miR-185对黑色素瘤的抑制作用和辐射增敏作用研究

发布时间：2018-01-18 12:22

本文关键词：miR-185对黑色素瘤的抑制作用和辐射增敏作用研究　出处：《兰州大学》2016年硕士论文　论文类型：学位论文

【摘要】：黑色素瘤是发生率、死亡率增长速度最快的皮肤病癌症,一直以来都受到人们的广泛关注。放疗作为目前临床上最常用的抗癌常规治疗手段之一,是治疗晚期转移型黑色瘤的主要方法。但是,研究发现黑色素瘤细胞普遍对放疗具有一定程度的耐受性,这使得常规放疗疗效受限,而通过增大放疗剂量提高疗效时又会带来较大的副作用。目前,临床上开发了一系列放疗增敏剂用以提高放疗的治疗效果,主要包括化学类、中草药类药物,然而这些放疗增敏剂均存在一定的弊端。因此,寻求高效、低毒的放疗增敏剂对于临床黑色素瘤的放射治疗意义重大。microRNA(mi RNA)是一类长度约为22个核苷酸的小RNA分子,通过与靶基因mRNA的3'-UTR区结合而引起其降解或翻译抑制,在转录后水平发挥调控功能。miRNA不仅可以调节细胞增殖、分化、周期和凋亡等细胞进程,而且参与调控细胞的辐射应答通路,影响细胞对射线的敏感性。多项研究证实,miR-185通过诱导肿瘤细胞的周期阻滞、促进细胞的凋亡以及改变细胞基因组表观遗传等方式在多种肿瘤中发挥抑瘤因子的功能。另一方面,我们的前期研究发现X射线辐照后的肾癌组织中mi R-185的表达水平发生显著下调,在体外和体内水平miR-185均能显著增强肾癌细胞的辐射敏感性;此外,miR-185还能降低胃癌细胞对化疗药物的耐受性。这些研究表明miR-185在调控肿瘤细胞对放疗和化疗的应激响应进程中发挥着重要的作用,能够影响肿瘤细胞对辐射及对化疗药物的敏感性。然而,miR-185是否在黑色素瘤中同样能够发挥肿瘤抑制因子的功能以及是否能够增强黑色素瘤细胞的辐射敏感性仍不清楚。为了探究miR-185对黑色素瘤发生和发展的影响,我们通过转染的方法在小鼠黑色素瘤细胞系B16中过表达miR-185,并利用细胞生长曲线、划痕及克隆形成实验检测miR-185对B16细胞增殖、迁移和克隆能力的影响,结果显示miR-185能够显著地抑制细胞增殖、降低克隆形成和削弱细胞迁移。此外,我们将B16细胞接种到C57小鼠皮下成瘤,构建了同种移植瘤模型,利用PEI载体直接在瘤体内转染miR-185,发现外源性过表达miR-185能显著抑制瘤体生长,而瘤体组织Ki-67及PCNA免疫组化结果也表明过表达miR-185显著降低了瘤体内增殖期细胞的比率。在明确了miR-185的肿瘤抑制功能后,我们继续深入探索了miR-185对黑色素瘤细胞辐射敏感性的影响。我们发现4Gy的X射线能显著降低B16细胞的增殖和迁移能力。更为重要的是与对照组相比,辐照对过表达miR-185的B16细胞具有更好的杀伤效果,过表达miR-185能显著增强辐照导致的细胞增殖抑制和迁移抑制以及促凋亡水平。小鼠移植瘤实验同样表明miR-185能增强X射线辐照对瘤体生长的抑制效果,使得瘤细胞凋亡水平明显增加。综上所述,我们的研究首次证实了无论在体外还是体内水平miR-185都能够发挥抑制黑色素瘤生长的作用,是一个强有力的抑癌因子;并且miR-185还能够增强黑色素细胞对电离辐射的敏感性,是一个有效的放疗增敏分子。我们的研究为miR-185的抑癌及辐射增敏增加了新的基础数据,有助于推动miR-185的临床应用。
[Abstract]:Melanoma is the incidence of skin cancer mortality is the fastest growing, has been widely concerned by people. Radiotherapy as one of the most commonly used clinical anticancer conventional treatment method at present, is the main method for the treatment of advanced metastatic melanoma type. However, the study found that the tolerance of melanoma cells generally have a certain the degree of radiation, which makes the limited conventional radiotherapy, and by increasing the radiation dose to improve the curative effect and will bring more side effects. At present, the clinical development of a series of radiosensitizer to improve the therapeutic effect of radiotherapy, including chemical, herbal drugs, but these radiosensitizer there are some disadvantages. Therefore, looking for effective, low toxicity radiosensitizer for radiotherapy in the treatment of major clinical.MicroRNA melanoma (MI RNA) is a kind of about 22 nucleotides in length Small molecular RNA, through the mRNA and the 3'-UTR of target gene binding caused by the degradation or translation inhibition at the post transcriptional level play a regulatory function of.MiRNA can not only regulate cell proliferation, differentiation, cell cycle and apoptosis process, and the radiation response pathway involved in the regulation of cell sensitivity to radiation, affecting cell. A number of studies have confirmed that miR-185 through the induction of tumor cell cycle arrest, cell apoptosis and changes of cell genome epigenetic