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1 :02 中国细胞生物学学报 Chinese Journal of Cell Biology 2014, 6(5): DOI: /cjcb Lipoxin A 4 通过调节肿瘤相关巨噬细胞对肝癌细胞株 HepG2 microrna 表达谱的影响黄婉秋 1# 马明洋 2# 程雪王褚琳苗烁吴萍 * ( 1 华中科技大学 2010 级临床医学八年制, 武汉 4000; 2 华中科技大学附属同济医院肝脏外科, 武汉 4000; 华中科技大学同济医学院基础医学院病理生理学系, 武汉 4000) 摘要肿瘤与炎症密切相关, 作者以往已证实内源性促炎症缓解介质脂氧素 A 4 (Lipoxin A 4, LXA 4 ) 能在整体和细胞水平发挥抗肝癌细胞增殖和转移的作用为进一步探讨 LXA 4 通过调节肿瘤相关巨噬细胞对肝癌细胞株 HepG2 micrornas(mirnas) 表达谱的影响, 该文首先提取经脂多糖 (LPS) 或 LPS+LXA 4 作用 24 h 的人巨噬细胞株 U97 培养上清液, 分别称为 ACM 或 LCM, 以模拟肿瘤的炎症微环境, 并用此上清液刺激 HepG2 细胞, 24 h 后提取细胞总 RNA, 采用 microrna 芯片 mir- CURY TM LNA Array(V16.0) 检测, 计算各样本中的 mirnas 标准值及比值以两组间 Hy 荧光标记信号强度的比值 0.5 或 2 为标准判定差异表达 mirna Real-time PCR 检测 hsa-mir-62 的相对含量以验证基因芯片的结果结果发现, 与对照组细胞相比, 经过 ACM 作用 24 h 的 HepG2 细胞有 5 个 mirnas 上调 10 个 mirnas 下调 LCM 组与 ACM 组相比, HepG2 细胞有 185 个 mirnas 上调 71 个 mirnas 下调 Real-time PCR 检测的结果证实, hsa-mir-62 的变化与基因芯片趋势一致综上所述, LXA 4 能通过肿瘤相关巨噬细胞而间接发挥其调节 HepG2 细胞 mirnas 表达谱的作用关键词肝癌 ; 微小 RNA; 脂氧素 Effect of Lipoxin A 4 on MicroRNAs Expression Profile in HepG2 Cells through Modulating the Tumor Associated Macrophages Huang Wanqiu 1#, Ma Mingyang 2#, Cheng Xue, Wang Chulin, Miao Shuo, WU Ping * ( 1 Eight-year Program of Clinical Medicine, 2010 Session, Huazhong University of Science and Technology, Wuhan 4000, China; 2 Hepatic Surgery Center, Tongji Hospital, Huazhong University of Science and Technology, Wuhan 4000, China; Department of Pathophysiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 4000, China) Abstract On the basis that the pathologic process of tumor is closely related to uncontrolled inflammation, we have proved that Lipoxin A 4 (LXA 4 ) could inhibit the proliferation and metastasis of HCC cells both in vitro and in vivo. This study further investigated the indirect effect of Lipoxin A 4 (LXA 4 ) on the microrna (mirna) expression profile in HepG2 cells through modulating the tumor-associated macrophages. Conditioned cell culture media from LPS-stimulated and LPS/LXA 4 co-stimulated U97 cells, as ACM and LCM respectively, 收稿日期 : 接受日期 : 国家自然科学基金 ( 批准号 : ) 和中央高校基本科研业务费华中科技大学 ( 批准号 2012QN ) 资助的课题 # 共同第一作者 * 通讯作者 Tel: , wpingwp@mails.tjmu.edu.cn Received: October 10, 201 Accepted: January 20, 2014 This work was supported by the National Natural Science Foundation of China (Grant No ) and the Fundamental Research Funds for the Central Universities, HUST (Grant No.2012QN2, 201-1) # These authors contributed equally to this work *Corresponding author. Tel: , wpingwp@mails.tjmu.edu.cn

