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摘要:目的
探讨受体酪氨酸激酶样孤儿受体-1（receptor tyrosine kinase-like orphan receptor，ROR1）诱导人肺癌A549细胞上皮-间质转化（epithelial-mesenchymal transition, EMT）的作用及可能机制。
方法构建ROR1慢病毒表达载体，感染A549细胞筛选稳定过表达ROR1的A549细胞，采用划痕实验、Transwell实验检测A549细胞的侵袭和迁移能力；real-time PCR、Western blot分别检测ROR1、EMT相关标志物的表达。
结果过表达ROR1能够促进A549细胞向间质样细胞表型转化，Western blot结果显示Vimentin、N-cadherin表达上调，E-cadherin表达下调；划痕实验、Transwell结果显示细胞侵袭迁移能力显著增强（P=0.0023）；Western blot结果显示过表达ROR1能够上调EMT转录因子Snail的表达，干扰Snail能够逆转ROR1诱导的EMT，即下调Vimentin、N-cadherin表达，上调E-cadherin表达；划痕实验、Transwell结果显示干扰Snail显著降低细胞侵袭迁移能力（P=0.013）；进一步研究发现ROR1通过激活AKT信号而促进Snail的表达。
结论ROR1能够促进人肺癌A549细胞EMT转化，其机制可能与ROR1调控AKT/Snail信号有关。
Abstract:ObjectiveTo investigate the effect and molecular mechanism of receptor tyrosine kinase-like orphan receptor (ROR1) on epithelial-mesenchymal transition(EMT) in human lung cancer A549 cells.
MethodsLung cancer A549 cells were transfected with ROR1 lentiviral expression vector; the abilities of invasion and migration were detected by wound healing assay and Transwell assay. The expression of ROR1 and EMT-associated markers were analyzed by real-time PCR and Western blot.
ResultsThe overexpression of ROR1 could induce morphological alteration of A549 cells from epithelial morphology to mesenchymal morphology. Western blot results demonstrated that the expression of mesenchymal makers N-cadherin and Vimentin were increased but epithelial marker E-cadherin was decreased. Wound healing and Transwell assay showed that the number of invasive and migrated A549 cells was significantly increased (P=0.0023). Moreover, ROR1 overexpression significantly increased the expression of EMT-related transcription factor Snail. The knockdown of Snail reversed ROR1-induced EMT, decreasing the expression of N-cadherin and Vimentin, but increasing E-cadherin expression. Wound healing and Transwell assay showed that the knockdown of Snail significantly decreased the invasion and migration of A549 cells(P=0.013). Furthermore, the activation of AKT contributed to ROR1-mediated regulation of Snail.
ConclusionROR1 could promote EMT in A549 cells, which is related to the activation of AKT/Snail signaling.
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Key words:
- Lung cancer /
- ROR1 /
- Epithelial-mesenchymal transition /
- Snail /
- AKT
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0 引言
肺癌在中国是发病率和死亡率最高的恶性肿瘤，肺癌的转移是导致肺癌患者死亡的重要原因[1]。上皮细胞间质化过程（epithelial-mesenchymal transition, EMT）是肺癌转移的关键起始步骤，在肺癌的发生发展中起着重要的作用[2-3]。
