STAT3抑制剂下调miR-21增加人舌鳞癌对顺铂化疗敏感性的实验研究

发布时间：2018-02-03 07:53

  本文关键词： 人舌鳞状细胞癌 STAT3miR-21 顺铂 化疗耐药　出处：《天津医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：头颈部恶性肿瘤是世界范围内常见的6大常见的恶性肿瘤之一,其中舌鳞状细胞癌是头颈部恶性肿瘤最常见的病理类型。晚期舌鳞状细胞癌多数呈浸润性生长并伴有颈部淋巴结转移,手术很难完全切除已经浸润周围组织和发生远处转移的肿瘤。众所周知,顺铂(DDP)是被广泛应用于治疗人舌鳞状细胞癌的化疗药物,但DDP的不良反应以及肿瘤对其耐药性的产生严重影响了其治疗效果,因此逆转化疗耐药、提高舌鳞癌对其敏感性越来越受到关注。 STAT3在肝癌、乳腺癌、肺癌、结肠癌、头颈部鳞状细胞癌等上皮系统来源的恶性肿瘤中均有过表达,并参与多种细胞增殖、凋亡、侵袭及转移等相关基因转录调控。在多发性骨髓瘤及脑胶质瘤中均发现STAT3能够直接调控miR-21的转录。本课题选用TCA8113、TCA8113/DDP舌鳞癌细胞系,采用WP1066下调STAT3表达,并选用临床常用舌鳞癌化疗药物DDP,就WP1066联合DDP影响人舌鳞癌细胞体内外生长能力的作用效果和机制进行了以下体内外实验研究。课题研究共分为三部分： 第一部分：首先运用MTT法测定两舌癌细胞WP1066、DDP的半数抑制浓度(IC50)；Western blot、实时定量PCR法检测耐药及敏感舌鳞癌细胞株STAT3、 miR-21表达差异；Western blot与免疫荧光法检测检测WP1066及DDP处理后舌鳞癌细胞STAT3/p-STAT3表达情况；运用实时定量PCR法、原位杂交法检测两舌癌细胞WP1066、DDP处理后miR-21表达情况。运用MTT法、平板克隆实验、Matrigel基质生长实验、Transwell体外侵袭实验、细胞划痕实验、流式细胞术等研究WP1066、DDP抑制舌癌细胞体外牛长的效果。 第二部分：rtPCR法、Western blot法检测miR-21靶基因及蛋白的变化；CHIP实验、荧光素酶报告基因实验验证STAT3与miR-21调控关系及其对舌鳞癌耐药的影响。 第三部分：建立裸鼠荷TCA8113/DDP舌鳞癌皮下动物模型,瘤内注射WP1066及DDP,动态观察肿瘤生长情况,检测WP1066抑制STAT3/pSTAT3表达后,miR-21表达情况,miR-21靶点蛋白及舌鳞癌生长相关蛋白的变化情况,并且使用TUNEL法检测细胞凋亡,对体外实验的结果进一步验证。 结果： TCA8113、TCA8113/DDP细胞WP1066的IC50分别为3.1和3.5μmol/L。 DDP的ICso分别为0.9μM/L和10.9t.tM/L；顺铂耐药细胞株STAT3、miR-21表达水平显著高于顺铂敏感细胞株。WP1066能有效抑制pSTAT3表达；WP1066组、DDP组及WP1066+DDP组均出现肿瘤细胞增殖、克隆形成能力明显下降,形成球形克隆、侵袭能力被抑制,迁移能力下降,凋亡率增加；联合治疗效果优于单一处理组。WP1066处理后Bcl-2、mTOR、Ki-67、MMP2/9蛋白表达水平明显下降,Caspase3表达水平上调,DDP不能改变其表达。WP1066组与WP1066+DDP组miR-21表达水平低于对照组及DDP组。各组miR-21靶基因表达均无明显变化,WP1066可抑制miR-21靶点蛋白表达；CHIP实验、荧光素酶报告基因实验证明miR-21是STAT3的调控靶点。肿瘤生长曲线显示WP1066及DDP治疗组肿瘤生长速度及体积相似,且均小于空白对照及DMSO组,WP1066+DDP组效果优于单一治疗；经WP1066治疗后肿瘤组织中STAT3/p-STAT3及miR-21表达水平均下降,DDP不能改变其表达；WP1066、 DDP治疗组细胞凋亡数显著均高于对照组,联合治疗后凋亡数明显高于单一治疗；Bcl-2、mTOR、Ki-67、MMP2/9表达下降,Caspase3、PTEN、PDCD4及TIMP3升高,DDP不能改变其表达。体内外实验结果一致。 结论： 1. STAT3、miR-21过表达与人舌鳞癌顺铂耐药相关。 2.WP1066可通过抑制STAT3磷酸化,转录抑制miR-21表达,进而减弱对miR-21靶点PTEN、PDCD4、TIMP3的转录后调控,分别在增殖、凋亡、侵袭及迁移方面抑制人舌鳞癌细胞的生长。 3.WP1066与DDP联合治疗人舌鳞癌细胞及荷瘤裸鼠模型效果较单一处理(单用WP1066或DDP)效果明显增强；STAT3作为人舌鳞癌中的新治疗靶点,其小分子抑制剂WP1066为逆转化疗耐药及其治疗提供了新的方向机可能性。
[Abstract]:Head and neck cancer is one of the most common in the world of the 6 common malignant tumors, including tongue squamous cell carcinoma is the most common pathological type of malignant tumor in head and neck. Most advanced tongue squamous cell carcinoma and infiltrative growth associated with cervical lymph node metastasis, surgical resection has been difficult to completely infiltrate the surrounding tissue and distant metastasis the tumor. As everyone knows, cisplatin (DDP) chemotherapy has been widely used in the treatment of squamous cell carcinoma of tongue, but the adverse reaction of DDP and tumor on the drug resistance seriously affect the treatment effect, thus reversing resistance to chemotherapy, improve the sensitivity of squamous cell carcinoma of the tongue has attracted more and more attention.