2 2 研究论文 were collected. HepG2 cells were separated into control group, ACM group and LCM group, which were cultured in normal culture media, ACM and LCM for 24 hours, respectively. mirnas were extracted, labeled with mircury TM Hy TM /Hy5 TM Power labeling kit and hybridized to microarray. It was considered to be up- or downregulated when the mirnas fluorescent intensity ration between two groups was over 2 or less than 0.5. Validation of microarray results was carried out by real-time quantitative RT-PCR of hsa-mir-62. Compared with control group, ACM treatment for 24h up-regulated 5 mirnas and down-regulated 10 mirnas in HepG2 cells, while, LCM group had 185 mirnas higher and 71 mirnas lower than ACM group. Hsa-miR-62 showed similar variation with microarray. Our study indicated that LXA 4 could indirectly regulate the mirnas expression profile in HepG2 cells through modulating the tumor-associated macrophages. Key words hepatic carcinoma; microrna; lipoxin 肿瘤是目前严重影响人类生命和健康的疾病之一经过多年的研究, 虽然在原癌基因抑癌基因以及肿瘤相关的信号转导通路等方面都取得了较多令人兴奋的成果, 但肿瘤发生发展的确切机制仍有许多未解之谜 [1] micrornas(mirnas) 是一类新近发现的非编码小分子 RNA, 包含 21~25 个核苷酸, 是哺乳动物基因组中最为丰富的调节基因之一迄今, mirna 的基因序列数据库 mirbase( microrna.sanger.ac.uk/) 已收录了约种人类 mi- RNA [2] 近年来的研究显示其在肿瘤的发生发展过程中发挥着重要的调节作用 [] 肿瘤相关巨噬细胞 (tumor-associated macrophages, TAMs) 处于炎症和肿瘤交叉路口这一特殊位置, 参与了肿瘤的发生转移基质重构血管新生等几乎各个环节 [4] 本课题组以往证实, 内源性促炎症缓解介质脂氧素 A 4 (Lipoxin A 4, LXA 4 ) 能抑制巨噬细胞的炎症反应 [5-6], 亦能在整体和细胞水平发挥抗肝癌细胞增殖和转移的作用 [7-8] 在此基础上, 我们还证实, 经过 LPS 活化的巨噬细胞其上清液作用于 HepG2 肝癌细胞株后, 对其增殖和迁移均有促进, 而 LXA 4 对上述作用具有明显的抑制那么, LXA 4 的这一作用与 HepG2 细胞的 mirnas 表达有何关联? 目前还未见报道本文采用 mirna 芯片技术, 初步研究了 LXA 4 通过调节 TAM 对肝癌细胞株 HepG2 mirnas 表达谱的影响 1 材料与方法 1.1 细胞与试剂人肝癌细胞株 HepG2 和人单核细胞株 U97 购自中科院上海细胞库 1640 培养基和胎牛血清 (FBS) 为 Gibco 公司产品 ; LXA 4 购自 Cayman 公司 ; 脂多糖 (lipopolysaccharide, LPS) 购自 Sigma 公司 ; Trizol 购自 Invitrogen 公司 ; mirna RT-PCR 相关试剂购自 QIAGEN 公司 ; mircury TM Hy TM /Hy5 TM Power labeling kit 和 mircury TM LNA Array kit 购自 Exiqon 公司 ; 0.45 μm 滤器为 Fisher Scientific 公司产品 1.2 试验方法细胞培养细胞在含有 10% 胎牛血清 2% 谷氨酰胺 100 U/mL 链霉素和 100 U/mL 青霉素的 1640 完全培养基中, 置于 7 C 5% CO 2 培养箱内常规培养, 细胞贴壁达到 80%~90% 时进行传代巨噬细胞上清液的制备和收集经常规培养无血清化过夜后, U97 巨噬细胞分别于 100 ng/ml LPS 或 100 ng/ml LPS+100 nmol/l LXA 4 中培养 24 h 弃培养基并用 PBS 洗涤 2 次无血清培养基中继续培养 4 h 以清除残余刺激物最后, 细胞在完全培养基中生长 24 h 后收集上清液, 0.45 μm 滤器过滤经 LPS 刺激的巨噬细胞上清液称为 (activated macrophageconditioned media, ACM), 经 LPS+LXA 4 作用的巨噬细胞上清液称为 LCM 1.2. 实验分组按本课题组建立的条件, HepG2 细胞分为空白对照组 ACM 和 LCM 组, 分别采用完全培养基 50% ACM+50% 完全培养基 50% LCM+50% 完全培养基作用 24 h 细胞总 RNA 的提取和鉴定参照 Trizol 说明书, 采用一步法收集细胞总 RNA 紫外分光光度计测定 RNA 的浓度和纯度取 1 μg 总 RNA 进行 1.5% 甲醛变性凝胶电泳, 分析 RNA 质量芯片分析差异表达的 mirna 应用丹麦 Exiqon 公司生产的 microrna 芯片 mircury TM LNA Array(V16.0) 进行检测, 由上海康成生物工程有限公司代理检测和分析该芯片检测了 mirbase( 版本