受体酪氨酸激酶样孤儿受体（receptor tyrosine kinase-like orphan receptor 1, ROR1）是Ⅰ型受体酪氨酸激酶家族中的成员，参与细胞间信号交流、胞内信号转导等过程，调节细胞增殖、分化和转移[4]。ROR1主要在胚胎发育过程中表达，而在成年人体细胞和组织中通常不表达[5]。然而新近研究发现，ROR1在乳腺癌、卵巢癌、结直肠癌、胰腺癌等多种恶性肿瘤中表达，提示ROR1可能参与了恶性肿瘤的发生发展过程[6-9]。另有研究[9-10]发现，ROR1在肺腺癌组织中显著高表达，且与临床分期、淋巴结转移、预后不良相关。然而，迄今ROR1在肺癌发生发展中的作用及机制尚不明确。本研究拟探讨过表达ROR1对肺癌A549细胞上皮-间质转化的影响。
1 材料与方法
1.1 材料
RPMI 1640培养液，胎牛血清，胰蛋白酶购自Gibco公司（上海）；E-cadherin、N-cadherin、Vimentin、ROR1、Snail、Twist、Zeb1、p-AKT、AKT抗体购自CST公司（美国）；HRP标记山羊抗鼠IgG、山羊抗兔IgG、β-actin购自Santa Cruz公司（美国），预染蛋白质Marker购自Fermentas公司（美国）；RIPA裂解液购自碧云天生物技术公司（上海）；ECL化学发光底物试剂盒购自Pierce公司（美国）；Quick Start Bradford蛋白定量试剂购自Bio-Rad公司（美国）；反转录试剂盒、RT-PCR试剂盒购自TaKaRa公司（大连）；PCR引物由上海生工公司合成；Lenti-Pac™ HIV慢病毒包装试剂盒购自复能基因（广州）；嘌呤霉素购自Sigma公司（美国）；Transwell小室购自Corning公司（美国），其余试剂均为国产分析纯以上。
1.2 细胞培养
人肺癌A549细胞购自中科院上海细胞库。人肺癌A549细胞用含10%胎牛血清（fetal calf serum, FBS）的RPMI 1640培养液，于37℃、5% CO2的培养箱内饱和湿度培养。
1.3 ROR1稳定细胞株筛选
ROR1慢病毒表达载体（LV-ROR1）及对照慢病毒表达载体（LV-Control）购自广州复能基因有限公司。接种293T细胞于10 cm的培养皿，参照Lenti-Pac™ HIV慢病毒包装试剂盒说明书包装慢病毒，48 h后收集含慢病毒颗粒的上清，离心去除细胞碎片，测定慢病毒颗粒滴度。
接种A549细胞于6孔板贴壁培养，慢病毒感染细胞24 h之后，更换RPMI 1640完全培养液。48 h后采用1 µg/ml嘌呤霉素筛选稳定表达ROR1的细胞株。
1.4 细胞划痕实验
接种细胞于6孔板贴壁培养，待细胞汇合时，用10 µl微量移液枪头垂直于板面划痕，PBS洗涤3次后加入含1%胎牛血清的RPMI 1640培养基，在倒置显微镜下观察划痕两侧细胞在0、48 h的迁移情况，并拍照分析。
1.5 Transwell侵袭实验
将Transwell小室放入培养板中，在上室加入300 µl预温的无血清培养液，室温下静置15~30 min，使基质胶再水化。消化细胞，用PBS洗2遍，用无血清培养液重悬。调整细胞密度至3×105个/毫升。取细胞悬液200 µl加入24孔板Transwell小室。24孔板下室加入500 µl含FBS的培养液。对24孔板Transwell小室的细胞进行处理。继续培养24 h后用4%多聚甲醛溶液固定小室30 min，结晶紫染色30 min，观察细胞并拍照，在高倍镜下分别选取4个视野统计细胞迁移数，结果取4个数值的平均值。
1.6 Real-time PCR
Real-time PCR按照TRIzol（Invitrogen，英杰生命技术有限公司，美国）说明书方法提取细胞总RNA，按Primescript RT reagent Kit反转录试剂盒（TaKaRa，宝生物工程有限公司，大连）说明书将RNA反转录为cDNA。引物由上海生工生物工程有限公司设计并合成，引物序列如下：ROR1上游引物：5’-AATGCAGAGTAACGTGGAAGTGGTC-3’，ROR1下游引物：5’-TGGTCGCTCAATCTCCAGGTC-3’；Snail上游引物：5’-GACCACTATGCCGCGCTCTT-3’，Snail下游引物：5’-TCGCTGTAGTTAGGCTTCCGATT-3’；GAPDH上游引物：5’-GCACCGTCAAGGCTGAGAAC-3’，GAPDH下游引物：5’-TGGTGAAGACGCCAGTGGA-3’。Real-time PCR反应体系及条件参照SYBR Premix Ex TapTM试剂盒（TaKaRa，大连），2–ΔΔCt法计算mRNA的相对表达量，以GAPDH作为内参。每个实验组重复3次。
1.7 蛋白提取和Western blot分析
收集细胞，PBS洗涤2次后用RIPA裂解液裂解，离心后收集上清液，用BCA法测定蛋白浓度；等量蛋白样品经10%的SDS-PAGE电泳分离后，转印至PVDF膜上，5%脱脂奶粉室温封闭2 h，ROR1、E-cadherin、N-cadherin、Vimentin、Snail、Twist、Zeb1、AKT、p-AKT、β-actin抗体均以1:1 000比例稀释，4℃孵育过夜。经PBST洗涤后，加入HRP标记的特异性二抗以1:5 000比例稀释，室温下孵育2 h，最后用ECL化学发光试剂对X光片显影，扫描图片。
1.8 统计学方法
应用软件GraphPad Prism 5.0进行统计学分析，实验所得数据用平均数±标准差（x±s）表示，独立样本采用t检验，组间比较采用单因素方差分析，P < 0.05为差异有统计学意义。
2 结果
2.1 过表达ROR1对A549细胞侵袭迁移能力的影响
