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摘要:
胃癌是全球范围内常见的恶性肿瘤之一，中国胃癌发病及死亡人数几乎占全球发病和死亡人数的50%。晚期胃癌生存预后差。随着个体化精准治疗的发展，靶向治疗及免疫治疗已成为综合治疗的热点，本文就近年来晚期胃癌的化疗、靶向治疗和免疫治疗研究进展作简要综述。
Abstract:Gastric cancer is one of the most common malignant tumors worldwide. The number of gastric cancer cases and related deaths in China accounts for almost half of the global data. The survival prognosis of advanced gastric cancer is poor. With the development of individualized precision therapy, targeted therapy and immunotherapy have become the focus of comprehensive therapy. This paper reviews the latest research progress of chemotherapy, targeted therapy, and immunotherapy for advanced gastric cancer.
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Key words:
- Gastric cancer /
- Chemotherapy /
- Targeted therapy /
- Immunotherapy
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0 引言
Yes相关蛋白（Yes-associated protein, YAP）参与调控细胞的生长、器官的发育，与肿瘤的发生有关[1]。研究显示，YAP在乳腺癌、卵巢癌等肿瘤中表达异常，并且其表达水平的高低与肿瘤患者分期及预后等有关[2-3]。在胃癌中发现YAP过度表达，并且与胃癌患者的病理特征等有关[4]。YAP敲低可以抑制多种肿瘤细胞的增殖，如胰腺癌、乳腺癌等，对于肿瘤细胞的凋亡也具有调控作用[5-6]。Wnt/β-catenin在真核生物体细胞内广泛存在，在正常的成熟细胞中激活水平极低，而在肿瘤细胞中过度激活，与肿瘤细胞的增殖、凋亡等有关[7]。研究表明，YAP可以通过作用于Wnt/β-catenin信号通路影响细胞的生长，提示两者之间存在潜在联系[8]。本实验以BGC823细胞为体外研究对象，通过小RNA干扰技术降低细胞中YAP的表达，明确YAP对胃癌BGC823细胞凋亡的影响及对Wnt/β-catenin信号通路的调控作用。
1 材料与方法
1.1 材料
BGC823细胞购自中国医学科学院基础医学研究所基础医学细胞中心；含半胱氨酸的天冬氨酸蛋白水解酶3（cysteinyl aspartate specific proteinase 3, Caspase-3）活性测定试剂盒和含半胱氨酸的天冬氨酸蛋白水解酶9（cysteinyl aspartate specific proteinase 9, Caspase-9）活性测定试剂盒为武汉艾美捷公司产品；Lipofectamine2000为美国Invitrogen公司产品；MMLV反转录试剂盒为美国Promega公司产品；qRT-PCR为大连宝生物公司产品；β-连环蛋白（β-catenin）抗体、c-myc抗体、细胞周期蛋白D1（CyclinD1）抗体、辣根过氧化物标记的二抗均为美国Abcam公司产品；YAP、β-actin引物由上海生工公司合成；YAP siRNA1、YAP siRNA2、siRNA control为美国Santa Cruz Biotech公司产品。YAP siRNA1：5’-GCAUCUUCGACAGUCUUCUTT-3’，5’-AGAAGACUGUCGAAGAUGCTT-3’。YAP siRNA2：5’-GGUGAUACUAUCAACCAAATT-3’，5’-UUUGGUUGAUAGUAUCACCTT-3’。
1.2 细胞分组
BGC823细胞中转染YAP siRNA1和YAP siRNA2，同时转染siRNA control，依次命名为si-YAP1、si-YAP2和si-NC，以不作任何处理的BGC823细胞记为Control。细胞转染用Lipofectamine2000，具体步骤参照转染试剂说明书。BGC823细胞培养参数为：饱和湿度、37℃、5%CO2培养箱。细胞为90%时，用0.25%的胰蛋白酶消化传代细胞。细胞培养于含有10%胎牛血清的RPMI1640中。