mirnas 标准值及比值以两组间 Hy 荧光标记信号强度的比值 0.5 或 2

3 黄婉秋等 : Lipoxin A 4 通过调节肿瘤相关巨噬细胞对肝癌细胞株 HepG2 microrna 表达谱的影响 16.0) 收录的个属于人小鼠或大鼠的 mirna 以及 66 个 Exiqon 公司独家的 microrna 分别取 1 μg 上述各组细胞样本的总 RNA, 采用 mircury TM Hy TM /Hy5 TM Power labeling kit 进行荧光标记芯片杂交其后进行图像扫描, 所得数据通过原始值减去背景值进行修正, 并用中值标准化, 分别计算各样本中的 mirnas 标准值及比值以两组间 Hy 荧光标记信号强度的比值 0.5 或 2 为标准判定差异表达 mirnas 定量 PCR 验证差异表达的 mirnas 为进一步验证芯片结果, 选取在芯片实验中发现的几组细胞之间含量有明显差异的 hsa-mir-62 进行实时荧光 PCR 验证按照操作指南, 首先以细胞总 RNA 为模板, 用 miscript II RT kit 逆转录生成 cdna; 进一步采用 hsa-mir-62 的特异引物 Hs_miR-62_1 miscript Primer Assay 和通用引物 The miscript Universal Primer, 利用 miscript SYBR Green PCT kit 进行实时荧光 PCR 内参为 U6, 其特异性引物采用 Hs_RNU6-2_11 miscript Primer Assay 反应条件为: 95 C 预变性 15 min; 94 C 15 s, 55 C 0 s, 70 C 0 s, 40 个循环 ; 反应结束后得到各组目的 mirna 和内参的 Ct 值以 ΔΔCt 法计算 hsa-mir-62 的相对表达量 1: 对照组 ; 2: ACM 组 ; : LCM 组 1: control group; 2: ACM group; : LCM group. From left to right, control group, ACM group and LCM group. 图 1 三组细胞的 RNA 甲醛变性琼脂糖凝胶电泳图 Fig.1 RNA agarose gel electrophoresis of HepG2 cells 2 结果 2.1 细胞总 RNA 质量检测从各组细胞中提取的 RNA 浓度分别为 , , mg/l D 260/280 值均在 2.0~2.04 之间其 18S 和 28S 条带清晰, 总 RNA 纯度达到实验要求且无明显降解 ( 图 1) 实验结果提示, 来自组细胞的总 RNA 质量较好, 能满足后续 mirna 芯片实验的需要 2.2 mirna 芯片检测结果来自组细胞的总 RNA 经过 Hy 荧光标记后, 同 mirna 芯片进行杂交结果显示, 与空白组细胞相比, 经过 ACM 作用 24 h 的 HepG2 细胞有 5 个 mirna 上调 10 个 mirna 下调 LCM 组与 ACM 组相比, HepG2 细胞有 185 个 mirna 上调 71 个 mirna 下调 ( 图 2) 表 1 列出部分相对含量变化明显的 mirnas, 并总结了其相应的功能 2. hsa-mir-62 Real-time PCR 结果为了进一步验证基因芯片的结果, 我们随机选取了在 mirna 芯片中具有差异表达的 hsa-mir-62 进行 RT-PCR 检测, 数据表明其变化趋势与基因芯片 1: 对照组 ; 2: ACM 组 ; : LCM 组细胞分别采用完全培养基 50% ACM+50% 完全培养基 50% LCM+50% 完全培养基作用 24 h, 然后进行 mirna 芯片检测 1: control group; 2: ACM group; : LCM group. Before analyzed with mirna microarray, cells were cultured in completed medium, 50% ACM+50% complete medium, 50% LCM+50% complete medium for 24 h, respectively. 图 2 各组 HepG2 细胞的 mirna 芯片扫描图 Fig.2 mirna microarray maps of HepG2 cells 相同 : ACM 作用后的 HepG2 细胞 hsa-mir-62 表达下调, 相反, LCM 组的表达较 ACM 增高 ( 表 2) 讨论自 186 年最初观察到肿瘤组织周围有炎性细胞浸润 [9], 其后大量证据提示肿瘤与炎症息息相关,