STAT3 in liver cancer, breast cancer, lung cancer, colon cancer, over expressed in head and neck squamous cell carcinoma and other malignant tumor derived from epithelial system, and participate in a variety of cell proliferation, apoptosis, invasion and metastasis related gene transcription. In multiple myeloma and glioma were found in STAT3 can directly regulate miR-21 the transcription. The project uses TCA8113 and TCA8113/DDP in tongue squamous cell carcinoma cell line, the WP1066 reduced the expression of STAT3, and the clinical commonly used chemotherapeutic drugs DDP tongue squamous cell carcinoma, the effect and mechanism of WP1066 combined with DDP of human tongue squamous cell carcinoma cells in vitro and in vivo growth ability were studied. The following in vivo research is divided into three parts:
The first part: firstly, the determination of two tongue cancer cell WP1066 by MTT method, DDP half inhibitory concentration (IC50); Western blot, real time quantitative PCR for the detection of resistant and sensitive tongue squamous cell carcinoma cell line STAT3, the difference of miR-21 expression in squamous cell carcinoma of the tongue; STAT3/p-STAT3 to detect the expression of WP1066 and DDP after treatment with Western blot and immunofluorescence detection method; application real time quantitative PCR method and in situ hybridization were used to detect two tongue cancer cell line WP1066, the expression of miR-21 after DDP treatment. Using MTT method, experimental plate cloning, Matrigel matrix growth experiment, Transwell in vitro invasion assay, cell scratch test of WP1066, flow cytometry, DDP inhibitory effect of tongue cancer cells in vitro bovine long.
The second part: rtPCR method, Western blot method to detect miR-21 target gene and protein changes; CHIP experiment, luciferase reporter gene experiment to verify the relationship between STAT3 and miR-21 and its impact on drug resistance of tongue squamous cell carcinoma.
The third part: the establishment of nude mice bearing subcutaneous TCA8113/DDP tongue squamous cell carcinoma animal model, intratumoral injection of WP1066 and DDP, the dynamic observation of tumor growth. The detection of WP1066 after inhibition of STAT3/pSTAT3 expression, miR-21 expression, changes in miR-21 target protein and growth associated protein in squamous cell carcinoma of tongue, and use the TUNEL method to detect apoptosis in vitro. The results of further validation.
Result锛，

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