转录因子Oct4对骨肉瘤增殖、侵袭的影响及其机制的研究

发布时间：2018-05-26 13:33

本文选题：骨肉瘤 + Oct4　；参考：《吉林大学》2017年博士论文

【摘要】：骨肉瘤(Osteosarcoma;OS)又被称作成骨肉瘤,是一种骨的恶性肿瘤,其发病年龄较早,大多常见于儿童或20岁以下的青少年,同时易于发生转移,因此可以说其严重危害了广大青少年和儿童的身体健康。此外,其发病率亦占据了原发性恶性肿瘤中的榜首。OS的恶性程度非常高,且预后相当差,甚至与OS发现的时间、采取的治疗措施等均无关。综上,OS给年轻的患者、家庭乃至社会带来了很重的负担和打击,故其一直是生命科学领域中研究的重点和热点,但尽管相关研究很多,其发病机制尤其是其易于生长和侵袭的机制,至今为止仍未给出很好的解释,而转录因子Oct4是一种重要的关键调控基因,密切参与多向性分化的调控,大量报道显示其非常可能参与了成体细胞和肿瘤的发生发展过程,但与OS的相关性如何至今鲜有研究。故本研究将以分子生物学技术为手段来探讨转录因子Oct4在OS发生发展中的具体作用机制,以便给OS发病机制的深入研究提供理论基础,具有重要的现实意义。目的检测Oct4在骨肉瘤中的表达情况,探讨Oct4对骨肉瘤的具体影响并研究这种影响产生的具体作用机制,为骨肉瘤的发病机制的研究奠定理论基础,具有重要的科学价值和研究意义。方法本论文的研究工作分为4个部分,具体如下:(1)Oct4基因在不同骨肉瘤细胞系和人类骨肉瘤组织表达分析对骨肉瘤细胞株SOSP-9607、MG63、F5M2和成骨细胞株h FOB1.19进行培养,同时收集吉林大中日联谊医院进行骨肉瘤手术治疗的10名患者于手术期间获得的骨肉瘤组织和正常组织。对上述样本组织进行总RNA和蛋白质的提取,分别用real-time RT-PCR和western blotting方式对样本进行Oct4基因和蛋白质表达的检测,检测结果采用单向方差分析和Bonferroni检测多重比较进行统计学分析。(2)Oct4基因对F5M2细胞株增殖和入侵的影响研究1)对骨肉瘤细胞株F5M2进行培养,采用Lipofectamine 2000转染试剂将三种Oct4 siRNAs和scramble分别转染至F5M2细胞中。转染三天后,分别用real-time RT-PCR和western blotting方式检测被Oct4 siRNAs干扰的F5M2细胞株的Oct4表达情况,以评价siRNAs和scramble的转染效果。2)对骨肉瘤细胞株F5M2分别加入Oct4 si RNA1和scramble进行干扰培养三天。采用MTT试验检测细胞增殖情况,分别测定Oct4 si RNA1和scramble干扰后的骨肉瘤细胞的吸光度。采用膜联蛋白V-FITC凋亡检测试剂盒检测,流式细胞仪监测骨肉瘤的细胞凋亡情况。通过体外侵袭实验对骨肉瘤细胞进行侵袭情况的评估。检测结果采用单向方差分析和Bonferroni检测多重比较进行统计学分析。(3)Oct4基因对AK055347表达影响对骨肉瘤细胞株F5M2进行培养,采用Lipofectamine 2000转染试剂将Oct4si RNA-1和scramble转染至骨肉瘤F5M2细胞株中。设计9种相关lnc RNAs引物,转染三天后采用real-time RT-PCR检测相关lnc RNAs的表达情况。(4)AK055347的下调表达对F5M2细胞增殖和入侵的影响研究1)对骨肉瘤细胞株F5M2进行培养,采用Lipofectamine 2000转染试剂将三种AK055347 siRNAs和scramble分别转染至F5M2细胞中。转染三天后,分别用real-time RT-PCR方式检测被AK055347 siRNAs干扰的F5M2细胞株的AK055347表达情况,以评价siRNAs和scramble的转染效果。2)对骨肉瘤细胞株F5M2分别加入AK055347 si RNA3和scramble进行干扰培养三天。采用MTT试验检测细胞增殖情况,分别测定AK055347 si RNA3和scramble干扰后的骨肉瘤细胞的吸光度。采用膜联蛋白V-FITC凋亡检测试剂盒检测,流式细胞仪监测骨肉瘤的细胞凋亡情况。通过体外侵袭实验对骨肉瘤细胞进行侵袭情况的评估。检测结果采用单向方差分析和Bonferroni检测多重比较进行统计学分析。结果(1)Oct4基因在不同骨肉瘤细胞系的表达显著增加;同时相较于癌旁周围正常组织,Oct4基因在人类骨肉瘤组织中的表达显著增加。(2)1)三种Oct4 siRNAs干扰骨肉瘤细胞株F5M2三天后,均显著降低Oct4 m RNA和蛋白的表达。其中,Oct4 si RNA-1表现出最大抑制作用,提示Oct4si RNA-1具有最佳的Oct4基因沉默效果。2)MTT试验检测结果显示Scramble组与空白组吸光度增加明显,而Oct4 si RNA1干扰组吸光度增加不明显,说明后者的细胞增殖情况明显受到抑制;膜联蛋白的血细胞计数检测结果表明Oct4si RNA-1干扰后,癌细胞凋亡率明显升高;侵袭小室实验结果显示与scramble组和空白对照组相比,Oct4下调的F5M2细胞里只有少量细胞穿越了涂有聚碳酸酯膜的人工基底膜,提示入侵减少。(3)F5M2细胞被Oct4 si RNA1和scramble干扰培养三天后,通过Real-time RT-PCR检测Oct4下调对lnc RNAs表达的影响,其中只有显著降低了AK055347的表达,其它8种并没有显著影响。(4)1)三种AK055347 siRNAs干扰骨肉瘤细胞株F5M2三天后,均显著降低AK055347 m RNA的表达。其中,AK055347 si RNA-3表现出最大抑制作用,提示AK055347 si RNA-3具有最佳的AK055347基因沉默效果。2)等量F5M2细胞被AK055347 si RNA3和scramble转染并培养3天,AK055347的表达下调与scramble组相比显著降低了MTT的吸光度值,表明了AK055347的表达下调降低了F5M2细胞的增殖;采用流式细胞仪检测膜联蛋白V的细胞表面表达,结果显示,AK055347的表达下降显著增加了膜联蛋白V表面表达,所以同时增加了癌细胞的凋亡;F5M2细胞分别加入AK055347 si RNA-3和scramble干扰培养三天后,通过侵袭小室实验检测细胞入侵能力,结果表明与scramble组相比,AK055347下调的F5M2细胞里只有少量细胞穿越了涂有聚碳酸酯膜的人工基底膜,即入侵减少,提示了AK055347下调降低了F5M2细胞的入侵能力。结论(1)Oct4参与骨肉瘤的发生发展过程,与其细胞增殖密切相关。(2)靶向Oct4的RNAi导致F5M2细胞增殖抑制,其原因与细胞死亡相关联,且死亡形式以凋亡为主而非坏死。(3)Oct4可能参与骨肉瘤细胞的侵袭和转移。(4)转录因子Oct4通过调节lnc RNA AK055347的表达促进骨肉瘤的发生发展创新率先发现Oct4基因在骨肉瘤组织及细胞系中表达,且呈现增高表达。提出Oct4可能是骨肉瘤中的重要转录因子之一,对其发生、发展起到巨大的推动作用。
[Abstract]:Osteosarcoma (OS), also known as osteosarcoma, is a malignant tumor of bone. The age of the osteosarcoma is early, most common in children or young people under 20 years of age, and it is easy to metastasize. Therefore, it can be said that it seriously endangers the body health of young people and children. In addition, the incidence of the disease also occupies the primary malignant tumor. .OS, at the top of the list, has a high degree of malignancy, and the prognosis is very poor, even with the time that OS has been found and the treatment measures are taken. To sum up, OS has brought heavy burden and shock to young patients, family and society, so it has been the focus