KCTD12基因过表达对脉络膜黑色素瘤细胞生物学功能的影响

发布时间：2018-03-18 20:09

本文选题：KCTD12　切入点：OCM-1　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：人类钾离子通道四聚体结构域蛋白12(KCTD12,也称为Pfetin)是KCTD家族26个成员之一。KCTD12调节GABAB受体信号的激动剂效力和动力学效应。蛋白质组学分析表明KCTD12可能是判断胃肠道间质瘤预后潜在的生物标志物。然而,很少有报道关于KCTD12对其他肿瘤类型的作用。葡萄膜黑色素瘤通常指发生在脉络膜,睫状体或虹膜上的黑色素瘤。葡萄膜黑色素瘤是最常见的成人原发性眼内恶性肿瘤。在美国,这些癌症的发生率是5%,其中85%是葡萄膜原发。葡萄膜黑色素瘤患者早期表现为无痛性视力下降,后期则可导致视网膜脱离。此外,葡萄膜黑色素瘤具有高度转移性,患者黑色素瘤可能转移到肝脏,肺或者皮肤。因此,葡萄膜黑色素瘤患者有高达一半的死亡率。葡萄膜黑色素瘤肿瘤的发生和转移是复杂过程。到目前为止,尚无研究KCTD12对葡萄膜黑素瘤影响的相关研究。本课题研究旨在探讨过表达KCTD12基因对脉络膜黑色素瘤细胞OCM-1的生物学功能的影响。本研究设计并亚克隆KCTD12全长和部分片段基因。构建了真核过表达载体PC3.1-KCTD12,瞬转人类脉络膜黑素瘤细胞系(OCM-1),发现过表达KCTD12可以上调肿瘤上皮组织标志蛋白E-cad的表达及降低肿瘤上皮组织间质化(EMT)标志蛋白Vimitin的表达。构建慢病毒核心质粒p LVX-Puro-KCTD12,筛选出稳定表达KCTD12的OCM-1细胞系。我们将得到的细胞系进行RNA-Sequencing检测,并对数据进行生物信息学分析。发现基因表达水平变化主要包括增殖,细胞黏附,细胞凋亡,血管生成等方面。我们也针对以上结果进行实验验证。通过MTS实验、平板克隆实验及软琼脂克隆实验,结果表明KCTD12可有效抑制OCM-1细胞的增殖。通过划痕实验、transwell侵袭迁移实验及相关蛋白的表达情况的检测,发现上皮化标志物E-cadherin,β-Catenin表达量上调,而间质化标志物Vimentin的表达量则下降,Snail及slug作为E-cadherin的调节因素,表达量也有所下降。实验结果说明过表达KCTD12可以限制OCM-1细胞的侵袭及迁移。通过流式细胞仪对细胞周期及凋亡进行检测,并对细胞周期相关的关健蛋白进行检测。蛋白检查结果发现cyclin B表达量明显下降。结果表明过表达KCTD12的OCM-1细胞中G2/M到G1期的过程时间延长。另外,KCTD12过表达OCM-1细胞抑制了裸鼠体内异种移植物的生长。最后,实验还构建了PGEX-4T-KCTD12全长和部分片段的载体,进行原核表达,并纯化了带GST及HIS标签的KCTD12全长及部分片段的融合蛋白。用全长部分蛋白验证对OCM-1细胞的生物学作用,并用部分片段蛋白免疫小鼠制备了鼠源性抗体。总的来说,过表达KCTD12具有抑制OCM-1细胞生长的作用,未来有可能作为治疗脉络膜黑色素瘤的新型治疗靶标。
[Abstract]:Human potassium channel tetramer domain protein 12KCTD12, also known as Pfetinin, is one of the 26 members of the KCTD family. KCTD12 regulates the agonist potency and kinetic effects of GABAB receptor signal. Proteomics analysis indicates that KCTD12 may be the judge of gastrointestinal tract. Potential biomarkers for tumor prognosis. However, The role of KCTD12 in other types of tumors is rarely reported. Uveal melanoma usually refers to melanoma occurring in the choroid, ciliary body, or iris. Uveal melanoma is the most common primary intraocular malignant tumor in adults. The incidence of these cancers is 5%, of which 85% are primary uveal. Patients with uveal melanoma show painless visual acuity at an early stage, which can lead to retinal detachment later. In addition, uveal melanoma is highly metastatic. Patients with melanoma may metastasize to the liver, lungs, or skin. As a result, uveal melanoma patients have a high rate of death. The occurrence and metastasis of uveal melanoma tumors is a complex process. No study has been done on the effect of KCTD12 on uveal melanoma. This study aims to investigate the effect of overexpression of KCTD12 gene on the biological function of OCM-1 in choroidal melanoma cells. This study designed and subcloned the full length and part of KCTD12. Fragment gene. Eukaryotic expression vector PC3.1-KCTD12 was constructed and transformed into human choroidal melanoma cell line (OCM-1). It was found that overexpression of KCTD12 could up-regulate the expression of E-cad, and decrease the expression of mesenchymal marker of tumor epithelium. Construction of lentivirus core plasmid pLVX-Puro-KCTD12 and screening of OCM-1 cell lines stably expressing KCTD12. The obtained cell lines will be detected by RNA-Sequencing. Bioinformatics analysis of the data shows that the changes of gene expression level mainly include proliferation, cell adhesion, apoptosis, angiogenesis and so on. The results of plate cloning and soft Agar cloning showed that KCTD12 could effectively inhibit the proliferation of OCM-1 cells, and the expression of E-cadherin and 尾 -Catenin was up-regulated by scratching test, transwell invasion and migration assay and the expression of related proteins. However, the expression of interstitial marker Vimentin decreased. Snail and slug were the regulatory factors of E-cadherin. The results showed that overexpression of KCTD12 could limit the invasion and migration of OCM-1 cells. Flow cytometry was used to detect cell cycle and apoptosis. The results showed that the expression of cyclin B was significantly decreased. The results showed that the process time from G2 / M to G1 phase was prolonged in OCM-1 cells with overexpression of KCTD12. In addition, KCTD12 over-expressed OCM-1 cells. Inhibit the growth of xenografts in nude mice. Finally, The full-length and partial fragments of PGEX-4T-KCTD12 were constructed and expressed in prokaryotic expression, and the fusion proteins of KCTD12 with GST and HIS tags were purified. The biological effects on OCM-1 cells were verified by full-length partial proteins. In general, overexpression of KCTD12 could inhibit the growth of OCM-1 cells and might be a new therapeutic target for choroidal melanoma in the future.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R739.7

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