Research Progress on Combination of Radiotherapy and Immunotherapy on Advanced Non-small Cell Lung Cancer with Negative Driver Genes
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摘要:
非小细胞肺癌（NSCLC）患者中有很大比例在发现时被诊断为转移性和不可治愈的晚期肺癌，以致于失去了手术的机会，5年生存率较低。免疫治疗时代，众多种类的免疫检查点抑制剂被批准用于驱动基因阴性的晚期NSCLC患者的一/二线治疗，同时与放疗联合作为晚期NSCLC患者的重要治疗策略。放射治疗与免疫治疗相结合的创新策略在临床研究中获得了大量的可行性证据。在分子和细胞水平上观察免疫机制的临床前实验初步揭示了肿瘤、辐射和免疫系统之间的相互作用。本文对放免联合治疗在驱动基因阴性晚期NSCLC治疗中的进展进行简要综述。
Abstract:A large proportion of patients with non-small cell lung cancer (NSCLC) are diagnosed with metastatic and incurable advanced lung cancer at the time of discovery, so these patients are given no surgical opportunity and have a low 5-year survival rate. In the era of immunotherapy, many kinds of immune checkpoint inhibitors (ICIs) have been approved as the first/second-line treatment for patients with advanced NSCLC with negative driving genes and have been combined with radiotherapy as an important treatment strategy for patients with advanced NSCLC. The innovative strategy of combining radiotherapy and immunotherapy has shown feasibility as supported by practical evidence in clinical research. A preclinical experiment of observing the immune mechanism at the molecular and cellular levels preliminarily revealed the interaction among tumor, radiation, and immune system. This paper briefly reviews the progress of combined radiotherapy and immunotherapy in the treatment of advanced NSCLC with negative drvier genes.
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0 引言
胶质瘤是一种中枢神经系统肿瘤[1]。世界卫生组织（WHO） 将胶质瘤分为四个等级，其中Ⅳ级胶质瘤恶性程度最高，也称为胶质母细胞瘤（GBM），其两年生存率仅26%，中位生存期仅14.6个月[2-4]。探索胶质瘤进展的分子机制对于胶质瘤治疗具有重要意义。
各种转录因子可参与调控人类疾病的进展，目前可表达转录因子的基因约占所有致癌基因的20%[5-8]。E-twenty six（ETS）蛋白是转录因子家族中最大的一个家族之一，在调节多种生物学过程中发挥重要作用[9]。ETS原癌基因1（ETS proto-oncogene 1, ETS1）是RAS/RAF/ERK通路的下游效应子[10]。据报道ETS1可参与多种实体瘤的发生发展，如结直肠癌[11]、宫颈癌[12]、非小细胞肺癌[13]。在胶质瘤中，ETS1可通过激活长链非编码RNA（long non-coding RNA, lncRNA）SNHG10转录发挥促癌作用[14]。然而，ETS1在胶质瘤中的生物学功能和下游调控机制尚未完全阐明。
LncRNA XIST高表达可促进胶质瘤的恶性生物学表型[15]。本研究中，我们发现ETS1在胶质瘤组织和细胞内高表达，调控肿瘤细胞的增殖和凋亡，且能激活XIST的转录。这些发现揭示了ETS1新的生物学功能，并提示ETS1可能是胶质瘤的治疗靶点。
1 材料与方法
1.1 组织样本收集
收集2019年9月至2022年12月在三峡大学第一临床医学院神经外科收治的58例初诊胶质瘤手术患者的肿瘤组织及其相应的癌旁组织。所有组织样本取出后立即放入液氮中冷冻，随后转移至冰箱中（−80℃）保存。所有患者术前未接受放疗或化疗，患者或家属在组织样本收集前均签署了知情同意书。所有实验方案经本院伦理委员会批准，实验流程严格遵守赫尔辛基宣言。