1.3 qRT-PCR测定转染后细胞YAP mRNA水平
Control、si-NC、si-YAP1、si-YAP2细胞培养48 h以后，提取总RNA。用MMLV反转录试剂盒进行反转录，程序为42℃ 65 min；70℃ 5 min；合成的cDNA保存于-80℃。以cDNA为模板，用qRT-PCR扩增。引物序列如下：β-actin F5’-CGTCTTCCCCTCCATCGT-3’，β-actin R5’-GAAGGTGTGGTGCCAGATTT-3’。YAP F5’-ACCCACAGCTCAGCATCTTCG-3’，YAP R5’-TGGCTTGTTCCCATCCATCAG-3’。结果以2-ΔΔCt法计算。
1.4 蛋白质印迹法测定转染后细胞YAP蛋白水平
Control、si-NC、si-YAP1、si-YAP2细胞培养48 h以后，提取细胞中的总蛋白，蛋白保存在-80℃。取蛋白样品，用BCA法对蛋白进行定量。以每个泳道添加40 μg蛋白进行电泳，电泳前将蛋白样品和上样缓冲液以等体积混合煮沸5 min。在浓缩胶中以90 V的低压进行电泳，在分离胶中以120 V的电压电泳，观察染料进入到分离胶的底部之后关闭电源。把蛋白凝胶上的蛋白转移到PVDF膜上，以100 mA电流进行转膜，转膜在4℃进行。转膜结束后把膜放在用TBST稀释的牛血清白蛋白中封闭2 h。取出膜，放入含有YAP一抗（1:200稀释）的平皿中，4℃反应过夜后，再把膜放入含有HRP标记的二抗（1:3 000稀释）中，室温孵育2 h。取出膜，用ECL显色试剂盒显色，凝胶成像仪拍照，分析灰度值，以β-actin为内参。
1.5 MTT法测定细胞增殖
Control、si-NC、si-YAP1细胞种植到96孔板中，分别培养24、48、72、96 h以后，用MTT法测定各组胃癌细胞增殖情况。步骤如下：在每孔中添加20 μl的MTT，把细胞放在37℃培养箱内继续培养约4 h，取出培养板，在每个孔中依次添加150 μl的DMSO溶液，置于振荡仪上10 min，放在酶标仪上测定每孔492 nm的A值。每个组设置5个重复孔，测定前以空白孔调零，空白孔中不加细胞。
1.6 平板克隆检测细胞克隆形成能力
Control、si-NC、si-YAP1细胞以每个培养皿200个细胞接种到细胞培养皿中，每个培养皿中含有10 ml的培养液。放在37℃、5%CO2培养箱中孵育14 d后，肉眼可以观察到形成的细胞克隆。把培养液吸弃后，用PBS洗涤2次，加入4%的多聚甲醛固定各组细胞15 min。用吉姆萨染色液染约20 min后，用水把染液冲洗掉，放在空气中干燥。计数细胞克隆数目，用（细胞克隆数目÷细胞总数）×100%表示细胞克隆形成率。
1.7 流式细胞术测定细胞凋亡
Control、si-NC、si-YAP1细胞分别培养48 h后，用胰蛋白酶消化，收集细胞，用PBS把细胞沉淀洗涤2次。在细胞中添加195 μl的结合缓冲液、5 μl的Annexin V-FITC，混合后再加入10 μl的PI，充分混合，流式细胞仪测定细胞凋亡。
1.8 分光光度法检测Caspase-3、Caspase-9活性
Control、si-NC、si-YAP1细胞分别培养48 h后，收集各组细胞，分别用Caspase-3、Caspase-9活性检测试剂盒测定细胞中Caspase-3、Caspase-9活性。用酶标仪测定各组样品在405 nm的A值，用实验组A值÷对照组A值表示Caspase-3、Caspase-9活性。同时用蛋白质印迹法测定细胞中β-catenin、c-myc、cyclinD1、Cleaved Caspase-3、Cleaved Caspase-9蛋白表达，步骤同上，β-catenin、c-myc、cyclinD1、Cleaved Caspase-3、Cleaved Caspase-9一抗分别以1:600稀释。
1.9 Wnt/β-catenin激活剂对敲低YAP后的胃癌细胞增殖和凋亡的影响
以20 mmol/L的Wnt/β-catenin激活剂LiCl处理在0 h加入转染YAP siRNA1后的细胞，记为si-YAP1+LiCl。用MTT实验测定si-YAP1+LiCl、si-YAP1细胞48 h的增殖情况，步骤同1.5。平板克隆实验测定克隆形成能力，步骤同1.6。流式细胞术测定48 h细胞凋亡，步骤同1.7。分光光度法检测48 h细胞中Caspase-3、Caspase-9活性，步骤同1.8。蛋白质印迹法测定48 h细胞中β-catenin、c-myc、cyclinD1、Cleaved Caspase-3、Cleaved Caspase-9蛋白水平，步骤同1.4。
1.10 统计学方法
实验数据用SPSS21.0软件统计分析，计量资料以（x±s）表示，多组差异比较用单因素方差，组间比较用LSD-t检验，以P < 0.05为差异有统计学意义。
