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肺癌是全球范围内最常见的恶性肿瘤之一，严重影响人类健康[1]。非小细胞肺癌（non-small cell lung cancer，NSCLC）占肺癌的85%[2]。放疗是其主要的非手术治疗方法，但放射抵抗往往会导致局部肿瘤复发和预后不良[3-4]，目前尚无逆转NSCLC放射抵抗的有效靶点，亟需探究。
环状RNA（circular RNA，circRNA）是一类在真核生物中广泛表达的环状非编码RNA，其由mRNA前体通过反向剪接产生。不同于其他线性非编码RNA，circRNA不具备5′端帽和3′端尾结构，对核酸外切酶具有抵抗性，故circRNA极具稳定性[5-6]。2011年，Salmena等[7]提出，circRNA具有微小RNA（microRNA，miRNA）反应元件，可作为竞争性内源性RNA海绵吸附并抑制miRNA，从而改变细胞的基因表达。circRNA还可以通过结合RNA结合蛋白、调节基因转录等方式来发挥作用，故其可参与基因转录、转录后调控、细胞内RNA调控网络及蛋白质翻译等生理过程，进而影响细胞周期进程、细胞衰老和凋亡等多种生物学过程[8]。
研究结果证实，circRNA在肺癌、胃癌、膀胱癌和乳腺癌等多种肿瘤中表达异常，其参与调控肿瘤细胞增殖、侵袭和转移等多种生物学过程，因此circRNA被认为是一种潜在的肿瘤生物标志物及治疗靶点[9]。Wang等[10]发现，circRNA_0008305可诱导NSCLC细胞发生上皮间充质转化，进而促进肺癌转移。circRNA_0000199通过抑制miR-198上调磷脂酰肌醇-3激酶调节亚基1，以此增强胃癌细胞对铂类药物的耐药性[11]。另有研究结果证实，circRNA可能参与肿瘤的放射抵抗过程[12]。Ma等[13]发现，circRNA_0122683在食管癌组织中高表达，其通过调控miR-186-5p/腺苷二磷酸核糖聚合酶9（PARP9）轴调控细胞恶性程度并促进放射抵抗。circRNA_100367通过吸附miR-217增强食管癌细胞的放射抗性[14]。目前，针对circRNA与NSCLC细胞放射敏感性关系的研究尚不多见，circRNA_0128846对NSCLC细胞放射敏感性的影响不清楚。本研究探讨circRNA_0128846对人NSCLC细胞放射敏感性的调控作用及分子机制，以期为解决临床NSCLC放射抵抗问题提供潜在的治疗靶点。
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人支气管上皮细胞Beas-2B、人NSCLC细胞A549、H460、H1299、H1975和人胚肾细胞293T均购自中国科学院上海生命科学研究院细胞资源中心，培养代次为10。DMEM培养基、DMEM/F12培养基、DMEM高糖培养基、胎牛血清、0.25%胰蛋白酶（不含EDTA）、0.25%胰蛋白酶、表皮细胞生长因子、β-成纤维细胞生长因子、2% B27添加物均购自美国Gibco公司；青霉素和链霉素均购自浙江吉诺生物医药技术有限公司；Lipofectamine3000、Trizol试剂均购自美国Invitrogen公司；SYBR Green PCR试剂盒购自日本Takara公司；RIPA裂解液购自北京普利莱基因技术有限公司；CCK-8试剂盒、RNA反转录试剂盒均购自上海碧云天生物技术有限公司；miR-1183抑制剂、miR-1183模拟物及阴性对照均购自苏州吉玛基因股份有限公司。NanoDrop2000分光光度计、酶联免疫分析仪均购自美国Thermo公司；X-RAD 320直线加速器购自美国North Branford公司。质粒（LUC）购自上海和元生物技术股份有限公司；引物购自广州生工生物工程股份有限公司。双荧光素酶报告基因分析系统购自美国Promega公司。
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人支气管上皮细胞Beas-2B和人NSCLC细胞A549、H460、H1299、H1975分别培养于含10%胎牛血清、100 U/ml青霉素、100 g/ml链霉素的DMEM培养基中；人胚肾细胞293T培养于含10%胎牛血清的DMEM高糖培养基中，培养条件均为37℃、5% CO2。放射抵抗细胞系是本实验室前期经分割辐射（2 Gy×30次）诱导而成的，已建立具有稳定放射抵抗能力的人NSCLC细胞A549（命名为A549R），并每2周给予低剂量（2 Gy）照射以维持细胞的辐射抗性，照射条件为6 MV X射线，剂量率为4 Gy/min。
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在GEO数据库（
https://www.ncbi.nlm.nih.gov/geo ）中下载微阵列数据集GSE101684，筛选出在4对癌组织与癌旁正常组织中差异表达最显著的10种circRNA[筛选标准为log2FC（折叠变化）≥1.5，且P<0.05]，选择在亲本细胞和放射抵抗细胞中表达差异最显著的circRNA作为目标circRNA。利用人类环状RNA数据库（http://www.circbank.cn ）和circinteractome数据库（https://circinteractome.nia.nih.gov ）预测目标circRNA下游的miRNA，筛选出沉默目标circRNA后，表达水平上升最显著的miRNA作为目标miRNA。 -
将人NSCLC细胞A549、A549R接种于6孔培养板中（1×105个/孔），培养至50%~70%融合度后转染，转染操作按照Lipofectamine3000说明书进行。对A549R细胞进行转染，目标circRNA沉默载体为pLKD-CMV-G&PR-U6-shRNA，干扰序列为5′-TCCAAGCTGGCCAGCAGCCAGC-3′；对A549细胞进行转染，目标circRNA过表达载体为pIRES-hrGFP-1a，目标circRNA过表达序列见“
http://www.circbank.cn/search.html?selectValue=has_circ_0128846 ”。使用miRNA抑制剂转染A549细胞以实现miRNA沉默；使用miRNA模拟物转染A549R细胞以实现miRNA过表达，转染对照组均为空载体。 -
