YY1和miR-186及其相互作用网络在非小细胞肺癌发展中的作用

发布时间：2018-04-16 12:00

本文选题：非小细胞肺癌 + miR-186　；参考：《南方医科大学》2017年博士论文

【摘要】：在中国乃至世界人群内,肺癌的发病率与死亡率在所有癌症中居于首位。非小细胞肺癌是肺癌的主要临床类型,约占肺癌总数的80%。临床上对肺癌的手术治疗和化疗治疗手段一直在改进,可是肺癌患者的预后依然不容乐观。近几年肺癌分子生物学的研究进展为寻找新的肺癌治疗方式及新的药物研发提供了重要的思路,也为肺癌患者带来了新的希望。在分子生物学水平上对肺癌的发生、发展的研究有助于我们发现新的肺癌治疗的靶标和生物标志物。为此我们研究了转录因子Yin Yang-1(YY1),microRNAmiR-186,lncRNAPVT1及其相互作用网络在肺癌中的作用。YY1是一种多功能转录因子,能够正负调节大量细胞内基因和病毒基因。大量研究表明,PVT1在人类多种类型的癌症组织中均存在异常表达。miR-186在多种人类恶性肿瘤中下调,发挥肿瘤抑制剂的作用。但是,miR-186在非小细胞肺癌的发展和代谢过程中的调节机制目前仍不明确。在本研究中,我们收集了非小细胞肺癌患者的癌组织和癌旁组织,及人类不同肺癌细胞系A549、95-D、HCC827、NCI-H1650和肺上皮HPAEpiC细胞系。首先我们采用qRT-PCR或者western blot的方法检测这些因子在肺癌组织、癌旁组织及肺癌细胞系中的表达水平。通过CCK-8实验检测细胞的增殖情况,流式细胞仪检测细胞的凋亡情况,Transwell检测细胞的迁移和侵袭能力。通过荧光素酶报告试验、电泳迁移实验以及染色质免疫沉降试验验证各因子之间的相互靶向作用。结果发现YY1在肺癌肿瘤组织以及肺癌细胞系中的表达量显著上调;且YY1的过表达促进了癌细胞增殖,迁移和侵袭,并抑制了细胞凋亡。YY1能够直接结合于lncRNA-PVT1启动子,并激活其转录。PVT1也能促进肺癌细胞的细胞增殖、迁移以及侵袭。miR-186在肺癌组织和细胞中显著下调;且miR-186的过表达抑制了肺癌细胞的增殖、迁移和侵袭,诱导细胞凋亡。MAP3K2在肺癌组织中高表达,且MAP3K2可促进肺癌细胞的增殖,迁移和侵袭,并抑制了细胞凋亡;miR-186能靶向MAP3K2并抑制MAP3K2的表达。此外,MAP3K2过表达在一定程度上反转了 miR-186对肺癌细胞系增殖和代谢的抑制效果。miR-186与YY1在肺癌组织和肺癌细胞系中的表达呈现负相关,miR-186能直接靶向作用于YY1。YY1siRNA在抑制细胞增殖、细胞侵袭和细胞迁移,以及诱导细胞凋亡所发挥的作用要显著强于miR-186。综上,我们的研究表明,YY1在肺癌发生过程中在一定程度上是通过影响其下游靶点PVT1而发挥肿瘤促进作用。miR-186可通过抑制MAP3K2表达来调节肺癌组织生长和代谢;也可通过直接靶向作用YY1而诱导非小细胞肺癌细胞凋亡。因此,针对非小细胞肺癌,miR-186-MAP3K2及miR-186-YY1可能是一种全新的、有用的、潜在的临床治疗和诊断靶点,为非小细胞肺癌的治疗提供新的手段。
[Abstract]:Lung cancer is the leading cause of morbidity and mortality in China and the world.Non-small cell lung cancer is the main clinical type of lung cancer, accounting for about 80% of the total number of lung cancer.The surgical and chemotherapeutic methods for lung cancer have been improved, but the prognosis of lung cancer patients is still not optimistic.Recent advances in molecular biology of lung cancer have provided important ideas for finding new treatments and new drugs for lung cancer, as well as new hopes for lung cancer patients.The research on the occurrence and development of lung cancer at the level of molecular biology helps us to find new targets and biomarkers for the treatment of lung cancer.Therefore, we have studied the role of the transcription factor Yin Yang-1 (YY1) microRNAmiR-186ncRNAPVT1 and its interaction network in lung cancer. YY1 is a multifunctional transcription factor which can positively and negatively regulate a large number of intracellular and viral genes.A large number of studies have shown that PVT1 has abnormal expression in various types of human cancer tissues. MiR-186 is down-regulated in a variety of human malignant tumors and plays the role of tumor inhibitor.However, the regulatory mechanism of miR-186 in the development and metabolism of non-small cell lung cancer is still unclear.In this study, we collected the cancer tissues and adjacent tissues of patients with non-small cell lung cancer (NSCLC), as well as different human lung cancer cell lines A549NC827NCI-H1650 and lung epithelial HPAEpiC cell lines.First, qRT-PCR or western blot were used to detect the expression of these factors in lung cancer tissues, paracancerous tissues and lung cancer cell lines.CCK-8 assay was used to detect cell proliferation and flow cytometry was used to detect cell apoptosis. Transwell was used to detect cell migration and invasion.Luciferase report test, electrophoresis migration test and chromatin immuno deposition test were used to verify the mutual targeting of each factor.The results showed that the expression of YY1 was significantly up-regulated in lung cancer tumor tissues and lung cancer cell lines, and the overexpression of YY1 promoted the proliferation, migration and invasion of cancer cells, and inhibited apoptosis. YY1 could directly bind to lncRNA-PVT1 promoter.Activation of its transcription. PVT1 also promoted the proliferation of lung cancer cells, migration and down-regulation of invasion. MiR-186 in lung cancer tissues and cells, and the overexpression of miR-186 inhibited the proliferation, migration and invasion of lung cancer cells.Apoptosis. MAP3K2 was highly expressed in lung cancer tissues, and MAP3K2 could promote the proliferation, migration and invasion of lung cancer cells, and inhibit the apoptosis of miR-186 targeting MAP3K2 and inhibiting the expression of MAP3K2.In addition, the overexpression of MAP3K2 reversed to some extent the inhibitory effect of miR-186 on proliferation and metabolism of lung cancer cells. MiR-186 was negatively correlated with the expression of YY1 in lung cancer tissues and lung cancer cell lines. MiR-186 could directly target YY1.YY1siRNA to inhibit the proliferation of lung cancer cells.The effect of cell invasion and migration and apoptosis induction was stronger than that of miR-1866.In conclusion, our study shows that YY1 plays a role in promoting tumor growth and metabolism by inhibiting the expression of MAP3K2 in lung cancer, to a certain extent, by affecting its downstream target PVT1.Apoptosis of non-small cell lung cancer cells can also be induced by direct targeting of YY1.Therefore, for non-small cell lung cancer, miR-186-MAP3K2 and miR-186-YY1 may be a new, useful and potential clinical treatment and diagnostic target, and provide a new method for the treatment of non-small cell lung cancer.
【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】：R734.2

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