2 结果
2.1 转染后胃癌细胞中YAP的表达
Control、si-NC、si-YAP1、si-YAP2细胞中YAP mRNA水平分别为1.00、（1.01±0.12）、（0.36±0.05）、（0.47±0.03），蛋白水平分别为（0.38±0.04）、（0.39±0.06）、（0.14±0.02）、（0.21±0.04）。si-NC胃癌细胞中的YAP mRNA和蛋白水平与Control比较没有变化。si-YAP1、si-YAP2胃癌细胞中的YAP mRNA和蛋白水平明显低于Control，差异有统计学意义（t1=22.170, P=0.000; t2=30.599, P=0.000; t3=9.295, P=0.000; t4=5.205, P=0.000）。si-YAP1胃癌细胞中的YAP mRNA和蛋白水平明显低于si-YAP2，差异有统计学意义（t1=3.268, P=0.001; t2=2.711, P=0.007），见图 1。YAP siRNA1对胃癌细胞中YAP的转录和表达抑制作用更强，后续选用si-YAP1继续研究。
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图 1 蛋白质印迹法测定转染后的胃癌细胞中YAP蛋白水平Figure 1 YAP protein level in gastric cancer cells after transfection detected by Western blot2.2 YAP下调抑制胃癌细胞增殖和克隆形成能力
Control、si-NC、si-YAP1细胞24 h的A值分别为0.35±0.04、0.36±0.03、0.24±0.01；48 h的A值分别为0.56±0.06、0.54±0.05、0.28±0.03；72 h的A值分别为0.86±0.09、0.87±0.09、0.31±0.02；96 h的A值分别为0.98±0.07、0.99±0.12、0.35±0.04。克隆形成率分别为（39.62±3.24）%、（38.49±4.51）%、（25.14±2.36）%。si-NC胃癌细胞A值和克隆形成率与Control比较没有变化。si-YAP1胃癌细胞A值和克隆形成率明显低于Control，差异有统计学意义（t1=4.621, P=0.000; t2=7.230, P=0.000; t3=10.333, P=0.000; t4=13.535, P=0.000; t5=6.257, P=0.000）。YAP敲低可以降低胃癌细胞增殖和克隆形成能力，见图 2。
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图 2 YAP表达下调对细胞生长影响Figure 2 Effect of down regulation of YAP expression on growth of gastric cancer cells*:P < 0.052.3 YAP下调诱导胃癌细胞凋亡和Caspase-3、Caspase-9活化
Control、si-NC、si-YAP1细胞Caspase-3活性分别为1.00、1.03±0.14、2.61±0.22，Caspase-9活性分别为1.00、0.99±0.11、3.25±0.30，凋亡率分别为（6.32±0.58）%、（6.53±0.71）%、（32.71±2.64）%，Cleaved Caspase-3蛋白水平依次为0.15±0.06、0.16±0.03、0.62±0.05，Cleaved Caspase-9蛋白水平依次为0.52±0.07、0.54±0.09、0.97±0.12。si-NC胃癌细胞凋亡率和Caspase-3、Caspase-9活性与Control比较没有变化。si-YAP1胃癌细胞凋亡率、Caspase-3活性、Caspase-9活性和Cleaved Caspase-3、Cleaved Caspase-9蛋白水平明显高于Control，差异有统计学意义（t1=12.676, P=0.000; t2=12.990, P=0.000; t3=16.911, P=0.000; t4=10.423, P=0.000; t5=5.610, P=0.000），见图 3。YAP敲低可以诱导胃癌细胞凋亡，增加Caspase-3、Caspase-9活性。
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图 3 YAP下调对胃癌细胞凋亡的影响Figure 3 Effect of down regulation of YAP expression on apoptosis of gastric cancer cellsA: the apoptosis of Control, si-NC, si-YAP1 groups; B: the protein expression of Cleaved Caspase-3 and Cleaved Caspase-9 in control, si-NC and si-YAP1 groups2.4 YAP下调抑制胃癌细胞中β-catenin、c-myc、cyclinD1蛋白表达
