肾细胞癌中原肌球蛋白1的表达水平及其临床和肿瘤学意义

发布时间：2018-05-03 16:54

本文选题：肾细胞癌 + 原肌球蛋白　；参考：《吉林大学》2015年博士论文

【摘要】：原肌球蛋白（tropomyosin, TPM）是组成细胞骨架结构微丝的重要成分，主要调节肌细胞的收缩和非肌细胞的细胞骨架稳定性。以往的观点都认为这些TPM蛋白与多种良性肌肉病变的发生密切相关，比如重症肌无力和家族性肥厚性心肌病等。但近年，越来越多的研究开始关注TPM1在肿瘤的发生发展过程中的作用，进而发现TPM1在乳腺癌等肿瘤中扮演了肿瘤抑制基因（tumorsuppressor gene, TSG）的角色。这种抑癌作用具体表现在，TPM1参与了细胞变形、侵袭、迁移、抗凋亡等一系列恶性肿瘤的生物学行为。对其作用和调控机理的深入认识会使TPM1运用到肿瘤的诊断和治疗，并将使之发展为新的肿瘤标志物或药物治疗靶点成为可能。最近研究发现TPM1可能是miRNA-21的靶基因之一，而miRNA-21是在各种实体肿瘤中研究得最多的miRNA，它在各种实体肿瘤包括肾癌中都是高表达的。然而，对于TPM1在肾细胞癌中的表达水平如何以及TPM1在肾癌中是否也发挥抑癌基因的作用等问题，由于以前没有进行过系统的研究，目前这些情况仍然是不清楚的。但根据现有的事实与理论推测TPM1在肾细胞癌中应该是被下调的，并且在肾细胞癌中发挥着抑癌基因的作用，可能参与了肿瘤细胞的转移和侵袭等恶性生物学行为。为了证实以上推测，本实验运用实时定量PCR、免疫蛋白印记（Westernblotting）和免疫组织化学染色的方法检测了TPM1在肾癌组织和肾癌细胞系OSRC-2及786-O中的表达水平。结合肾细胞癌患者的临床资料，分析了TPM1的表达水平与病理类型、肿瘤分期、肿瘤核分级和术后生存率等临床特征的相关性。构建pcDNA3.1-TPM1重组真核表达质粒，运用lipofectamine2000做媒介瞬时转染OSRC-2和786-O肾癌细胞，研究TPM1在肾癌中的肿瘤生物学功能。使用CCK-8试剂盒检测TPM1转染后的细胞增殖状态；利用划痕实验以及Transwell迁移实验检测TPM1对肾癌细胞迁移能力的影响；使用Transwell基质胶侵袭实验检测TPM1转染前后肾癌细胞侵袭能力的变化；使用流式细胞术PI/AnnexinV双染色和PI单染色法分别检测了TPM1对肾癌细胞凋亡和细胞周期的调控作用。实验结果显示：与正常肾组织相比，，TPM1在肾细胞癌中的mRNA和蛋白质表达水平显著且特异的下调。TPM1的表达水平与肾细胞癌患者的肿瘤大小、Fuhrman核分级和疾病特异性生存率相关。在肾癌细胞系中增加TPM1的表达会显著地抑制肿瘤细胞的迁移和侵袭，TPM1还可以促进肾细胞癌细胞的凋亡和非特异性的细胞周期阻滞，这些都提示TPM1是肾细胞癌中潜在的肿瘤抑制基因。
[Abstract]:Tropomyosin (TPM) is an important component of cytoskeletal microfilaments, which mainly regulates the contraction of myocytes and the cytoskeleton stability of non-myocytes. It has been suggested that these TPM proteins are closely related to the occurrence of various benign muscle diseases, such as myasthenia gravis and familial hypertrophic cardiomyopathy. However, in recent years, more and more studies have begun to focus on the role of TPM1 in tumorigenesis and development, and it has been found that TPM1 plays the role of tumor suppressor gene in breast cancer and other tumors. TPM1 is involved in the biological behavior of a series of malignant tumors, such as cell deformation, invasion, migration, anti-apoptosis and so on. Further understanding of its role and regulation mechanism will make it possible for TPM1 to be used in the diagnosis and treatment of tumors and to develop into new tumor markers or drug therapy targets. Recent studies have found that TPM1 may be one of the target genes of miRNA-21, while miRNA-21 is the most widely studied miRNAs in various solid tumors, including renal cell carcinoma. However, the expression level of TPM1 in renal cell carcinoma and whether TPM1 also plays the role of tumor suppressor gene in renal cell carcinoma have not been systematically studied before, so these conditions are still unclear. However, according to the existing facts and theories, we speculate that TPM1 may be down-regulated in renal cell carcinoma and play an important role in tumor suppressor gene, which may be involved in malignant biological behavior such as metastasis and invasion of tumor cells. In order to confirm the above conjecture, the expression of TPM1 in renal cell carcinoma (RCC) and renal cell lines (OSRC-2 and 786-O) was detected by real-time quantitative PCR, Western blotting and immunohistochemical staining. Based on the clinical data of renal cell carcinoma (RCC), the correlation between the expression of TPM1 and pathological type, tumor stage, tumor nuclear grade and postoperative survival rate was analyzed. PcDNA3.1-TPM1 recombinant eukaryotic expression plasmid was constructed and transient transfection of OSRC-2 and 786-O RCC cells was carried out using lipofectamine2000 as a medium to study the tumor biological function of TPM1 in RCC. CCK-8 kit was used to detect the cell proliferation after TPM1 transfection, and the effect of TPM1 on the migration ability of renal cancer cells was detected by scratch test and Transwell migration test. Transwell matrix gel invasion assay was used to detect the invasion ability of renal cancer cells before and after TPM1 transfection, and the effects of TPM1 on apoptosis and cell cycle of renal cell carcinoma cells were detected by flow cytometry PI/AnnexinV double staining and Pi single staining, respectively. The results showed that the expression level of mRNA and protein in renal cell carcinoma was significantly and specifically down-regulated compared with normal renal tissue. The expression level of TPM1 was correlated with the tumor size and the disease specific survival rate in renal cell carcinoma patients. Increasing the expression of TPM1 in renal cell carcinoma cell lines significantly inhibited the migration and invasion of tumor cells and promoted the apoptosis and non-specific cell cycle arrest of renal cell carcinoma cells. These results suggest that TPM1 is a potential tumor suppressor gene in renal cell carcinoma.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.11

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