and hot spot in the field of life science. Mechanism, especially its mechanism that is easy to grow and invasion, has still not been well explained, and the transcription factor Oct4 is an important key regulatory gene, closely involved in the regulation of multidirectional differentiation. A large number of reports show that it is very likely to be involved in the development of adult cells and tumors, but how the correlation with OS is so far It is rarely studied. Therefore, this study will study the specific mechanism of the transcription factor Oct4 in the development of OS by means of molecular biology technology, so as to provide theoretical basis for the in-depth study of the pathogenesis of OS. The purpose of this study is to detect the appearance of Oct4 in osteosarcoma and to explore the specific effects of Oct4 on osteosarcoma. And study the specific mechanism of this effect to lay a theoretical foundation for the study of the pathogenesis of osteosarcoma. It has important scientific value and research significance. The research work of this paper is divided into 4 parts: (1) the expression analysis of Oct4 gene in different osteosarcoma cell lines and human osteosarcoma tissue is fine for osteosarcoma The cells SOSP-9607, MG63, F5M2 and osteoblast h FOB1.19 were cultured, and the osteosarcoma tissues and normal tissues obtained during the operation were collected by 10 patients undergoing osteosarcoma operation in Jilin Dazhong friendship hospital. The total RNA and protein were extracted from the above samples with real-time RT-PCR and Western blotting respectively. Methods to detect the expression of Oct4 gene and protein in the sample, the results were analyzed statistically by one-way ANOVA and Bonferroni detection. (2) the effect of Oct4 gene on the proliferation and invasion of F5M2 cell line 1) culture of osteosarcoma cell line F5M2, and three Oct4 Si using Lipofectamine 2000 transfection reagent. RNAs and scramble were transfected into F5M2 cells respectively. After three days, real-time RT-PCR and Western blotting were used to detect Oct4 expression of Oct4 siRNAs interfering F5M2 cell lines. The cell proliferation was detected by MTT test, and the absorbance of osteosarcoma cells after Oct4 Si RNA1 and scramble interference were measured respectively. The apoptosis of osteosarcoma cells was detected by the membrane associated protein V-FITC apoptosis detection kit and the flow cytometry was used to evaluate the invasion of osteosarcoma cells through the invasion test in vitro. The results were statistically analyzed by one-way ANOVA and Bonferroni detection. (3) the effect of Oct4 gene on osteosarcoma cell line F5M2 was cultured on AK055347 expression, and Oct4si RNA-1 and scramble were transfected to F5M2 cell line of osteosarcoma by Lipofectamine 2000 transfection reagent. 9 kinds of LNC RNAs primers were designed and transfected for three days. Then real-time RT-PCR was used to detect the expression of related LNC RNAs. (4) the effect of AK055347 down expression on the proliferation and invasion of F5M2 cells. 1) the osteosarcoma cell line F5M2 was cultured and three AK055347 siRNAs and scramble were transfected into F5M2 cells respectively with Lipofectamine 2000 transfection reagent. The transfection for three days was used respectively. The expression of AK055347 in F5M2 cell lines interfered by AK055347 siRNAs was detected by -time RT-PCR, and the transfection effect of siRNAs and scramble was evaluated for three days. The absorbance of osteosarcoma cells after e interference. The apoptosis of osteosarcoma was detected by the flow cytometry with V-FITC apoptosis detection kit. The invasion of osteosarcoma cells