将转染后24 h的人NSCLC细胞A549、A549R各分为3组进行照射，照射剂量分别为0、4、8 Gy，照射后将细胞接种于6孔低吸附培养板中（1×104个/孔），使用DMEM/F12无血清培养基培养，培养基中加入50 ng/ml表皮细胞生长因子、10 ng/ml β-成纤维细胞生长因子和2% B27添加物。培养7 d后在显微镜下拍照分析，以细胞克隆最大径>20 μm为判定标准，统计细胞克隆形成率（细胞克隆形成率=细胞克隆数/细胞总数×100%）[15]。
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使用Trizol试剂从细胞中提取总RNA，使用NanoDrop2000分光光度计检测纯化的总RNA的质量和浓度。按照RNA反转录试剂盒说明书提取RNA，并进行反转录合成互补DNA（cDNA）。采用SYBR Green PCR试剂盒检测细胞中在人NSCLC癌组织与配对的癌旁正常组织中差异表达的10种circRNA以及可能与目标circRNA结合的miRNA的表达水平，操作按照说明书进行。以甘油醛-3-磷酸脱氢酶（GADPH）作为目标circRNA的参考基因，U6作为目标miRNA的参考miRNA。PCR反应条件：94℃预变性1 min，94℃变性10 s，60℃退火30 s，72℃延伸30 s，40个循环。引物序列如表1所示。
引物名称 引物序列 GADPH F: 5′-CACCCACTCCTCCACCTTTG-3′ R: 5′-CCACCACCCTGTTGCTGTAG-3′ circRNA_0128846 F: 5′-GACCTCTGTCAGCGAGTTCC-3′ R: 5′-GCTACTGGAGCCTGATGGAC-3′ circRNA_0116061 F: 5′-GAGATGGAACAGCACGGATT-3′ R: 5′-GGCAGATTTGCAAAAGATGA-3′ circRNA_0116961 F: 5′-TCGAATTGTCAAGATGTGGAA-3′ R: 5′-AAAAACCTGATTGCGAATGAA-3′ circRNA_0106711 F: 5′-TAATCCCAGGAGTCCAGCTC-3′ R: 5′-ACGAGTGCTGCTTTGGTCTT-3′ circRNA_0106714 F: 5′-GGACAGCAGGCATTACCAAT-3′ R: 5′-CCAATTTCCAAAGCCACAGT-3′ circRNA_0094088 F: 5′-CCTTTGAAGATGGCCTCTGA-3′ R: 5′-CACAGACGAGTGGGTTCACA-3′ circRNA_0096041 F: 5′-TTGGTCCAATCTGCTTCACC-3′ R: 5′-GTTCACATCGGGATCCTTGT-3′ circRNA_0046264 F: 5′-TTGGTCCAATCTGCTTCACC-3′ R: 5′-GTTCACATCGGGATCCTTGT-3′ circRNA_0096042 F: 5′-CCCGCTGGTGAAGTCTCTAT-3′ R: 5′-TGGCTACTGAACCAGTCACG-3′ circRNA_0096049 F: 5′-GGATGTTTCTCTGCCCAAAA-3′ R: 5′-TTTGGGTCCCTTGAATTTACC-3′ miR-646 F: 5′-ACACTCCAGCTGGGAAGCAGCTGCCTC-3′ R: 5′-CTCAACTGTGCTGCATTAGTTAGCTCAGA-3′ miR-885-3p F: 5′-GAGCACGAGGCAGTAGGCAAAGTGT-3′ R: 5′-GAGGCAGCGGGGTGTAGTGGATAGA-3′ miR-578 F: 5′-GTGCAGGGTGTTAGGA-3′ R: 5′-GAAGAACACGTCTGGT-3′ miR-647 F: 5′-GTGTTGGCCTGTGGCTG-3′ R: 5′-CTGACCCTCCCTCCTGC-3′ miR-892a F: 5′-GCCGAGCACTGTGTCCTTT-3′ R: 5′-CAGTGCAGGGTCCGAGGTAT-3′ miR-1183 F: 5′-ACTGACCACTGTAGGTGATGG-3′ R: 5′-GCGAGCACAGAATTAATACGACTCACTATAGG-3′ miR-1827 F: 5′-GGTGAGGCAGTAGATTGAATCTC-3′ R: 5′-CTCAACTGGTGTCGTGGAGTC-3′ miR-532-3p F: 5′-GCCCATGCCTTGAGTGTAG-3′ R: 5′-GTGCGTGTCGTGGAGTCG-3′ miR-626 F: 5′-CTTATTTGAGAGCTGAGGA-3′ R: 5′-CGGACTAGTACATCATCTATACTG-3′ U6 F: 5′-GCTTCGGCAGCACATATACTAAAAT-3′ R: 5′-CGCTTCACGAATTTGCGTGTCAT-3′ 注：GADPH为甘油醛-3-磷酸脱氢酶；circRNA为环状RNA；miR为微小RNA 表 1 荧光实时定量聚合酶链反应的引物序列
Table 1. Primers sequences used in fluorescence real-time quantitative polymerase chain reaction
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质粒在荧光素酶基因启动子（LUC-目标circRNA）下游插入序列5′-CUUACAGUAC-3′（目标circRNA中miRNA的潜在结合序列）。将293T细胞接种于24孔培养板中（5×104个/孔），将LUC-目标circRNA与miRNA模拟物或miRNA阴性对照共转染293T细胞，48 h后裂解细胞，使用双荧光素酶报告基因分析系统测定荧光素酶活性。
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应用SPSS 23.0软件、GraphPad Prism 7.0软件进行统计学分析。符合正态分布的计量资料以
$\bar x \pm s $ 表示，2组间数据的比较采用独立样本t检验（方差齐）。P<0.05为差异有统计学意义。 -