4 4 研究论文 [10] 肿瘤相关炎症甚至被称为肿瘤的另一半 LX 是花生四烯酸经过脂加氧酶 (lipoxygenase, LOX) 代谢途径的产物, 已被证实为体内最重要的抗炎及促炎 [11] 症消退脂质, 具有炎症刹车信号之称我们以往针对移植 H22 肝癌细胞的小鼠研究证实, LXA 4 及其类似物不仅能减缓移植部位肿瘤的 [8] 生长, 还显著抑制了肿瘤的肺转移 ; 其机制可能与 [8] LXA 4 抑制肿瘤内部血管新生减弱肝癌细胞的侵 [12] 袭能力有关我们进一步发现, LXA 4 处理后的移植肝癌小鼠的癌组织里, TAMs 数量减少, 且由弥漫 [8] 分布于肿瘤内部转向肿瘤周围 ; 在细胞水平, LXA 4 能通过巨噬细胞间接抑制肝癌细胞迁移和增殖 TAMs 处于炎症和肿瘤交叉路口这一特殊位 [4] 置, 为肿瘤微环境的重要组成部分, 也是目前肿瘤治疗策略的研究热点为深入探讨 TAM 在 LXA 4 抑制肝癌细胞中的作用, 本实验中我们将 mirnas 作为研究的靶点 mirna 是一个在进化上高度保守的小分子 RNA 家族, 主要通过结合于靶 mrna 的末端非翻译区介导蛋白翻译阻抑, 或者通过诱导靶 mrna 的端去腺苷酸化或 5 端去帽而介导靶 mrna 的快速降解, 对靶基因的表达进行调节其在调节肿瘤细胞增殖分化凋亡等方面扮演了重 [] 要的角色以往对 mirnas 的研究多集中于比较其在肿瘤组织与正常组织的差异, 本文采用已发表的方法, 以 LPS 活化巨噬细胞并收集其培养上清液以模拟肿瘤的炎症微环境, 进而用此 ACM 刺激 HepG2 细胞以期探讨肿瘤微环境对肝癌细胞 mirna 的影响诚然, 肿瘤微环境中的细胞种类很多, 除了 TAM 还包括其他如血管内皮细胞, 白细胞纤维母细胞甚至是脂肪细胞等 ; 同时, 肿瘤的微环境也并非单由炎症环境构成, 其他如缺氧等微环境也影响了肿瘤的发展因此, 本文所采用的 LPS 刺激的模型只能帮助我们从一个侧面研究 LXA 4 的作用结果发现 LPS 能通过活化巨噬细胞而改变肝癌细胞的 mirnas 表达谱 ( 表 1): 如能上调 hsa-mir-181a, 后者被发现在慢性 [1] 淋巴细胞白血病细胞中高表达 ; 上调 hsa-mir-125, [14] 后者能抑制抑癌基因 p5 从而促进肿瘤生长 ; 上调 mir-0a, 后者与肿瘤对细胞外基质的黏附有关 ; 上调促进 EMT 的 mir-141 和 mir-216 同时, 为了探讨 LXA 4 通过肿瘤微环境的间接作用, 我们也收集了 LXA 4 和 LPS 共同作用的巨噬细胞上清液刺激 HepG2 细胞, 数据显示 LXA 4 亦能通过对活化的巨噬细胞发挥作用, 间接改变肝癌细胞的 mirnas 一项涉及 8 例肝细胞肝癌的临床调查显示, 肝癌组织的 mir-4a 明显低于正常组织, 且 TNM III 期和 IV 期的患者低于 I 期和 II 期患者, 同时, 发生转移的患者低于未发生转移的患者 ; 在细胞水平还证实转染了 mir-4a 的肝癌细胞其增殖减缓转移和 [15] 浸袭能力减弱本文中, 芯片结果显示, ACM 刺激组 mir-4a 仅为对照组细胞的 45%, 而 LXA 4 处理过的巨噬细胞上清液明显增加了 HepG2 细胞的 mir- 4a 的表达这有可能是 LXA 4 抑制肝癌的原因之一除此之外, 结果还发现, LCM 能下调其他促癌的 mirnas 如 mir-27a, 上调抑癌 mirnas 如 mir-22 等肿瘤内部的血管新生是实体瘤发展的必需, 本 ACM groups vs control group mirnas 表 1 各组 HepG2 细胞部分 mirnas 变化情况 Table 1 Change of mirnas in HepG2 cells with different treatments 倍数功能 mirnas Ratio Function hsa-mir-216b Down-regulated the expression of CKIIα in colon cancer [16] hsa-mir-181a 12.9 Highly expressed in chronic CLL cells [1] and gastric cancer tissue [17] ; In malignant hsa-mirplus-g1267-5p 9.84 Patent product of Exiqon glioma cells, has-mir-181a was down-regulated by radiation treatment [18] hsa-mir-0a 8.91 Down-regulated in colon cancer. mir-0a suppresses cell migration and invasion through downregulation of PIKCD in colorectal carcinoma [19] hsa-mir mir-62 was up-regulated in transient depletion of TIA-proteins in HeLa cells [20] hsa-mir Differently expressed between decidualized and undecidualized endometrial stromal cells [21] hsa-mir Expressed in human sarcomas [22] hsa-mir-10-5p 0.02 Not reported hsa-mir-2114* 0.01 Not reported

5 黄婉秋等 : Lipoxin A 4 通过调节肿瘤相关巨噬细胞对肝癌细胞株 HepG2 microrna 表达谱的影响 5 LCM group vs ACM group ( 续表 1) hsa-mir-1224-p 0.62 The combination of mirs-15b/15b/1224-p detected bladder cancer with a high sensitivity (94.1%) [2] hsa-mir-1909* 22.1 Significantly upregulated in TRG1 patients [24] hsa-mir mir-62 was up-regulated in transient depletion of TIA-proteins in HeLa cells [20] hsa-mir Expressed in human sarcomas [22] hsa-mir Regulates