play a tumor suppressor function in a variety of tumors. On the other hand, our previous study found that the expression level of X ray irradiation in renal cell carcinoma mi R-185 was significantly down regulated, can significantly enhance the radiation the sensitivity of renal cell carcinoma cells in vitro and in vivo levels of miR-185; moreover, miR-185 can reduce the resistance of gastric cancer cells to chemotherapeutic agents. These studies show that miR-185 in the Stress regulation of tumor cells to radiotherapy and chemotherapy response plays an important role in the process, can affect the sensitivity of tumor cells to radiation and chemotherapeutic drugs. However, whether miR-185 in melanoma can also play a tumor suppressor function and whether it can enhance the radiation sensitivity of melanoma cells to remain unclear. To explore the influence of miR-185 on the occurrence and development of melanoma, we transfected in mouse melanoma cell line B16 overexpression of miR-185, and the cell growth curve, colony formation assay and scratch and miR-185 on B16 cell proliferation, migration and effect of cloning ability, the result shows that miR-185 can significantly inhibit cell proliferation reduce, clone formation and weaken cell migration. In addition, we B16 cells were inoculated subcutaneously into C57 mice tumor, construct transplantation tumor model by P EI vector transfection in vivo directly in the miR-185, found that overexpression of miR-185 can inhibit the growth of tumor, and the tumor tissue Ki-67 and PCNA immunohistochemistry results also showed that overexpression of miR-185 significantly reduced the rate of tumor cell proliferation period. The tumor suppressor function of miR-185, we continue to explore the effect of miR-185 on radiation sensitivity of melanoma cells. We found that X - 4Gy can significantly reduce the proliferation and migration of B16 cells. The more important is compared with the control group, irradiation on the expression of miR-185 B16 cells has a good killing effect, overexpression of miR-185 can significantly enhance the irradiation induced cell inhibition of proliferation and migration inhibition and apoptosis level. Mouse xenograft experiments also showed that miR-185 can enhance X radiation inhibition effect on tumor growth, the tumor cell apoptosis level was significantly increased Plus. In summary, our study confirms for the first time to play a role in inhibiting the growth of melanoma are either in vitro or in vivo levels of miR-185, is a powerful anti cancer factor; and miR-185 can also increase the sensitivity to ionizing radiation of melanoma cells, is an effective radiosensitizer molecule. Our study the miR-185 tumor suppressor and radiation sensitivity increases the basic data, contribute to the clinical application to promote the miR-185.

【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R739.5

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