由图1A可见，经GEO数据库微阵列数据集GSE101684（包括4对癌组织与配对的癌旁正常组织）筛选，在人NSCLC癌组织中表达上调的前10种circRNA分别为circRNA_0116061、circRNA_0116961、circRNA_0106711、circRNA_0106714、circRNA_0128846、circRNA_0094088、circRNA_0046264、circRNA_0096041、circRNA_0096042和circRNA_0096049。qRT-PCR结果显示，上述10种circRNA中，circRNA_0116961、circRNA_0106714、circRNA_0128846和circRNA_0046264在A549R细胞中的相对表达水平均高于A549细胞（均P<0.05），其中circRNA_0128846表达差异最显著（图1B）。故本研究选择circRNA_0128846作为目标circRNA。qRT-PCR检测circRNA_0128846在正常人支气管上皮细胞Beas-2B以及人NSCLC细胞A549、H460、H1299、H1975中的相对表达水平，结果显示，circRNA_0128846在A549、H460、H1299、H1975细胞中的相对表达水平均高于Beas-2B细胞（均P<0.01，图1C）。以上4株人NSCLC细胞的克隆形成实验结果显示，经0、4、8 Gy的X射线照射后，随着circRNA_0128846表达水平的升高，4株人NSCLC细胞的克隆形成能力逐渐增强（图2），这提示circRNA_0128846的高表达可能与人NSCLC细胞的放射抵抗呈正相关。
图 1 筛选目标circRNA并检测其在正常人支气管上皮细胞Beas-2B及人非小细胞肺癌细胞A549、H460、H1299、H1975中的表达情况
Figure 1. Screening target circRNA and detecting its expression in normal human bronchial epithelial cell Beas-2B and human non-small cell lung cancer cell A549, H460, H1299, H1975
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细胞克隆形成实验结果显示，在4、8 Gy X射线的照射下，A549R细胞的辐射抗性显著高于亲本A549细胞（均P<0.05，图3）。qRT-PCR检测结果表明，在A549细胞中成功过表达了circRNA_0128846（图4A），与对照组相比，过表达circRNA_0128846显著增强了A549细胞的克隆形成能力（0.22%对0.45%，图4B）；在A549R细胞中成功沉默了circRNA_0128846（图5A），与对照组相比，沉默circRNA_0128846显著降低了A549R细胞的克隆形成能力（0.23%对0.10%，图5B），该结果提示circRNA_0128846能够促进人NSCLC细胞的放射抵抗。
图 2 不同剂量X射线照射后正常人支气管上皮细胞Beas-2B和人非小细胞肺癌细胞A549、H460、H1299、H1975的克隆形成情况（×200）
Figure 2. Clone formation in normal human bronchial epithelial cells Beas-2B and human non-small cell lung cancer cell A549, H460, H1299, H1975 after irradiation with different doses of X-ray radiation (×200)
图 3 不同剂量X射线照射后人非小细胞肺癌细胞A549及其放射抵抗细胞（A549R）的克隆形成情况（×200）
Figure 3. Clone formation in human non-small cell lung cancer cell A549 and its radioresistant cell A549R after irradiation with different doses of X-ray radiation (×200)
图 4 circRNA_0128846过表达载体转染人非小细胞肺癌细胞A549 48 h后的相对表达水平（A）及8 Gy X 射线照射后的克隆形成能力（B，×200）
Figure 4. Relative expression level of circRNA_0128846 overexpression vector transfected human non-small cell lung cancer cell A549 for 48 h (A) and clone formation ability after 8 Gy X-ray irradiation (B, ×200)
图 5 circRNA_0128846沉默载体转染人非小细胞肺癌细胞A549的放射抵抗细胞（A549R） 48 h后的相对表达水平（A）及8 Gy X射线照射后的克隆形成能力（B，×200）
Figure 5. Relative expression level of circRNA_0128846 silencing vector transfected the radioresistant cell (A549R) of human non-small cell lung cancer cell A549 for 48 h (A) and clone formation ability after 8 Gy X-ray irradiation (B, ×200)
