miR-149靶向SP1抑制人舌鳞状细胞癌细胞增殖和迁移机制的研究

发布时间：2018-06-09 23:10

本文选题：舌鳞状细胞癌 + miR-149　；参考：《南方医科大学》2017年博士论文

【摘要】：研究背景:舌鳞状细胞癌是常见的口腔恶性肿瘤。临床上通常采用手术为主综合治疗,但死亡率仍然维持在较高水平。从基因水平探索舌鳞癌发生和发展机制,有可能在提高舌鳞癌患者的治愈率、降低死亡率等方面具有十分重要的科学意义。miR-149在多种不同类型的实体恶性肿瘤中具有抑癌基因的功能,SP1在包括胃癌、胰腺癌等恶性肿瘤细胞中的表达量均异常增高,且SP1高表达的患者其预后情况通常较差,这两者在舌癌中的研究还相对较少,所以希望通过研究miR-149和SP1在舌恶性肿瘤中的表达及及两者关系在舌癌发生和发展过程中的作用,为舌恶性肿瘤的治疗提供一些新的思路。目的本研究拟检测miR-149在舌鳞癌患者肿瘤组织中的表达,探讨其与患者临床病例参数的相关性,分析miR-149的表达对舌鳞癌细胞增殖及转移过程可能发生的影响,探究miR-149信号活化对于舌鳞癌细胞中SP1表达的调控作用及对舌鳞癌细胞生物学行为的影响,有利于了解舌鳞癌的发生发展机制,同时为舌鳞癌的临床诊断以及靶向治疗提供依据。方法(1)采集2014.1-2014.9期间我院口腔颌面外科舌鳞癌手术患者癌组织样本及相应癌旁组织样本,共62例。荧光定量PCR检测上述组织样本中miR-149的表达并分析其与患者临床参数的相关性。(2)构建miR-149mimic质粒并转染Tca8113和CAL-27细胞。荧光定量PCR检测转染后细胞中miR-149的表达。MTT法检测转染后细胞增殖情况,流式细胞术分别检测细胞周期和细胞凋亡情况,Transwell小室法检测细胞侵袭能力。(3)采用生物学软件分析miR-149的靶基因并筛选、构建野生型和突变型SP1质粒载体分别与miR-149 mimic共转染Tca8113和CAL-27细胞,双荧光素酶检测试剂盒验证miR-149是否靶向调控SP1基因的3'UTR。荧光定量 PCR 和 western blot 法检测转染 miR-149 mimic 后 Tca8113 和 CAL-27细胞中SP1 mRNA和蛋白的表达。结果(1)荧光定量PCR检测结果显示舌鳞癌组织中miR-149的表达量明显低于癌旁相应正常组织(P0.01)。(2)荧光定量PCR检测结果表明,成功构建miR-149过表达Tca8113和CAL-27细胞系。转染miR-149 mimic后Tca8113和CAL-27细胞增殖率明显低于相应对照组(P0.01)、侵袭细胞数量明显低于对照组(P0.05)、细胞凋亡率明显高于对照组(P0.01)。细胞周期检测G1/G0期细胞所占比例明显高于对照组(P0.05),而S期细胞比例则显著低于对照组(P0.05)。(3)SP1被预测为miR-149的靶基因,其在SP1上所绑定的位置位于miR-149的 3'UTR 区域。转染 miR-149 mimic 后 Tca8113 和 CAL-27 细胞中 SP1 mRNA的表达量均明显低于对照组(P=0)、SP1蛋白的表达量均明显低于对照组(P0.05)。结论miR-149在舌鳞癌组织中呈低表达,且其表达量与舌鳞癌患者的组织学分级、临床分期以及远处转移密切相关。miR-149能够通过直接抑制SP1蛋白的表达来实现对舌鳞癌细胞增殖、侵袭以及细胞周期调控等一系列生物学过程的影响。
[Abstract]:Background: tongue squamous cell carcinoma is a common oral malignant tumor. Surgery is usually used in clinical treatment, but the mortality rate is still at a high level. To explore the mechanism of occurrence and development of squamous cell carcinoma of tongue at the gene level may improve the cure rate of patients with squamous cell carcinoma of tongue. It is of great scientific significance to reduce mortality. MiR-149 has the function of suppressor gene in various types of solid malignant tumors. The expression of SP1 in malignant tumor cells, including gastric cancer and pancreatic cancer, is abnormally high. The prognosis of patients with high expression of SP1 is usually poor, and there are relatively few studies on the expression of miR-149 and SP1 in malignant tumor of tongue, and the relationship between miR-149 and SP1 in the carcinogenesis and development of tongue cancer, so we hope to study the role of miR-149 and SP1 in the carcinogenesis and development of tongue cancer. To provide some new ideas for the treatment of malignant tumor of tongue. Objective to investigate the expression of miR-149 in the tumor tissue of tongue squamous cell carcinoma (SCC), and to explore the correlation between miR-149 expression and clinical parameters, and to analyze the effect of miR-149 expression on the proliferation and metastasis of tongue squamous cell carcinoma (LSCC) cells. To investigate the effect of miR-149 signal activation on the expression of SP1 and the biological behavior of tongue squamous cell carcinoma cells, it is helpful to understand the mechanism of development of tongue squamous cell carcinoma, and to provide the basis for clinical diagnosis and targeted therapy of tongue squamous cell carcinoma. Methods A total of 62 patients with oral and maxillofacial squamous cell carcinoma of tongue were collected from January to April 2014.The samples of cancer tissues and adjacent tissues were collected. The expression of miR-149 was detected by fluorescence quantitative PCR and the correlation between miR-149 and clinical parameters was analyzed. The miR-149 mimic plasmid was constructed and transfected into Tca8113 and CAL-27 cells. The expression of miR-149 in transfected cells was detected by fluorescence quantitative PCR. MTT assay was used to detect the proliferation of transfected cells. Cell cycle and apoptosis were detected by flow cytometry. The wild-type and mutants SP1 plasmids were cotransfected with miR-149 mimic into Tca8113 and CAL-27 cells, respectively. The expression of SP1 mRNA and protein in Tca8113 and CAL-27 cells after transfection of miR-149 mimic was detected by fluorescence quantitative PCR and western blot. Results the results of fluorescence quantitative PCR showed that the expression of miR-149 in tongue squamous cell carcinoma was significantly lower than that in adjacent normal tissue (P0.01U. 2) the results of fluorescence quantitative PCR showed that miR-149 overexpression Tca8113 and CAL-27 cell lines were successfully constructed. After transfection of miR-149 mimic, the proliferation rate of Tca8113 and CAL-27 cells was significantly lower than that of control group (P 0.01), the number of invasive cells was significantly lower than that of control group (P 0.05), and the apoptosis rate of Tca8113 and CAL-27 cells was significantly higher than that of control group. The percentage of cells in G _ 1 / G _ 0 phase was significantly higher than that in control group (P _ (0.05), while the proportion of S phase cells was significantly lower than that of control group (P _ (0.05) P _ (0.05). The target gene was predicted to be miR-149. The binding position on SP1 was located in the 3UTR region of miR-149. After transfection of miR-149 mimic, the expression of SP1 mRNA in Tca8113 and CAL-27 cells was significantly lower than that in control group. Conclusion the expression of miR-149 is low in tongue squamous cell carcinoma, and its expression level is closely related to histological grade, clinical stage and distant metastasis of tongue squamous cell carcinoma. MiR-149 can directly inhibit the expression of SP1 protein to realize the proliferation of tongue squamous cell carcinoma cells. A series of biological processes, such as invasion and cell cycle regulation.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R739.86

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