对照组RNA vs. ROR1过表达组RNA：1 vs.（4.8±1.1）（P=0.0009），见图 1A~1B。倒置相差显微镜观察细胞形态发现，A549/ROR1细胞呈梭形“间质细胞样”，散在生长，见图 1C。划痕实验发现过表达ROR1能够明显促进A549细胞的迁移，见图 1D。Transwell实验发现A549/ROR1细胞穿过Transwell小室的数量比对照组A549/Control显著增多，对照组vs. ROR1过表达组：（61±12）vs.（185±23）（P=0.0023），见图 1E。说明过表达ROR1能显著增强A549细胞的侵袭迁移能力。
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图 1 过表达ROR1对A549细胞侵袭迁移能力的影响Figure 1 Effect of ROR1 overexpression on invasion and migration abilities of A549 cellsA: the expression of ROR1 mRNA was detected by real-time PCR; B: the expression of ROR1 protein was detected by Western blot; C: the effect of ROR1 overexpression on A549 cells morphology by inverted microscopy; D: the effect of ROR1 overexpression on A549 cells migration was detected by wound healing assay; E: the effect of ROR1 overexpression on A549 cells invasion was detected by Transwell assay2.2 过表达ROR1对A549细胞EMT相关标志物表达的影响
过表达ROR1能明显抑制E-cadherin的表达、促进Vimentin、N-cadherin的表达。进一步检测过表达ROR1对EMT相关转录因子表达的作用，结果发现过表达ROR1能够促进转录因子Snail的表达，而对Twist、Zeb1的表达没有明显的影响，见图 2。
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图 2 过表达ROR1对A549细胞EMT相关标志物表达的影响Figure 2 Effect of ROR1 overexpression on expression of EMT-related markers in A549 cells2.3 干扰Snail对ROR1诱导A549细胞EMT的影响
在过表达ROR1的A549细胞进一步干扰Snail，对照组RNA vs. Snail干扰组RNA：1 vs.（0.25±0.13）（P=0.0007），见图 3A。48 h后取细胞总蛋白，Western blot结果显示转染Snail siRNA能明显上调E-cadherin蛋白的表达，下调Vimentin、N-cadherin蛋白的表达，见图 3B。说明干扰Snail能够逆转ROR1对A549细胞EMT的诱导作用。
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图 3 干扰Snail对ROR1诱导A549细胞EMT的影响Figure 3 Effect of Snail knockdown on ROR1-induced EMT in A549 cells1: LV-Control; 2: LV-ROR1; 3: LV-ROR1+Snail siRNA; A: the expression of Snail mRNA was detected by real-time PCR; B: the expressions of Snail and EMT-related markers were detected by Western blot2.4 干扰Snail对ROR1诱导A549细胞侵袭迁移的影响
在过表达ROR1的A549细胞进一步干扰Snail，细胞划痕实验结果显示干扰Snail能明显抑制ROR1对A549细胞迁移的诱导作用，见图 4A。Transwell实验结果发现Snail干扰组穿过基质胶的侵袭细胞数明显少于ROR1过表达组，ROR1过表达组vs. Snail干扰组：（182±10）vs.（110±22），差异有统计学意义（P=0.0130），见图 4B。
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图 4 干扰Snail对ROR1诱导A549细胞侵袭迁移的影响Figure 4 Effect of Snail knockdown on ROR1-induced invasion and migration of A549 cells1: LV-Control; 2: LV-ROR1; 3: LV-ROR1+Snail siRNA; A: the effect of Snail knockdown on ROR1-induced migration of A549 cells was detected by wound healing assay; B: the effect of Snail knockdown on ROR1-induced invasion of A549 cells was detected by Transwell assay2.5 AKT介导ROR1对A549细胞EMT的诱导作用