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在circinteractome数据库和人类环状RNA数据库中取交集得到9种可能与circRNA_0128846结合的潜在miRNA。qRT-PCR检测A549R细胞中circRNA_0128846沉默后各miRNA的表达，结果显示，沉默circRNA_0128846可上调miR-1183和miR-1827的表达（t=5.095、3.464，P=0.007、0.026），其中miR-1183表达上调最显著，该结果提示circRNA_0128846可能与miR-1183结合，并下调其表达。采用生物信息学预测circRNA_0128846与miR-1183潜在的结合位点如图6所示。qRT-PCR检测结果表明，在A549细胞中过表达circRNA_0128846可显著下调miR-1183的表达（t=6.002，P=0.004）。在人胚肾293T细胞中进行双荧光素酶报告基因分析的结果显示，过表达miR-1183可显著降低circRNA_0128846的表达（t=4.562，P=0.010），这提示miR-1183可以与circRNA_0128846结合并下调其表达。经qRT-PCR验证，miR-1183在A549R细胞中的表达水平显著低于A549细胞（t=6.025，P=0.004），这提示miR-1183可能与人NSCLC细胞的放射抵抗有关。
图 6 生物信息学预测circRNA_0128846与miR-1183的结合位点
Figure 6. Bioinformatics prediction of the binding site of circRNA_0128846 and miR-1183
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经qRT-PCR验证，使用miR-1183抑制剂成功对A549细胞进行了miR-1183沉默，沉默miR-1183显著增强了8 Gy X射线照射后A549细胞的克隆形成能力（0.21%对0.31%，图7）；同时，使用miR-1183模拟物成功对A549R细胞进行了miR-1183过表达，过表达miR-1183显著降低了8 Gy X射线照射后A549R细胞的克隆形成能力（0.26%对0.15%，图8），这提示miR-1183可抑制人NSCLC细胞的放射抵抗。过表达circRNA_0128846可显著增强8 Gy X射线照射后A549细胞的克隆形成能力（P<0.01），而过表达miR-1183逆转了circRNA_0128846诱导的放射抵抗（1.90%对1.20%，图9），这表明circRNA_0128846可以通过介导miR-1183促进人NSCLC细胞的放射抵抗。
图 7 miR-1183抑制剂转染人非小细胞肺癌细胞A549 48 h后的相对表达水平（A）及8 Gy X射线照射后的克隆形成能力（B，×200）
Figure 7. Relative expression level of miR-1183 inhibitor transfected human non-small cell lung cancer cell A549 for 48 h (A) and clone formation ability after 8 Gy X-ray irradiation (B, ×200)
图 8 miR-1183模拟物转染人非小细胞肺癌细胞A549的放射抵抗细胞（A549R） 48 h后的相对表达水平（A）及8 Gy X射线照射后的克隆形成能力（B，×200）
Figure 8. Relative expression level of miR-1183 mimic transfected the radioresistant cell (A549R) of human non-small cell lung cancer cell A549 for 48 h (A) and clone formation ability after 8 Gy X-ray irradiation (B, ×200)
图 9 人非小细胞肺癌细胞A549过表达circRNA_0128846和miR-1183后的克隆形成能力（×200）
Figure 9. Clonal formation ability of human non-small cell lung cancer cell A549 after overexpression of circRNA_0128846 and miR-1183 (×200)
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越来越多的研究结果表明，circRNA在许多恶性肿瘤的发生发展中起着关键的调节作用[16]。随着高通量测序及先进的生物信息学技术的发展，许多circRNA被鉴定出来，这使得circRNA与肿瘤的关系比以往更受关注。circRNA的异常表达已被证实与NSCLC的进展有关，如circRNA_100876在NSCLC组织中的表达水平明显高于邻近的非肿瘤组织，circRNA_100876的表达上调与NSCLC患者的淋巴结转移和肿瘤分期有关[17]。此外，circRNA_100876表达水平较高的NSCLC患者的总生存时间明显短于circRNA_100876表达水平较低的NSCLC患者[17]，故某些circRNA对NSCLC的发生、发展及诊断治疗可能有重大意义。目前，关于circRNA_0128846与肿瘤的关系仅有1篇文献报道，其研究结果显示，circRNA_0128846在结直肠癌组织中的表达水平显著高于正常组织，circRNA_0128846通过下调miR-1184的表达来增加LIM结构域蛋白（AJUBA）的表达，使得Hippo/Yes相关蛋白（YAP）通路信号失活，从而促进结直肠癌的发生[18]。在本研究中，我们通过生物信息学分析发现circRNA_0128846在人NSCLC细胞中显著高表达。
目前，关于circRNA与肿瘤放射敏感性关系的报道还不多见。研究结果表明，circZNF609在前列腺癌组织和细胞系中异常上调，其通过调控miR-501-3p/已糖激酶2（HK2）轴促进糖酵解代谢，从而增强前列腺癌细胞的放射抵抗[19]。circ_0086720通过靶向miR-375/纺锤体蛋白1（SPIN1）轴促进NSCLC放射抵抗[20]。下调circ_CCNB2可抑制miR-30b-5p/驱动蛋白家族成员18A（KIF18A）轴介导的细胞自噬，增强前列腺癌细胞的放射敏感性[21]。在本研究中，我们发现circRNA_0128846在人NSCLC放射抵抗细胞中显著高表达，沉默circRNA_0128846能显著增强人NSCLC细胞的放射敏感性，过表达circRNA_0128846能显著降低人NSCLC细胞的放射敏感性，circRNA_0128846可作用于miR-1183，促进人NSCLC细胞的放射抵抗。