Control、si-NC、si-YAP1细胞β-catenin水平分别为（0.32±0.03）、（0.33±0.02）、（0.10±0.01），c-myc水平分别为（0.48±0.05）、（0.49±0.08）、（0.05±0.02），cyclinD1水平分别为（0.66±0.06）、（0.67±0.05）、（0.14±0.06）。si-NC胃癌细胞中β-catenin、c-myc、cyclinD1蛋白水平与Control比较没有变化。si-YAP1胃癌细胞中β-catenin、c-myc、cyclinD1蛋白水平明显低于Control，差异有统计学意义（t1=12.050, P=0.000; t2=13.830, P=0.000; t3=10.615, P=0.000）。YAP敲低可以抑制胃癌细胞中β-catenin、c-myc、cyclinD1蛋白表达，抑制Wnt/β-catenin信号通路激活，见图 4。
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图 4 蛋白质印迹法检测敲低YAP对胃癌细胞中β-catenin、c-myc、cyclinD1蛋白的影响Figure 4 Effect of YAP knockdown on expression of betacatenin, c-myc and cyclinD1 proteins in gastric cancer cells detected by Western blot2.5 Wnt/β-catenin信号通路激活剂对敲低YAP的胃癌细胞中β-catenin、c-myc、cyclinD1蛋白表达影响
si-YAP1、si-YAP1+LiCl细胞中β-catenin水平分别为（0.12±0.03）、（0.29±0.05），c-myc水平分别为（0.16±0.03）、（0.54±0.06），cyclinD1水平分别为（0.15±0.04）、（0.71±0.07）。si-YAP1+LiCl胃癌细胞中β-catenin、c-myc、cyclinD1蛋白水平明显高于si-YAP1，差异有统计学意义（t1=5.450; P=0.000, t2=9.812, P=0.000; t3=12.031, P=0.000）。Wnt/β-catenin信号通路激活剂可以促进YAP敲低后的胃癌细胞中β-catenin、c-myc、cyclinD1蛋白表达，激活Wnt/β-catenin信号通路，见图 5。
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图 5 蛋白质印迹法检测Wnt/β-catenin信号通路激活剂对敲低YAP的胃癌细胞中β-catenin、c-myc、cyclinD1蛋白的影响Figure 5 Effects of Wnt/β-catenin signaling pathway activator on expression of β-catenin, c-myc and cyclinD1 proteins in gastric cancer cells with YAP knockdown detected by Western blot2.6 Wnt/β-catenin信号通路激活剂对敲低YAP的胃癌细胞增殖凋亡的影响
si-YAP1、si-YAP1+LiCl细胞A值分别为0.26±0.04、0.34±0.03；克隆形成率分别为（23.51±2.30）%、（31.14±2.69）%；凋亡率分别为（34.26±3.28）%、（15.69±1.27）%；Caspase-3活性分别为1.00、（0.62±0.06），Caspase-9活性分别为1.00、（0.76±0.05），Cleaved Caspase-3水平为（0.53±0.04）、（0.32±0.06）；Cleaved Caspase-9水平为（0.94±0.11）、（0.43±0.05）。si-YAP1+LiCl胃癌细胞A值和克隆形成率明显高于si-YAP1，而细胞凋亡率和Caspase-3、Caspase-9活性、Cleaved Caspase-3水平、Cleaved Caspase-9水平明显低于si-YAP1，差异有统计学意义（t1=2.771, P=0.006, t2=3.734, P=0.000, t3=9.145, P=0.000, t4=10.970, P=0.000, t5=8.314, P=0.000, t6=5.044, P=0.000, t7=7.311, P=0.000），见图 6。Wnt/β-catenin信号通路激活剂可以降低YAP敲低诱导的胃癌细胞凋亡和增殖抑制作用。