1.2 细胞培养
胶质瘤细胞系（U251、U87、LN229、LN308）和人星形胶质细胞（NHA）均购自中国典型培养物保藏中心（武汉）。所有细胞系培养于含10%胎牛血清（美国西格玛-奥德里奇公司）、100 U/ml青霉素、100 μg/ml链霉素的培养基中，并在5%CO2、37℃的培养箱中孵育。取对数生长期的细胞用于实验。
1.3 细胞转染
ETS1小干扰RNA（si-ETS1#1和si-ETS1#2），XIST过表达质粒（XIST），ETS1过表达质粒（ETS1）和空白质粒（NC）均购自吉满生物科技有限公司（上海），当细胞融合达60%~80%时，使用LipofectamineTM2000（美国英杰公司）将以上siRNA或载体转染U87和LN229细胞系中，转染48 h后，采用实时定量聚合酶链反应（qRT-PCR）检测转染效率，收集细胞用于后续实验。
1.4 免疫组织化学
将石蜡包埋的组织标本切成4 μm厚的切片，二甲苯脱蜡，磷酸盐缓冲液（PBS）洗涤，在柠檬酸溶液中煮沸进行抗原修复，然后用3%过氧化氢处理。随后将组织标本与1%FBS孵育，加入抗ETS1抗体（1∶100），4℃下孵育过夜，随后加入辣根过氧化物酶标记的二抗，室温孵育30 min后在室温下用DAB孵育5 min进行显色，苏木精对比染色，光学显微镜下观察并拍照。
1.5 实时定量PCR
TRIzol试剂提取组织和细胞中的RNA，ABI High-Capacity cDNA反转录试剂盒对RNA进行反转录，ABI StepOnePlus实时PCR系统进行实时定量PCR，GAPDH作为内参， 2-ΔΔCt法计算结果。引物序列：ETS1（正向：5’-GATAGTTGTGATCGCCTCACC-3’； 反向：5’-GTCCTCTGAGTCGAAGCTGTC-3’）；XIST（正向：5’-AGCTCCTCGGACAGCTGTAA-3’； 反向：5’-CTCCAGATAGCTGGCAACC-3’）； GAPDH （正向：5’-TCGACAGTCAGCCGCATCTTCTTT-3’，反向：5’-ACCAAATCCGTTGACTCCGACCTT-3’）。
1.6 CCK-8实验
CCK-8试剂盒检测U87细胞系和LN229细胞活性，将细胞以密度为1×103细胞/孔接种于96孔板上，转染细胞培养24、48和72 h后每孔分别加入10 μl CCK-8试剂，之后在37℃、5%CO2条件下孵育2 h。使用酶标仪检测每孔在450 nm波长处的吸光度值。
1.7 5-乙炔基-2'-脱氧尿苷实验
EdU试剂盒检测U87和LN229细胞的增殖。取对数生长期细胞，以2×103个细胞接种于96孔板中，每孔加入200 μl浓度为5 μmol/L EdU溶液，培养2 h，PBS冲洗，随后4%多聚甲醛固定30 min，加入Apollo®荧光染色液，避光孵育30 min，DAPI反应液染色30 min。荧光显微镜下观察细胞，并计算EdU阳性细胞的百分比。
1.8 Western blot实验
RIPA裂解液从细胞中提取蛋白，二喹啉甲酸法（BCA法）定量蛋白。十二烷基硫酸钠聚丙烯酰胺凝胶电泳后，将蛋白转移至聚偏二氟乙烯薄膜上，之后将PVDF膜用5%脱脂牛奶封闭。然后，将膜与下列一抗于4℃条件下孵育过夜：anti-Bax（1:
1000 ）、anti-Bak（1:1000 ）、anti-Bcl-2（1:1000 ）或anti-β-actin （1:1000 ），随后将膜与二抗（1:2000 ）于室温孵育2 h后使用ECL化学发光检测试剂盒检测蛋白条带，β-actin为内参。1.9 双荧光素酶报告基因实验
将ETS1与XIST启动子区域上的结合位点克隆到萤火虫荧光素酶报告载体pGL3中，构建XIST野生型载体（XIST-WT）和XIST突变型载体（XIST-MUT）。随后根据制造商的说明，LipofectamineTM 2000将上述载体和ETS1过表达质粒（ETS1）或空白质粒（NC）共转染到U87和LN229细胞中。转染48 h后，使用双荧光素酶报告系统检测荧光素酶活性，以海肾荧光素酶活性为内参。
1.10 染色质免疫共沉淀-PCR
EZ-ChIPTM试剂盒进行ChIP实验。将U87和LN229细胞用1%甲醛固定10 min，加入甘氨酸终止固定。刮取细胞并离心以获得细胞沉淀，加入含PMSF的细胞裂解缓冲液悬浮细胞，离心后去掉上清液获得核沉淀物，冰浴超声裂解后，将裂解物分别与一抗 IgG（1∶100）或ETS1一抗在4℃下过夜。随后，蛋白质琼脂糖沉淀DNA-蛋白质复合物，并在4℃下离心5 min。最后，根据EZ-ChIPTM试剂盒操作说明提取和处理DNA，qRT-PCR检测XIST的DNA水平。
1.11 生物信息学分析
GEPIA（http://gepia.cancer-pku.cn/）分析ETS1在胶质瘤组织中的差异表达。UALCAN数据库（ualcan.path.uab.edu/home）分析ETS1在胶质瘤组织以及正常脑组织中的表达。PROMO数据库（http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo）预测ETS1与XIST启动子之间的结合位点。cBioPortal数据库（https://www.cbioportal.org/）分析ETS1与XIST在胶质瘤组织中表达的相关性。