circRNA发挥生物学功能的重要途径之一就是作为分子海绵吸附miRNA。大量研究结果证实miRNA参与炎症[22]、心脏病[23]、肿瘤[24]等多种疾病的发生、发展。目前，关于miR-1183的研究还较少。miR-1183可通过调控B细胞淋巴瘤基因2（Bcl2）的表达参与风湿性心脏病的发生、发展过程[25]。在肿瘤中，miR-1183是circRNA_0073239和circRNA_0005273调控肿瘤细胞增殖、迁移、糖酵解的重要下游靶标[26-28]。Zhu等[26]的研究结果表明，miR-1183是circRNA_0073239介导胶质瘤放射抵抗的下游靶标。本研究通过人类环状RNA数据库和circinteractome数据库预测并验证了circRNA_0128846的下游靶标miR-1183，并发现其可能是circRNA_0128846介导人NSCLC细胞放射抵抗的重要靶标。我们发现，通过过表达miR-1183显著增强了人NSCLC细胞的放射敏感性，而沉默miR-1183显著降低了人NSCLC细胞的放射敏感性。
关于miR-1183下游靶标的研究不多，B细胞淋巴癌基因2（Bcl2）和磷脂酰肌醇依赖性激酶1（pPDPK1）是miR-1183的下游靶标。在前期，我们通过TargetScan和miRbase数据库分析预测了miR-1183可能的下游靶标，其中包括鼠双微体2（mouse double minute 2，MDM2）和缺氧诱导因子1（hypoxia inducible factor-1，HIF-1），这些基因已被证实参与肿瘤的放射抵抗[29-30]。MDM2作为一种癌基因，能调节抑癌基因p53对细胞周期和凋亡的调控[31]。多项研究结果表明，MDM2在肿瘤对辐射反应的过程中发挥关键作用[32]。HIF-1是一种关键的转录因子，可使肿瘤细胞适应缺氧条件[33]。放疗可以加速HIF-1的激活，而HIF-1反过来可通过多条信号途径影响辐射反应[34-35]。这些基因可能是miR-1183调控肿瘤放射敏感性的重要靶标。本研究中，circRNA_0128846在人NSCLC放射抵抗细胞中的表达显著上调，而miR-1183的表达下降，circRNA_0128846可通过靶向结合并抑制miR-1183的表达，进而降低人NSCLC细胞的放射敏感性，这提示circRNA_0128846在NSCLC的放射抵抗中发挥重要作用，其可能成为一种潜在的治疗靶点，但具体的作用机制还需进一步探究。
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circRNA_0128846靶向miR-1183介导人非小细胞肺癌细胞放射抵抗的研究
The study of circRNA_0128846 targeting miR-1183 to mediate radioresistance in human non-small cell lung cancer cells
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摘要:
目的 探讨环状RNA（circRNA）_0128846对人非小细胞肺癌（NSCLC）细胞放射抵抗的影响及机制。 方法 应用GEO数据库筛选、采用荧光实时定量聚合酶链反应（qRT-PCR）检测在人NSCLC细胞A549及其放射抵抗细胞（A549R）中差异表达最显著的circRNA作为目标circRNA，qRT-PCR检测其在人支气管上皮细胞Beas-2B及人NSCLC细胞A549、H460、H1299、H1975中的表达水平。在A549细胞中转染目标circRNA过表达载体，在A549R细胞中转染目标circRNA沉默载体，并检测以上细胞经8 Gy X射线照射后的克隆形成能力。应用人类环状RNA数据库和circinteractome数据库预测目标circRNA下游的miRNA，qRT-PCR检测A549R细胞中目标circRNA沉默后表达水平上升最显著的miRNA作为目标miRNA。qRT-PCR检测A549细胞中过表达目标circRNA后目标miRNA的表达情况；通过双荧光素酶报告基因分析人胚肾细胞293T中过表达目标miRNA后目标circRNA的表达情况；qRT-PCR检测目标miRNA在A549、A549R细胞中的表达水平。在A549细胞中转染目标miRNA抑制剂以沉默目标miRNA，在A549R细胞中转染目标miRNA模拟物以过表达目标miRNA，并检测以上细胞经8 Gy X射线照射后的克隆形成能力；将过表达目标circRNA的A549细胞进行8 Gy X射线照射并在该细胞中过表达目标miRNA，检测细胞的克隆形成能力。组间数据的比较采用独立样本t检验。 结果 qRT-PCR检测结果显示，circRNA_0128846为目标circRNA，且其在人NSCLC细胞A549、H460、H1299、H1975中的相对表达水平均高于人支气管上皮细胞Beas-2B（t=6.200、7.903、6.010、6.132，均P<0.01）。过表达circRNA_0128846的A549细胞经照射后的克隆形成能力增强（0.22%对0.45%，t=4.427，P<0.05）；沉默circRNA_0128846后A549R细胞经照射后的克隆形成能力降低（0.23%对0.10%，t=3.780，P<0.05）。qRT-PCR检测结果显示，miR-1183为目标miRNA，在A549细胞中过表达circRNA_0128846显著下调miR-1183的表达（t=6.002，P<0.01）；双荧光素酶报告基因分析证实过表达miR-1183可显著下调circRNA_0128846的表达（t=4.562，P<0.05）；miR-1183在A549R细胞中的相对表达水平显著低于亲本A549细胞（t=6.025，P<0.01）。过表达miR-1183显著降低A549R细胞经照射后的克隆形成能力，沉默miR-1183显著增强A549细胞经照射后的克隆形成能力（0.26%对0.15%、0.21%对0.31%，t=3.671、3.293，均P<0.05），且过表达miR-1183显著降低了过表达circRNA_0128846对A549细胞克隆形成能力的促进作用（1.90%对1.20%，t=6.325，P<0.01）。 结论 circRNA_0128846作为miR-1183的吸附海绵，可抑制miR-1183的表达进而抑制其放疗增敏作用，从而促进人NSCLC细胞的放射抵抗。 -