was evaluated by invasive test in vitro. The detection results were statistically analyzed by one-way ANOVA and Bonferroni detection. Results (1) the expression of Oct4 gene in different osteosarcoma cell lines increased significantly, and the expression of Oct4 gene in human osteosarcoma tissue was significantly increased. (2) 1) three Oct4 siRNAs interfered osteosarcoma cell line F5M2 for three days, and the expression of Oct4 m RNA and protein was significantly reduced. Oct4 Si RNA-1 The maximum inhibitory effect showed that Oct4si RNA-1 had the best Oct4 gene silencing effect.2) MTT test results showed that the absorbance of the Scramble group and the blank group increased obviously, while the increase in the absorbance of the Oct4 Si RNA1 interference group was not obvious, indicating that the cell proliferation of the latter was obviously inhibited; the blood cell count detection junction of the annexin was found. The results showed that the apoptosis rate of cancer cells increased significantly after Oct4si RNA-1 interference, and the results of invasive chamber experiment showed that only a few cells in Oct4 down regulated F5M2 cells passed the artificial basement membrane coated with polycarbonate membrane compared with the scramble group and the blank control group, suggesting that the invasion was reduced. (3) F5M2 cells were disturbed by Oct4 Si RNA1 and scramble three. Real-time RT-PCR was used to detect the effect of down regulation of Oct4 on the expression of LNC RNAs, which only significantly reduced the expression of AK055347, and the other 8 had no significant influence. (4) 1) three AK055347 siRNAs interfered osteosarcoma cell lines, which significantly decreased the expression of AK055347 m RNA. It was suggested that AK055347 Si RNA-3 had the best AK055347 gene silencing effect.2) and the F5M2 cells were transfected and cultured for 3 days by AK055347 Si RNA3 and scramble, and the downregulation of AK055347 expression decreased significantly compared with the scramble group, indicating that the expression lowered the proliferation of the cells; flow cytometry was used. The expression of the cell surface of annexin V was detected. The results showed that the decrease of AK055347 expression significantly increased the expression of the membrane associated protein V, so the apoptosis of cancer cells was increased at the same time. F5M2 cells were added to AK055347 Si RNA-3 and scramble for three days, and the invasion ability of the cells was detected by invasive chamber experiment, and the results showed that the cells were with scram. Compared with the ble group, only a few cells in the AK055347 down F5M2 cells passed through the artificial basement membrane coated with polycarbonate membrane, that is, the invasion decreased, suggesting that the AK055347 downregulation reduced the invasion ability of F5M2 cells. Conclusion (1) Oct4 is involved in the development process of osteosarcoma and is closely related to its fine cell proliferation. (2) RNAi of the target to Oct4 leads to F5M2 fine. The inhibition of cell proliferation is associated with cell death, and the death form is mainly apoptosis and not necrotic. (3) Oct4 may be involved in the invasion and metastasis of osteosarcoma cells. (4) the transcription factor Oct4 first found the expression of Oct4 gene in osteosarcoma tissue and cell lines by regulating the expression of LNC RNA AK055347. It is suggested that Oct4 may be one of the important transcription factors in osteosarcoma, which may play an important role in the development of osteosarcoma.
【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】：R738.1

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