1.12 统计学方法
采用SPSS 22.0软件进行统计分析。所有实验进行3次，结果以均数±标准差表示，组间比较使用Student's t test或单因素方差分析，Pearson进行相关性分析。P<0.05为差异有统计学意义。
2 结果
2.1 ETS1在胶质瘤中高表达
首先使用GEPIA数据库分析ETS1在胶质瘤中的差异表达，结果表明ETS1在胶质母细胞瘤（GBM）和低级别胶质瘤（LGG）组织中表达均显著上调，见图1A。另外，UALCAN数据库同样显示ETS1在GBM组织中表达上调，见图1B。IHC染色检测58例胶质瘤组织及其对照正常组织样本中ETS1的表达，结果表明与正常脑组织相比，ETS1在胶质瘤组织中的表达显著上调（χ2=8.838，P=0.003），见图1C。qRT-PCR分析ETS1 mRNA在58例胶质瘤患者肿瘤组织以及正常脑组织中的表达水平，结果显示ETS1 mRNA在肿瘤组织中的表达水平显著高于正常脑组织见图1D。根据胶质瘤组织中ETS1表达水平的中位数，将58例胶质瘤患者分为高表达组和低表达组，分析ETS1的表达与患者临床病理特征之间的关系，结果显示ETS1高表达与胶质瘤患者TNM分期增加有关，见表1。此外，与人星形胶质细胞（NHA）相比，ETS1 mRNA在4种胶质瘤细胞系（U251、 U87、 LN229、 LN308）中表达水平均显著升高 ，见图1E。
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图 1 ETS1在胶质瘤中的表达特征Figure 1 Expression characteristics of ETS1 in glioma*: P<0.05, ***: P<0.001; A: the differential expression of ETS1 in glioblastoma and low-grade glioma was analyzed using GEPIA online database; B: the expression of ETS1 in glioma and its adjacent tissues was analyzed using UALCAN database; C: the expression of ETS1 in 58 glioma tissues and adjacent tissues was detected by immunohistochemistry; D-E: the ETS1 mRNA expression levels in 58 glioma tissues and cell lines were detected by qRT-PCR.表 1 ETS1在胶质瘤组织中的表达与临床病理资料的关系Table 1 Relationship between ETS1 expression in glioma tissues and clinicopathological data of patientsCharacteristics ETS1 χ2 P High expression
(n=29)Low expression
(n=29)Gender 0.633 0.426 Male 15 18 Female 14 11 Age(years) 1.105 0.293 ≥40 13 17 <40 16 12 Diameter of
tumors (cm)0.624 0.430 < 2 12 15 ≥2 17 14 No 6 17 WHO staging
system5.695 0.017 3-4 21 12 1-2 8 17 2.2 敲低ETS1抑制胶质瘤细胞增殖、促进细胞凋亡
为探究ETS1在胶质瘤细胞中的生物学功能，我们将两种ETS1 siRNA（si-ETS1#1、si-ETS1#2）转染入U87和LN229细胞系中，qRT-PCR结果显示转染成功，见图2A。CCK-8实验显示，与si-NC组相比，si-ETS1#1和si-ETS1#2组中胶质瘤细胞的活性显著降低，见图2B。EdU实验同样显示si-ETS1#1和si-ETS1#2组中胶质瘤细胞增殖能力显著低于si-NC组，见图2C。Western blot实验结果显示，与si-NC组相比，si-ETS1#1和si-ETS1#2组细胞中促凋亡蛋白Bax和Bak的表达水平显著升高，而抗凋亡蛋白Bcl-2的表达显著降低，见图2D。因此，以上结果显示ETS1可促进胶质瘤细胞增殖，并抑制凋亡。