关键词:
- 癌，非小细胞肺 /
- RNA，环状 /
- circRNA_0128846 /
- miR-1183 /
- 放射抵抗
Abstract:Objective To investigate the effect and mechanism of circular RNA (circRNA)_0128846 on the radioresistance of human non-small cell lung cancer (NSCLC) cells. Methods The circRNA with the most significant differential expression in the human NSCLC cell line A549 and its radioresistant cell line (A549R) was considered as the target circRNA. It was screened by using the GEO database and detected through fluorescence real-time quantitative polymerase chain reaction (qRT-PCR). qRT-PCR was utilized to detect the expression of the target circRNA in the human bronchial epithelial cell strain Beas-2B and NSCLC cells A549, H460, H1299, and H1975. A549 cells were transfected with a silencing vector, and A549R cells were transfected with an overexpression vector. Clone formation assay was used to detected the clone formation ability of the cells after 8 Gy X-ray irradiation. The downstream miRNA of the target circRNA was predicted with the human circular RNA database and circinteractome database. qRT-PCR was applied to detect the miRNA with the expression level that had increased most significantly after the target circRNA was silenced in the A549R cells, and regarded it as the target miRNA. The expression of the target miRNA after the overexpression of the target circRNA in A549 cells was detected with qRT-PCR. A dual-luciferase reporter gene system was employed to analyze the expression of the target circRNA after the target miRNA was overexpressed in human embryonic kidney cells 293T. The expression level of the target miRNA in A549 and A549R cells was detected through qRT-PCR. The silencing and overexpression vectors of the target miRNA were transfected into A549 and A549R cells, respectively. Then, clone formation ability was detected after 8 Gy X-ray irradiation. The A549 cells overexpressing the target circRNA were irradiated with 8 Gy X-ray, and the target miRNA was overexpressed in the cells to detect their clone formation ability. Data were compared between groups by using independent sample t test. Results qRT-PCR results revealed that circRNA_0128846 was the target circRNA, and its relative expression level in human NSCLC cells A549, H460, H1299, and H1975 was higher than that in the human bronchial epithelial cell line Beas-2B (t=6.200, 7.903, 6.010, 6.132; all P<0.01). After circRNA_0128846 was overexpressed, the clone formation ability of irradiated A549 cells was enhanced (0.22% vs. 0.45%, t=4.427, P<0.05). After circRNA_0128846 was silenced, the clone formation ability of irradiated A549R cells decreased (0.23% vs. 0.10%, t=3.780, P<0.05). qRT-PCR