分别提取A549/ROR1和A549/Control细胞总蛋白，Western blot法检测AKT的表达及磷酸化水平。结果发现过表达ROR1能够促进AKT的磷酸化，但是对AKT蛋白表达无明显的影响，见图 5A，结果说明ROR1能够促进AKT信号通路的活化。为进一步观察ROR1激活AKT信号是否介导ROR1对EMT的诱导作用，采用AKT抑制剂LY294002处理A549/ROR1细胞后发现，LY294002能够明显抑制Snail和Vimentin的表达，但是能促进E-cadherin的表达，见图 5B，结果说明ROR1激活AKT信号参与了ROR1对EMT的诱导作用。
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图 5 AKT介导ROR1对A549细胞EMT的诱导作用Figure 5 AKT contributed to ROR1-mediated induction of EMT in A549 cells1: LV-Control; 2: LV-ROR1; A: the effect of ROR1 on the expression and phosphorylation of AKT in A549 cells; B: A549/ROR1 cells were treated with LY294002 for 48h, and the expressions of Snail and EMT-related markers were detected by Western blot3 讨论
近年来，ROR1在多种恶性肿瘤中异常表达已引起广泛关注。MacKeigan等最先发现ROR1具有原癌基因的作用[11]。随后，多项研究表明ROR1能够体内外促进肿瘤生长，在肿瘤恶性进展中发挥着促进作用[6-9]。研究发现，ROR1在转移潜能较高的低分化肿瘤中高表达，且与EMT相关标志物的表达相关[6, 12]。靶向抑制ROR1能够明显抑制乳腺癌细胞迁移与浸润能力[12]。然而，ROR1是否参与肺癌EMT发生尚不明确。本研究转染ROR1过表达质粒至肺癌A549细胞中，结果显示过表达ROR1能够诱导肺癌细胞EMT，上调间质性标志物Vimentin、N-cadherin的表达，而下调上皮间质转化标志物E-cadherin的表达。细胞划痕、Transwell实验进一步证明过表达ROR1能够促进肺癌细胞侵袭迁移能力。
肿瘤EMT发生主要受一类转录抑制因子超家族成员所调控，转录因子Snail是肿瘤细胞EMT的关键调控因子[13]。Snail是一种锌指DNA结合蛋白，主要通过与钙黏素（E-cadherin）启动子区的E-box连接基序结合，抑制E-cadherin的转录表达，从而参与调控EMT过程。此外，Snail也能通过其他方式参与调控EMT，如增强间质细胞/成纤维细胞标志物表达（N-cadherin、vimentin、Fibronectin等）[14]。我们发现ROR1处理显著促进A549细胞Snail蛋白的表达，而干扰Snail能够逆转ROR1对A549细胞EMT的诱导作用。
已知Snail的表达与活性受细胞内多条信号通路调节，如TGF-β1/Smad、TNF-α/NF-κB、AKT等信号[15-16]。此外，研究发现AKT信号参与了TGF-β促进EMT发生，AKT通过磷酸化失活GSK-3β而上调Snail的表达[17]。我们发现过表达ROR1能够促进AKT的磷酸化，而采用AKT抑制剂能够逆转ROR1对Snail的表达调控作用。提示ROR1可通过AKT/Snail信号促进肺癌细胞EMT转化。与本研究结果类似，Fernández等[18]同样发现ROR1能够激活PI3K/AKT信号而促进黑色素瘤细胞生长和迁移。新近研究发现，ROR1能够调控EGFR/ErbB3异源二聚化，并进一步诱导c-Src的磷酸化/活化[19]。已知Src是一种非受体酪氨酸蛋白激酶，活化的Src可通过激活AKT、MAPK等多种信号参与细胞内信号转导的过程[20]。另有研究表明ROR1能够与蛋白激酶CK1结合而直接激活PI3K/AKT[6]。因此，ROR1促进AKT磷酸化可能与ROR1调控c-Src或CK1的活性有关，然而具体作用机制还有待进一步研究。
总之，本研究发现ROR1能通过调节AKT/Snail信号促进肺癌A549细胞EMT转化，进而增强细胞浸润和转移能力，为靶向ROR1抑制肺癌EMT及转移提供重要的理论基础。
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图 1 过表达ROR1对A549细胞侵袭迁移能力的影响
Figure 1 Effect of ROR1 overexpression on invasion and migration abilities of A549 cells
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图 2 过表达ROR1对A549细胞EMT相关标志物表达的影响
Figure 2 Effect of ROR1 overexpression on expression of EMT-related markers in A549 cells
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图 3 干扰Snail对ROR1诱导A549细胞EMT的影响
Figure 3 Effect of Snail knockdown on ROR1-induced EMT in A549 cells
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图 4 干扰Snail对ROR1诱导A549细胞侵袭迁移的影响
Figure 4 Effect of Snail knockdown on ROR1-induced invasion and migration of A549 cells
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