carcinogenesis in malignant peripheral nerve sheath tumor [25] hsa-mir-191* 9.27 Up-regulated in breast cancer [26] and hepatocellular carcinoma [27] ; hsa-mir-667-5p 0.08 Not reported hsa-mir-27b 0.08 Up-regulated in breast cancer [28] hsa-mir-0a 0.07 Down-regulated in colon cancer. mir-0a suppresses cell migration and invasion through downregulation of PIKCD in colorectal carcinoma [19] hsa-mir-19b 0.06 Highly expressed in basal cell carcinoma [29] hsa-mir-181a 0.0 Highly expressed in chronic CLL cells [1] and gastric cancer tissue [17] ; In malignant glioma cells, has-mir-181a was down-regulated by radiation treatment [18] hsa-mir-78c 0.0 mir-78c was expressed after treated with X ray for 8 hours in TK6 cells [0] hsa-mir-27a 0.02 Significantly higher in breast cancer with lymph node metastasis [1], increase angiogenesis in tumor [2] hsa-mir-216b 0.00 Down-regulated the expression of CKIIα in colon cancer [16] 表 2 各组 HepG2 细胞 hsa-mir-62 表达情况 (n=8) Table 2 Expression of hsa-mir-62 in HepG2 cells with different treatments (n=8) 对照组 Con group ACM 组 ACM group LCM 组 LCM group Hsa-miR-62 Ct 22.4± ± ±0.2 U6 Ct 4.45± ± ±0.42 ΔCt ± ± ±0.94 ΔΔCt ACM/Con ACM vs Con LCM/Con LCM vs Con 2.2± ± ΔΔCt Relative value of Has-miR (0.18~0.26) Hsa-miR-62 的检测采用 Real-time PCR 法, 计算方法采用 ΔΔCt 法每组细胞重复 4 次, 每次做 2 个平行孔 0.72 (0.8~1.9) LCM/ACM LCM vs ACM -2.7± (.9~12.47) Hsa-miR-62 was measure by Real-time PCR and calculated with ΔΔCt method. The experiment was repeated for four times with 2 wells for each group every time. 试验中 LCM 能上调某些抑制血管新生的 mirna, 如 mir-20 和 mir-20b 另外, mirna 芯片结果还表明, 多种涉及到 EMT 的 mirnas 如 mir-0a, mir-20 亦受到了 LCM 的调节 ; 而对于能抑制肿瘤干性的 mir- 18, mir-148b, LCM 明显增加了其含量总之, 本文首次报道了促炎症缓解的介质 LXA 4 通过调节 TAM 而间接影响肝癌细胞的 mirnas 表达谱, 这不仅进一步阐明了 LXA 4 抗肝癌的作用机制, 更为肝癌的治疗提供了新的思路, 但具体的作用信号通路还有待进一步研究参考文献 (References) 1 Abdullah LN, Chow EK. Mechanisms of chemoresistance in cancer stem cells. Clin Transl Med 201; 2(1):. 2 Sita-Lumsden A, Dart DA, Waxman J, Bevan CL. Circulating micrornas as potential new biomarkers for prostate cancer. Br J Cancer 201; 108(10): Ceppi P, Peter ME. MicroRNAs regulate both epithelial-to-mesenchymal transition and cancer stem cells. Oncogene 201; (): Siveen KS, Kuttan G. Role of macrophages in tumour progression. Immunol Lett 2009; 12(2): Zhou XY, Wu P, Zhang L, Xiong W, Li YS, Feng YM, et al. Effects of lipoxin A 4 on lipopolysaccharide induced proliferation