results showed that miR-1183 was the target miRNA, and the overexpression of circRNA_0128846 in A549 cells significantly down-regulated the expression of miR-1183 (t=6.002, P<0.01). Dual-luciferase reporter gene analysis confirmed that the overexpression of miR-1183 could significantly reduce the expression of circRNA_0128846 (t=4.562, P<0.05). The relative expression level of miR-1183 in A549R cells was significantly lower than that in parental A549 cells (t=6.025, P<0.01). The overexpression of miR-1183 significantly reduced the clone formation ability of A549R cells after irradiation; the silencing of miR-1183 significantly enhanced the clone formation ability of A549 cells after irradiation (0.26% vs. 0.15%, 0.21% vs. 0.31%; t=3.671, 3.293; both P<0.05); and the overexpression of miR-1183 significantly reduced the promoting effect of the overexpression of circRNA_0128846 on the clone formation ability in A549 cells (1.90% vs. 1.20%, t=6.325, P<0.01). Conclusion In human NSCLC cells, circRNA_0128846 acts as a miR-1183 adsorption sponge that can inhibit the radiosensitization effect of miR-1183 by reducing miR-1183 expression, thereby promoting NSCLC radioresistance. -
Key words:
- Carcinoma, non-small-cell lung /
- RNA, circular /
- circRNA_0128846 /
- miR-1183 /
- Radioresistance
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图 1 筛选目标circRNA并检测其在正常人支气管上皮细胞Beas-2B及人非小细胞肺癌细胞A549、H460、H1299、H1975中的表达情况
Figure 1. Screening target circRNA and detecting its expression in normal human bronchial epithelial cell Beas-2B and human non-small cell lung cancer cell A549, H460, H1299, H1975
图 2 不同剂量X射线照射后正常人支气管上皮细胞Beas-2B和人非小细胞肺癌细胞A549、H460、H1299、H1975的克隆形成情况（×200）
Figure 2. Clone formation in normal human bronchial epithelial cells Beas-2B and human non-small cell lung cancer cell A549, H460, H1299, H1975 after irradiation with different doses of X-ray radiation (×200)
图 3 不同剂量X射线照射后人非小细胞肺癌细胞A549及其放射抵抗细胞（A549R）的克隆形成情况（×200）
Figure 3. Clone formation in human non-small cell lung cancer cell A549 and its radioresistant cell A549R after irradiation with different doses of X-ray radiation (×200)
图 4 circRNA_0128846过表达载体转染人非小细胞肺癌细胞A549 48 h后的相对表达水平（A）及8 Gy X 射线照射后的克隆形成能力（B，×200）
Figure 4. Relative expression level of circRNA_0128846 overexpression vector transfected human non-small cell lung cancer cell A549 for 48 h (A) and clone formation ability after 8 Gy X-ray irradiation (B, ×200)
图 5 circRNA_0128846沉默载体转染人非小细胞肺癌细胞A549的放射抵抗细胞（A549R） 48 h后的相对表达水平（A）及8 Gy X射线照射后的克隆形成能力（B，×200）
Figure 5. Relative expression level of circRNA_0128846 silencing vector transfected the radioresistant cell (A549R) of human non-small cell lung cancer cell A549 for 48 h (A) and clone formation ability after 8 Gy X-ray irradiation (B, ×200)
