6−7. DOI: 10.3969/j.issn.2095−1752.2017.06.012.[17] 张海嵩, 张旭, 王玉梅. 局部晚期非小细胞肺癌同期放化疗和序贯放化疗的临床疗效比较[J]. 现代肿瘤医学, 2011, 19(4): 694−697. DOI: 10.3969/j.issn.1672−4992.2011.04.23.
Zhang HS, Zhang X, Wang YM. Study of concurrent versus sequential chemoradiotherapy with gemcitabine and cisplatin in stage Ⅲ non-small cell lung cancer[J]. J Mod Oncol, 2011, 19(4): 694−697. DOI: 10.3969/j.issn.1672−4992.2011.04.23.[18] Prensner JR, Chinnaiyan AM. The Emergence of lncRNAs in Cancer Biology[J]. Cancer Discov, 2011, 1(5): 391−407. DOI: 10.1158/2159−8290.CD−11−0209. [19] Kang J, Kim W, Kwon T, et al. Plasminogen activator inhibitor-1 enhances radioresistance and aggressiveness of non-small cell lung cancer cells[J]. Oncotarget, 2016, 7(17): 23961−23974. DOI: 10.18632/oncotarget.8208. [20] Guo XG, Xiao HQ, Guo SH, et al. Long noncoding RNA HOTAIR knockdown inhibits autophagy and epithelial-mesenchymal transition through the Wnt signaling pathway in radioresistant human cervical cancer HeLa cells[J]. J Cell Physiol, 2019, 234(4): 3478−3489. DOI: 10.1002/jcp.26828. [21] Hu XG, Ding D, Zhang JY, et al. Knockdown of lncRNA HOTAIR sensitizes breast cancer cells to ionizing radiation through activating miR-218[J]. Biosci Rep, 2019, 39(4): BSR20181038. DOI: 10.1042/BSR20181038. [22] Yang XD, Xu HT, Xu XH, et al. Knockdown of long non-coding RNA HOTAIR inhibits proliferation and invasiveness and improves radiosensitivity in colorectal cancer[J]. Oncol Rep, 2016, 35(1): 479−487. DOI: 10.3892/or.2015.4397. [23] Da CL, Wu L, Liu YT, et al. Effects of irradiation on radioresistance, HOTAIR and epithelial-mesenchymal transition/cancer stem cell marker expression in esophageal squamous cell carcinoma[J]. Oncol Lett, 2017, 13(4): 2751−2757. DOI: 10.3892/ol.2017.5774. [24] Jiao DM, Yan L, Wang LS, et al. Exploration of inhibitory mechanisms of curcumin in lung cancer metastasis using a miRNA- transcription factor-target gene network[J/OL]. PLoS One, 2017, 12(2): e0172470[2019-12-03]. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0172470. DOI: 10.1371/journal.pone.0172470. [25] Lelli D, Pedone C, Majeed M, et al. Curcumin and Lung Cancer: the Role of microRNAs[J]. Curr Pharm Des, 2017, 23(23): 3440−3444. DOI: 10.2174/1381612823666170109144818. [26] Liu WL, Chang JM, Chong IW, et al. Curcumin Inhibits LIN-28A through the Activation of miRNA-98 in the Lung Cancer Cell Line A549[J/OL]. Molecules, 2017, 22(6): 929[2019-12-03]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6152786. DOI: 10.3390/molecules22060929. [27] Marley JE, Curram JB. General practice data derived tolerability assessment of antihypertensive drugs[J]. J Int Med Res, 1989, 17(5): 473−478. DOI: 10.1177/030006058901700510. [28] Ye MX, Zhang J, Zhang J, et al. Curcumin promotes apoptosis by activating the p53-miR-192-5p/215-XIAP pathway in non-small cell lung cancer[J]. Cancer Lett, 2015, 357(1): 196−205. DOI: 10.1016/j.canlet.2014.11.028. [29] Zhang J, Du YP, Wu CG, et al. Curcumin promotes apoptosis in human lung adenocarcinoma cells through miR-186* signaling pathway[J]. Oncol Rep, 2010, 24(5): 1217−1223. DOI: 10.3892/or_00000975. [30] Zhang W, Bai W, Zhang W. MiR-21 suppresses the anticancer activities of curcumin by targeting PTEN gene in human non-small cell lung cancer A549 cells[J]. Clin Transl Oncol, 2014, 16(8): 708−713. DOI: 10.1007/s12094−013−1135−9. [31] Wang AL, Wang JX, Zhang SM, et al. Curcumin inhibits the development of non-small cell lung cancer by inhibiting autophagy and apoptosis[J]. Exp Ther Med, 2017, 14(5): 5075−5080. DOI: 10.3892/etm.2017.5172. [32] Xu XD, Chen D, Ye B, et al. Curcumin induces the apoptosis of non-small cell lung cancer cells through a calcium signaling pathway[J]. Int J Mol Med, 2015, 35(6): 1610−1616. DOI: 10.3892/ijmm.2015.2167. [33] Ooi L, Wood IC. Chromatin crosstalk in development and disease: lessons from REST[J]. Nat Rev Genet, 2007, 8(7): 544−554. DOI: 10.1038/nrg2100. [34] Simon JA, Kingston RE. Mechanisms of polycomb gene silencing: knowns and unknowns[J]. Nat Rev Mol Cell Biol, 2009, 10(10): 697−708. DOI: 10.1038/nrm2763. [35] 宿希强. 药品安全成为质量“新焦点”[J]. 中国质量万里行, 2013, (5): 10−11. DOI: 10.3969/j.issn.1005−149X.2013.05.007.
Su XQ. Drug safety becomes the "new focus" of quality[J]. Chin Qual Lon Marc, 2013, (5): 10−11. DOI: 10.3969/j.issn.1005−149X.2013.05.007.[36] 李敏, 张楠, 樊赛军. 姜黄素具有肿瘤放射增敏和辐射损伤保护双向作用的研究进展[J]. 中华放射医学与防护杂志, 2013, 33(3): 326−330. DOI: 10.3760/cma.j.issn.0254−5098.2013.03.027.
Li M, Zhang N, Fan SJ. Research progress of Curcumin in tumor radiosensitization and radiation injury protection[J]. Chin J Radiol Med Prot, 2013, 33(3): 326−330. DOI: 10.3760/cma.j.issn.0254−5098.2013.03.027.[37] Lee JC, Kinniry PA, Arguiri E, et al. Dietary Curcumin Increases Antioxidant Defenses in Lung, Ameliorates Radiation-Induced Pulmonary Fibrosis, and Improves Survival in Mice[J]. Radiat Res, 2010, 173(5): 590−601. DOI: 10.1667/RR1522.1. [38] Dhandapani KM, Mahesh VB, Brann DW. Curcumin suppresses growth and chemoresistance of human glioblastoma cells via AP-1 and NFκB transcription factors[J]. J Neurochem, 2007, 102(2): 522−538. DOI: 10.1111/j.1471−4159.2007.04633.x.