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图 6 Wnt/β-catenin信号通路激活剂对敲低YAP的胃癌细胞凋亡影响Figure 6 Effect of Wnt/ β-catenin signaling pathway activator on apoptosis of gastric cancer cells with YAP knockdownA: the apoptosis of si-YAP1 and si-YAP1+LiCl; B: the protein expression of cleaved Caspase-3 and cleaved Caspase-9 in si-YAP1 and si-YAP1+LiCl groups3 讨论
人YAP基因定位在11q13染色体上，其编码的蛋白质与转录激活有关，其本身并不能同DNA特异性结合，可以通过与存在于细胞核内的相关因子结合从而影响基因的表达[9]。YAP是Hippo的下游效应分子，其参与调控细胞的生长过程，是一个潜在的癌因子[10]。多西环素可以调控YAP的表达影响小鼠肺癌的发生和发展，而抑制YAP以后，肺癌小鼠肿瘤恶性程度降低[11]。在乳腺癌、胰腺癌、肺癌等患者的癌组织中均检测到YAP的过度表达，并且与患者的预后有关[12-14]。YAP可以调控肿瘤细胞的生长，在视网膜母细胞瘤、卵巢癌等肿瘤细胞中均得到验证[15-16]。YAP在胃癌组织中高表达，并且与胃癌裸鼠成瘤能力有关[17]。本实验结果显示，YAP敲低后的胃癌细胞增殖能力下降，并且克隆形成能力也降低，说明YAP下调后可以发挥抑制胃癌的作用，这与上述研究报道相符合。
本实验结果还显示，YAP下调后的胃癌细胞凋亡增多，并且细胞中Caspase-3、Caspase-9活化水平也升高，YAP敲低诱导Caspase-3、Caspase-9介导的胃癌细胞凋亡。很多研究报道称，YAP不仅参与肿瘤细胞的生长，还与肿瘤细胞凋亡有关[18]。研究显示，在肝癌细胞SMMC-7721中转染YAP shRNA后，细胞的生长速度明显减慢，并且细胞凋亡增多[19]。Caspase级联反应参与肿瘤细胞的凋亡，Caspase-3是该级联反应的执行因子，以酶原的形式存在于正常细胞中，其氮端含有一个前区，可以与Caspase-9等起始因子结合，在受到激活后可以诱导细胞凋亡的发生，细胞凋亡主要分为线粒体途径、死亡受体途径，其中Caspase-9参与线粒体凋亡途径，受到线粒体内相关信号刺激以后，可以活化形成Cleaved Caspase-9，从而激活Caspase级联反应，诱导凋亡发生[20]。
Wnt/β-catenin是经典的Wnt信号通路的分支，在正常情况下，β-catenin可以由多个蛋白酶形成的泛素化蛋白酶体介导而处于降解状态，使得细胞内维持低水平的β-catenin，当细胞受到相关信号刺激以后，导致细胞内的β-catenin降解受阻，β-catenin大量聚集以后转移至细胞核内，影响下游基因的转录，调控细胞的生长[21-22]。Wnt/β-catenin与多种疾病的发生有关，在糖尿病心肌病、缺血再灌注、哮喘、肺炎等疾病中均有重要作用[23-24]。研究显示，Wnt/β-catenin在肿瘤中过度激活，其下游靶基因c-myc、cyclinD1转录和表达水平升高，是肿瘤细胞过度增殖和凋亡减少的重要原因[25]。YAP与Wnt/β-catenin共同作用影响胚胎的发育，组织器官的形成，在胶质瘤中发现，YAP可以影响胶质瘤细胞中Wnt/β-catenin的激活影响肿瘤细胞的生长[26-27]。本研究表明，YAP下调可以降低Wnt/β-catenin信号通路的激活水平，减少下游靶基因c-myc、cyclinD1的表达，而用Wnt/β-catenin信号通路激活剂处理后，YAP下调对胃癌细胞增殖凋亡的影响减弱，说明YAP可以通过抑制Wnt/β-catenin信号通路的激活再通过线粒体途径调控胃癌细胞的凋亡。
综上，YAP下调后可以诱导胃癌细胞的生长并促进胃癌细胞凋亡，并且其作用机制与抑制Wnt/β-catenin信号通路有关。YAP是潜在的治疗胃癌的基因靶点，沉默其表达后可以发挥抗肿瘤的作用，这为以后靶向YAP治疗胃癌提供了有力依据。本实验只在一株胃癌细胞系中进行了初步探讨，以后会在多株胃癌细胞和动物实验中进行验证，为明确YAP在胃癌中的作用提供可靠依据。
Competing interests: The authors declare that they have no competing interests.作者贡献：董彩霞：论文撰写袁瑛：论文审校 -
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