图 6 生物信息学预测circRNA_0128846与miR-1183的结合位点
Figure 6. Bioinformatics prediction of the binding site of circRNA_0128846 and miR-1183
图 7 miR-1183抑制剂转染人非小细胞肺癌细胞A549 48 h后的相对表达水平（A）及8 Gy X射线照射后的克隆形成能力（B，×200）
Figure 7. Relative expression level of miR-1183 inhibitor transfected human non-small cell lung cancer cell A549 for 48 h (A) and clone formation ability after 8 Gy X-ray irradiation (B, ×200)
图 8 miR-1183模拟物转染人非小细胞肺癌细胞A549的放射抵抗细胞（A549R） 48 h后的相对表达水平（A）及8 Gy X射线照射后的克隆形成能力（B，×200）
Figure 8. Relative expression level of miR-1183 mimic transfected the radioresistant cell (A549R) of human non-small cell lung cancer cell A549 for 48 h (A) and clone formation ability after 8 Gy X-ray irradiation (B, ×200)
图 9 人非小细胞肺癌细胞A549过表达circRNA_0128846和miR-1183后的克隆形成能力（×200）
Figure 9. Clonal formation ability of human non-small cell lung cancer cell A549 after overexpression of circRNA_0128846 and miR-1183 (×200)
表 1 荧光实时定量聚合酶链反应的引物序列
Table 1. Primers sequences used in fluorescence real-time quantitative polymerase chain reaction
引物名称 引物序列 GADPH F: 5′-CACCCACTCCTCCACCTTTG-3′ R: 5′-CCACCACCCTGTTGCTGTAG-3′ circRNA_0128846 F: 5′-GACCTCTGTCAGCGAGTTCC-3′ R: 5′-GCTACTGGAGCCTGATGGAC-3′ circRNA_0116061 F: 5′-GAGATGGAACAGCACGGATT-3′ R: 5′-GGCAGATTTGCAAAAGATGA-3′ circRNA_0116961 F: 5′-TCGAATTGTCAAGATGTGGAA-3′ R: 5′-AAAAACCTGATTGCGAATGAA-3′ circRNA_0106711 F: 5′-TAATCCCAGGAGTCCAGCTC-3′ R: 5′-ACGAGTGCTGCTTTGGTCTT-3′ circRNA_0106714 F: 5′-GGACAGCAGGCATTACCAAT-3′ R: 5′-CCAATTTCCAAAGCCACAGT-3′ circRNA_0094088 F: 5′-CCTTTGAAGATGGCCTCTGA-3′ R: 5′-CACAGACGAGTGGGTTCACA-3′ circRNA_0096041 F: 5′-TTGGTCCAATCTGCTTCACC-3′ R: 5′-GTTCACATCGGGATCCTTGT-3′ circRNA_0046264 F: 5′-TTGGTCCAATCTGCTTCACC-3′ R: 5′-GTTCACATCGGGATCCTTGT-3′ circRNA_0096042 F: 5′-CCCGCTGGTGAAGTCTCTAT-3′ R: 5′-TGGCTACTGAACCAGTCACG-3′ circRNA_0096049 F: 5′-GGATGTTTCTCTGCCCAAAA-3′ R: 5′-TTTGGGTCCCTTGAATTTACC-3′ miR-646 F: 5′-ACACTCCAGCTGGGAAGCAGCTGCCTC-3′ R: 5′-CTCAACTGTGCTGCATTAGTTAGCTCAGA-3′ miR-885-3p F: 5′-GAGCACGAGGCAGTAGGCAAAGTGT-3′ R: 5′-GAGGCAGCGGGGTGTAGTGGATAGA-3′ miR-578 F: 5′-GTGCAGGGTGTTAGGA-3′ R: 5′-GAAGAACACGTCTGGT-3′ miR-647 F: 5′-GTGTTGGCCTGTGGCTG-3′ R: 5′-CTGACCCTCCCTCCTGC-3′ miR-892a F: 5′-GCCGAGCACTGTGTCCTTT-3′ R: 5′-CAGTGCAGGGTCCGAGGTAT-3′ miR-1183 F: 5′-ACTGACCACTGTAGGTGATGG-3′ R: 5′-GCGAGCACAGAATTAATACGACTCACTATAGG-3′ miR-1827 F: 5′-GGTGAGGCAGTAGATTGAATCTC-3′ R: 5′-CTCAACTGGTGTCGTGGAGTC-3′ miR-532-3p F: 5′-GCCCATGCCTTGAGTGTAG-3′ R: 5′-GTGCGTGTCGTGGAGTCG-3′ miR-626 F: 5′-CTTATTTGAGAGCTGAGGA-3′ R: 5′-CGGACTAGTACATCATCTATACTG-3′ U6 F: 5′-GCTTCGGCAGCACATATACTAAAAT-3′ R: 5′-CGCTTCACGAATTTGCGTGTCAT-3′ 注：GADPH为甘油醛-3-磷酸脱氢酶；circRNA为环状RNA；miR为微小RNA 
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