6 6 研究论文 and reactive oxygen species production in RAW264.7 macrophages through modulation of G-CSF secretion. Inflamm Res 2007; 56(8): Zhang L, Wu P, Jin SW, Yuan P, Wan JY, Zhou XY, et al. Lipoxin A4 negatively regulates lipopolysaccharide-induced differentiation of RAW264.7 murine macrophages into dendriticlike cells. Chin Med J 2007; 120(11): Hao H, Liu M, Wu P, Cai L, Tang K, Yi P, et al. Lipoxin A4 and its analog suppress hepatocellular carcinoma via remodeling tumor microenvironment. Cancer Lett 2011; 09(1): Chen Y, Hao H, He S, Cai L, Li Y, Hu S, et al. Lipoxin A4 and its analogue suppress the tumor growth of transplanted H22 in mice: the role of antiangiogenesis. Mol Cancer Ther 2010; 9(8): Balkwill F, Mantovani A. Inflammation and cancer: Back to Virchow? Lancet 2001; 57(9255): Balkwill FR, Mantovani A. Cancer-related inflammation: Common themes and therapeutic opportunities. Semin Cancer Biol 2012; 22(1): Spite M, Serhan CN. Novel lipid mediators promote resolution of acute inflammation: Impact of aspirin and statins. Circ Res 2010; 107(10): Zhou XY, Li YS, Wu P, Wang HM, Cai ZY, Xu FY, et al. Lipoxin A(4) inhibited hepatocyte growth factor-induced invasion of human hepatoma cells. Hepatol Res 2009; 9(9): Marton S, Garcia MR, Robello C, Persson H, Trajtenberg F, Pritsch O, et al. Small RNAs analysis in CLL reveals a deregulation of mirna expression and novel mirna candidates of putative relevance in CLL pathogenesis. Leukemia 2008; 22(2): Zhang Y, Gao JS, Tang X, Tucker LD, Quesenberry P, Rigoutsos I, et al. MicroRNA 125a and its regulation of the p5 tumor suppressor gene. FEBS Lett 2009; 58(22): Dang Y, Luo D, Rong M, Chen G. Underexpression of mir- 4a in hepatocellular carcinoma and its contribution towards enhancement of proliferating inhibitory effects of agents targeting c-met. PLoS One 201; 8(4): e Kim SY, Lee YH, Bae YS. MiR-186, mir-216b, mir-7-p, and mir-760 cooperatively induce cellular senescence by targeting alpha subunit of protein kinase CKII in human colorectal cancer cells. Biochem Biophys Res Commun 2012; 429(/4): Lin Y, Nie Y, Zhao J, Chen X, Ye M, Li Y, et al. Genetic polymorphism at mir-181a binding site contributes to gastric cancer susceptibility. Carcinogenesis 2012; (12): Chen G, Zhu W, Shi D, Lv L, Zhang C, Liu P, et al. MicroRNA- 181a sensitizes human malignant glioma U87MG cells to radiation by targeting Bcl-2. Oncol Rep 2010; 2(4): Zhong M, Bian Z, Wu Z. mir-0a suppresses cell migration and invasion through downregulation of PIKCD in colorectal carcinoma. Cell Physiol Biochem 201; 1(2/): Sanchez-Jimenez C, Carrascoso I, Barrero J, Izquierdo JM. Identification