【作者】 曲珊珊；

【导师】 李荣贵；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 本研究以体外培养的人骨肉瘤U2OS细胞为实验模型,利用RNA干扰技术下调高迁移率蛋白A2 (HMGA2)表达,分析其对骨肉瘤细胞表型的影响,确定HMGA2在维持骨肉瘤细胞恶性表型中的作用;探讨其作用的分子机制及靶向HMGA2的RNA干扰技术在骨肉瘤治疗中的应用前景。结果:成功地构建了针对HMGA2的,基于DNA的shRNA干扰质粒载体(HMGA2-shRNA);将这一载体稳定转染人骨肉瘤U2OS细胞,明显地下调了其HMGA2的表达水平。细胞表型研究显示,抑制HMGA2表达明显地改变了该细胞的恶性表型;表现为细胞增殖和成集落能力明显下降;自发凋亡率则明显增加;同时细胞迁移速度、侵袭能力及粘附、铺展能力均明显受到抑制;并显著增加了细胞对化疗药物的敏感性。这些结果表明HMGA2在维持骨肉瘤U2OS细胞恶性表型及对化疗药物抗性方面起关键作用。应用实时定量RT-PCR对相关基因表达水平分析发现:下调HMGA2表达使细胞凋亡基因Caspase-3和Caspase-9表达上调,而凋亡抑制基因Bcl-2、Survivin表达水平降低;MMP-2和MMP-9表达降低,TIMP-3表达增高;CDKN1A表达增高,提示HMGA2是通过调控一系列靶基因表达水平来维持骨肉瘤U2OS细胞恶性表型的。本研究还首次发现下调HMGA2使该细胞的SOX2表达水平降低,PTEN表达水平提高。SOX2曾被报道为干细胞的标记物,这一发现提示在U2OS细胞群中有骨肉瘤干细胞样细胞的存在,且与HMGA2表达水平相关。而PTEN则曾被描述为肿瘤抑制基因,进一步证实了HMGA2在维持骨肉瘤细胞恶性表型的决定作用,提示靶向HMGA2的RNA干扰在骨肉瘤治疗中的应用前景。更多还原

【Abstract】 High mobility group protein A2 (HMGA2) is a member of the HMGA family. HMGA2 was composed with three AT-hook binding domain and an acidic C-terminal domain. AT-hook can help HMGA2 recognize and bind to the AT-rich regions of DNA small groove specifically, resulting in DNA bending, stretching, looping or melting, regulating replication, transcription and DNA repair, also known as architectural transcription factor. HMGA2 showed high expression at the embryonic period, whereas in the normal mature tissues almost no expression, but in some malignant tumors showed high expression, such as breast cancer, non-small cell lung cancer, pancreatic cancer, retinoblastoma, squamous cell carcinoma, suggesting that HMGA2 play an important role in malignant transformation of tumors. More and more studies have shown that cancer stem cells (CSC) are not sensitive to the chemotherapy and radiotherapy, resulting in cancer recurrence, metastasis and hard cure. It has been reported, overexpression HMGA2 and H-Ras gene can inhibit the differentiation of breast cancer stem cells in order to facilitate the maintenance of their self-renewal, but the miRNA let-7 can promote the differentiation of breast cancer stem cells to treat breast cancer by inhibiting the expression of HMGA2 and H-Ras gene.Osteosarcoma incidence growths rapidly and tends to younger, can occur lung metastasis within a few months. It has high degree of malignancy and poor prognosis, and chemotherapy or radiotherapy is ineffective. Therefore, searching for effective treatment for osteosarcoma is the focus of osteosarcoma study, and targeted gene therapy is considered one of the best treatments, so to find effective target genes for treatment are extremely important. In view of differential expression of HMGA2 in tumor cells and normal cells, RNAi gene therapy has certain advantages. In this study, we culture human osteosarcoma U2OS cells, utilize RNAi to inhibit the expression of HMGA2, and to observe the biological behaviors of U2OS cells after silencing of HMGA2, and to investigate the postential mechanisms, and to explore the possible relationship between HMGA2 and cancer stem cells.PartⅠHMGA2-shRNA transfected into U2OS cells reduced the expression of HMGA2Objective:To construct an effective HMGA2-shRNA silencing plasmid, and detect its expression in human osteosarcoma cell U2OS. Methods: According to the full cDNA sequence of HMGA2 (N0. 003483) in GenBank, select the specific sequence 5’-CGCCAACGTTCGA TTTCAT-3 ’for the target of the interference effect, and construct the sequence to the eukaryotic expression plasmid pGCsi-H1, containing the green fluorescent protein GFP. Select the scrambled plasmid which will not interfere with any human mRNA as negative control. HMGA2-shRNA and scrambled plasmids were transfected into U2OS cells, selected by G418 and obtained stable cell clone of the HMGA2-shRNA and scrambled respectively, RT-PCR detect silencing effect of HMGA2-shRNA. Results: Obtained stable transfection cell clones of HMGA2-shRNA and scrambled, RT-PCR results showed that HMGA2-shRNA plasmid reduced mRNA expression levels of HMGA2 significantly. Conclusion: Obtained effective stable cell clones of silencing HMGA2 gene successfull.PartⅡEffects on proliferation and apoptosis of HMGA2 in U2OS cellsObjective:To study the effects of silencing HMGA2 on cell morphology, growth curve, colony forming ability, cell apoptosis and cell cycle of human osteosarcoma U2OS cell, to detect mRNA levels of related gene, and to explore its possible mechanism. Methods: Utilize microscopy observe cell morphology; CCK-8 kit detect cell growth curve; soft agar colony forming experiment testing the ability of cell cloning;Hoechst33342 dyes detect cell apoptosis; PI staining with flow cytometry detect cell cycle changes. Utilized real-time RT-PCR detecting mRNA level of relative genes. Results: compared with untreated and scrambled group, HMGA2 gene silencing group cell proliferation activity was decreased significantly (P<0.01); capacity of clonal growth was decreased; apoptotic cells increased significantly (P <0.05); the cell number of S phase decreased, most of the cell were blocked in the G0/G1 phase. The same culture conditions, untreated and scrambled group cells were spindle-type, cell body were more prominent after adheren, the majority of HMGA2 silencing group cells in shape of irregular polygons, cell body relatively flat after adherent. Real-time RT-PCR results show that the expression of the apoptosis gene caspase-3 and caspase-9 are increased,and the expression of the apoptosis inhibitor gene Bcl-2 and suvivin are decreased, and the expression of CDKN1A is increased. Conlusion: HMGA2 through inducted the expression of CDKN1A, overcomed the G1/S block to promote the proliferation of U2OS cells. HMGA2 inhibited spontaneous apoptosis of U2OS cells by reducing the expression of caspase-3 and caspase-9 and increasing the expression of Bcl-2 and suvivin. PartⅢEffects on migration and invasion of HMGA2 in U2OS cellsObjective:To study the effects of HMGA2 gene on cell spreading, adhesion, migration, invasion in human osteosarcoma U2OS, to detect mRNA levels of related gene, and to explore its possible mechanism. Methods: Utilized microscope to observe cells spreading on FN; CCK-8 kit detect cells adhesion ability on FN and matrigel; scratch test detect cell migration; Boyden chamber observed cell invasion; Real-time RT-PCR detect related gene mRNA levels. Results: compared to untreated and scrambled group, HMGA2-shRNA group cells capacity of spreading on FN is diminished (P <0.05); and the capacity of adhesion on the FN (P <0.01) and Matrigel (P <0.05) were decreased significantly; migration ability of Cell injury repair and migrate to the chemokines were significantly reduced; Boyden chamber invasion assay results showed, the cell number of HMGA2-shRNA group reached the carbonate membrane is significantly lower than untreated and scrambled group(P <0.05). Real-time RT-PCR results showed that the expression of MMP-2 and MMP-9 were decreased significantly, while the expression of TIMP3 was increased. Conclusion: Reduced the expression of HMGA2 can decrease spreading and adhesion, migration and invasion of U2OS cell; real-time PCR results suggested that one important molecular mechanism of HMGA2 to maintain migration and invision of U2OS was increasing the expression of MMP-2 MMP-9 and reducing the expression of TIMP-3 expression.PartⅣthe role of HMGA2 in maintaining chemotherapy resistance of U2OS cellsObjective:To study the effects of HMGA2 gene on chemotherapy sensitivity of U2OS cells; to detecte the mRNA level of stem cells markers, and to explore the relationship between HMGA2 and cancer stem cells. Methods: CCK-8 detected the response of U2OS cells to chemotherapeutic drugs vincristine; Real-time RT-PCR detected related gene mRNA levele of stem cells. Results:compared with untreated group(IC50=136.39ng/ml) and scrambled group (IC50=148.26 ng/ml), HMGA2-shRNA group(IC50=71.73 ng/ml) cells increased the sensitivity to chemotherapy vincristine(P<0.05). Real-time RT-PCR results showed that the expression of PTEN was increased and the expression of SOX2 was reduced. Conclusion: HMGA2 played an important role in maintaining the resistant to chemotherapy vincristine in U2OS cells, the results provided an experimental basis for the treatment of osteosarcoma by RNA interference targeted to HMGA2. For the first time, we found that HMGA2 can induce the expression of stem cell marker SOX2, suggesting that in the osteosarcoma U2OS cell populations exist stem cell-like cells, and associated with the expression level of HMGA2. The tumor suppressor gene PTEN, further confirmed the important role of HMGA2 in maintaining malignant phenotype of osteosarcoma cells, suggesting that HMGA2 is a promsing target gene in the RNA interference treatment osteosarcoma.PartⅤIsolation and biological behaviour analysis of prostatic carcinoma PC3 cell subpopulationObjective:To identify the characteristic of cancer stem-like cells in prostatic carcinoma PC3 cell line, and analyze the morphous and clone growth, investigate the method of discriminating the different tumor cell subpopulation by Rhodamin123 dye. Methods:Flow cytometry analyze the PC3 cells stained with Rhodamin123 dye; using fluorescent microscope to observe cells morphous and clone growth stained with Rhodamin123 and DAPI. Results:Two different tumor cell subpopulation exist in PC3 cell line, and the morphous, reproductive activity and colon growth are significant different in the two cell subpopulation. Conclusion:Cancer stem-like cell subpopulation exist in prostatic carcinoma PC3 cell line, the character of some cells exclude Rhodamin123 dye can be used to discriminate tumor stem cells and other tumor cells.更多还原