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图 2 敲低ETS1抑制胶质瘤细胞增殖、促进细胞凋亡Figure 2 ETS1 knockdown inhibited glioma cell proliferation and promoted cell apoptosis*: P<0.05; **: P<0.01; ***: P<0.001; A: qRT-PCR was used to detect the expression level of ETS1 mRNA in U87 and LN229 cells after transfection with si-ETS1#1 or si-ETS1#2; B: CCK-8 assay was conducted to detect the activity of U87 and LN229 cells after transfection with si-ETS1#1 or si-ETS1#2; C: EdU assay was used to detect the proliferation of U87 and LN229 cells after transfection with si-ETS1#1 or si-ETS1#2; D: Western blot was adopted to detect the expression levels of apoptosis-related proteins (Bax, Bak, Bcl-2) in U87 and LN229 cells after transfection with si-ETS1#1 or si-ETS1#2.2.3 ETS1靶向结合XIST激活其转录
为探索ETS1的下游机制，我们检索了PROMO数据库，发现ETS1可能与XIST启动子序列结合，见图3A；cBioPortal数据库结果显示，在胶质瘤组织中XIST与ETS1 mRNA的表达呈正相关，见图3B。通过双荧光素酶报告基因实验验证ETS1与XIST的结合关系，结果显示在U87和LN229细胞系中，过表达ETS1均可显著提高XIST WT报告质粒的荧光素酶活性，见图3C。在U87和LN229细胞系中使用ETS1特异性抗体进行ChIP-qPCR检测进一步证实二者的结合关系，结果表明与IgG对照组相比，ETS1抗体可以显著富集XIST的启动子序列，见图3D。将si-ETS1#1、si-ETS1#2转染进U87和LN229细胞系中，qRT-PCR结果显示与对照组相比，敲低ETS1可显著降低XIST的表达，见图3E。此外，qRT-PCR结果显示，XIST在胶质瘤组织中高表达，见图3F；且Pearson分析发现在胶质瘤组织中XIST与ETS1 mRNA的表达呈正相关，见图3G；qRT-PCR结果显示，与正常细胞相比，XIST在胶质瘤细胞中表达上调，见图3H。这些数据表明，ETS1可靶向结合XIST促进其表达。
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图 3 ETS1靶向结合XISTFigure 3 ETS1 targeted XIST**: P<0.01; ***: P<0.001; A: PROMO database predicted the binding site of ETS1 to the XIST promoter; B: cBioPortal database was used to analyze the correlation between ETS1 mRNA and XIST expression in glioma tissues; C: dual-luciferase reporter assay was conducted to examine the effect of ETS1 overexpression on the luciferase activity of XIST-WT and XIST-MUT; D: ChIP-qPCR was used to detect the binding of ETS1 to the XIST promoter region; E: qRT-PCR was utilized to detect the expression level of XIST in U87 and LN229 cells after transfection with si-ETS1#1 or si-ETS1#2; F: qRT-PCR was used to detect the expression level of XIST in 58 glioma tissues and adjacent tissues; G: Pearson correlation analysis was performed on the expression of ETS1 mRNA and XIST in glioma tissues; H: the expression level of XIST in glioma cell lines and NHA cells was detected by qRT-PCR.2.4 过表达XIST可逆转敲低ETS1对胶质瘤细胞增殖和凋亡的影响
为进一步证实ETS1是否通过调控XIST促进胶质瘤细胞的增殖，我们将si-ETS1#1和XIST过表达质粒共转染U87和LN229细胞系，并通过qRT-PCR验证转染效率，见图4A。转染成功后，CCK-8和EdU实验结果显示，与单独转染si-ETS1#1组相比，共转染si-ETS1#1和XIST组中胶质瘤细胞增殖能力显著提高，见图4B、C。Western blot实验结果显示，与单独转染si-ETS1#1组相比，共转染si-ETS1#1和XIST组细胞中Bax、Bak的表达水平显著降低，而Bcl-2的表达显著升高，见图4D。结果表明过表达XIST可部分逆转敲低ETS1对胶质瘤细胞增殖的抑制作用，以及对细胞凋亡的促进作用，EST1可以依赖XIST调控胶质瘤细胞的增殖和凋亡。