of a set of mirnas differentially expressed in transiently TIA-depleted HeLa cells by genome-wide profiling. BMC Mol Biol 201; 14: Hu LL, Qian K, Li HX, Sun YP, Zhu GJ. MicroRNA expression and its role in the cell cycle regulation in decidualized endometrial stromal cells in vitro. Zhonghua Fu Chan Ke Za Zhi 2012; 47(2): Subramanian S, Lui WO, Lee CH, Espinosa I, Nielsen TO, Heinrich MC, et al. MicroRNA expression signature of human sarcomas. Oncogene 2008; 27(14): Miah S, Dudziec E, Drayton RM, Zlotta AR, Morgan SL, Rosario DJ, et al. An evaluation of urinary microrna reveals a high sensitivity for bladder cancer. Br J Cancer 2012; 107(1): Della Vittoria Scarpati G, Falcetta F, Carlomagno C, Ubezio P, Marchini S, De Stefano A, et al. A specific mirna signature correlates with complete pathological response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer. Int J Radiat Oncol Biol Phys 2012; 8(4): Gong M, Ma J, Li M, Zhou M, Hock JM, Yu X. MicroRNA-204 critically regulates carcinogenesis in malignant peripheral nerve sheath tumors. Neuro Oncol 2012; 14(8): Mar-Aguilar F, Luna-Aguirre CM, Moreno-Rocha JC, Araiza- Chavez J, Trevino V, Rodriguez-Padilla C, et al. Differential expression of mir-21, mir-125b and mir-191 in breast cancer tissue. Asia Pac J Clin Oncol 201; 9(1): He Y, Cui Y, Wang W, Gu J, Guo S, Ma K, et al. Hypomethylation of the hsa-mir-191 locus causes high expression of hsa-mir- 191 and promotes the epithelial-to-mesenchymal transition in hepatocellular carcinoma. Neoplasia 2011; 1(9): Jin L, Wessely O, Marcusson EG, Ivan C, Calin GA, Alahari SK. Prooncogenic factors mir-2b and mir-27b are regulated by Her2/Neu, EGF, and TNF-alpha in breast cancer. Cancer Res 201; 7(9): Sand M, Skrygan M, Sand D, Georgas D, Hahn SA, Gambichler T, et al. Expression of micrornas in basal cell carcinoma. Br J Dermatol 2012; 167(4): Chaudhry MA, Omaruddin RA, Brumbaugh CD, Tariq MA, Pourmand N. Identification of radiation-induced microrna transcriptome by next-generation massively parallel sequencing. 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864 现代药物与临床 Drugs & Clinic 第 31 卷第 6 期 2016 年 6 月 of apoptosis related factors, decrease the incidence of adverse reactions, which is of great

864 现代药物与临床 Drugs & Clinic 第 31 卷第 6 期 2016 年 6 月 of apoptosis related factors, decrease the incidence of adverse reactions, which is of great 现代药物与临床 Drugs & Clinic 第 31 卷第 6 期 2016 年 6 月 863 曲妥珠单抗联合多西紫杉醇治疗 Her-2 阳性乳腺癌的临床研究 * 周永安, 刘训碧黄石市中心医院 ( 普爱院区 ) 乳腺肿瘤外科, 湖北黄石 435000 摘要 : 目的探析注射用曲