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图 4 过表达XIST可逆转敲低ETS1对胶质瘤细胞增殖和凋亡的影响Figure 4 Overexpression of XIST reversed the effect of ETS1 knockdown on the proliferation and apoptosis of glioma cells*: P<0.05; **: P<0.01; ***: P<0.001; A: qRT-PCR was used to detect the expression level of XIST in U87 and LN229 cells after co-transfection of si-ETS1#1 and XIST; B: CCK-8 assay was performed to detect the activity of U87 and LN229 cells after co-transfection of si-ETS1#1 and XIST; C: EdU assay was used to detect the proliferation of U87 and LN229 cells after co-transfection of si-ETS1#1 and XIST; D: Western blot was employed to detect the expression levels of apoptosis-related proteins in U87 and LN229 cells after co-transfection of si-ETS1#1 and XIST.3 讨论
转录因子是一种DNA结合蛋白，通过结合特定区域来调节基因的表达[16]。长期以来，由于转录因子主要定位于细胞质和细胞核，除了配体诱导的核受体外，转录因子曾被认为是“不可成药”的靶点[17]。近年，很多研究发现特异性小分子可以阻断转录因子和DNA、蛋白质的相互作用，有些化合物可以诱导转录因子通过泛素化降解，这些发现为靶向转录因子治疗肿瘤开辟了新的可能性[18]。
最近的几项研究表明转录因子ETS1可参与胶质瘤的进展。如ETS1通过结合在SNHG10启动子区域促进其转录，从而上调FBXL19的表达，最终促进胶质瘤细胞的增殖、迁移和侵袭[14]；PAXIP1-AS1通过募集ETS1上调KIF14的表达，从而促进胶质瘤细胞的迁移和侵袭以及血管生成[19]。ETS1能作为果糖1,6二磷酸酶基因的转录抑制因子抑制其表达，介导糖酵解和肿瘤细胞侵袭迁移[20]。还有研究发现ETS1在胶质母细胞瘤衍生的内皮和间充质干细胞样细胞中高表达，且其表达与胶质母细胞瘤的侵袭性以及微血管密度相关[21]。本研究中，我们发现ETS1在胶质瘤组织和细胞系中高表达，且其高表达与胶质瘤患者不良临床病理指标有关。另外，敲低ETS1可显著抑制胶质瘤细胞的增殖，并促进细胞凋亡。我们的研究结果与既往的报道[14,19-21]均提示，ETS1可能成为胶质瘤的潜在治疗靶点。
本研究证实了ETS1可与XIST的启动子结合。lncRNA是一种长度超过200个核苷酸的非编码RNA，大量研究表明lncRNAs可参与多种疾病的发生发展[22-24]。XIST是lncRNA之一，研究发现其可调控肿瘤细胞的恶性表型[25]。XIST已被证实在许多肿瘤中上调，如在结直肠癌中，XIST充当致癌因子，通过抑制miR-132-3p的表达促进癌细胞的增殖[26]；甲状腺癌中，XIST在癌组织和细胞系中均显著上调，并通过调控miR-34a和MET/PI3K/AKT信号通路促进肿瘤细胞增殖[27]；非小细胞肺癌中，XIST在患者组织样本中高表达，敲低XIST可抑制癌细胞的生长，并促进肿瘤细胞对顺铂的敏感度[28]。研究表明XIST在胶质瘤中上调，且通过miR-133a/SOX4轴促进细胞的增殖和转移[15]；还有研究发现过表达XIST可通过调控miR-329/CREB1轴促进胶质瘤细胞增殖、侵袭，并抑制细胞凋亡及降低癌细胞的放射敏感度[29]；敲低XIST可通过上调miR-204-5p的表达，进而抑制胶质瘤细胞的增殖、迁移和侵袭，并促进细胞凋亡，从而抑制胶质瘤的进展[30]。本研究中，同样发现XIST在胶质瘤组织和细胞中显著上调，且生物信息学分析显示在胶质瘤组织中ETS1与XIST的表达呈正相关，且XIST的转录受ETS1调控，过表达XIST可部分逆转敲低ETS1对胶质瘤细胞恶性表型的抑制作用。本研究为XIST在胶质瘤中的异常表达提供了一种合理的解释。
总之，本研究证实ETS1在胶质瘤组织中高表达并与患者不良临床病理指标相关，过表达ETS1可以促进XIST的转录，这表明ETS1在胶质瘤进展中发挥潜在促癌作用。
Competing interests: The authors declare that they have no competing interests.利益冲突声明：所有作者均声明不存在利益冲突。作者贡献：李朋：文章撰写吴晓婷：文章修改宫晓梅：文章审阅 -
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