lncRNA ANRIL在骨肉瘤组织中的表达及其对骨肉瘤细胞增殖凋亡和侵袭迁移的影响

发布时间：2018-04-27 05:00

本文选题：lncRNA + ANRIL　；参考：《郑州大学》2017年硕士论文

【摘要】：研究背景和目的骨肉瘤是骨外科常见的恶性肿瘤,其起源于骨髓间充质干细胞,且好发青少年儿童。手术及放化疗联合应用是本病目前治疗的基本手段,伴随手术方式的改进和新型化疗药物的应用使得本病的预后有了较为明显的改善,但仍约有40%的患者治疗后出现肿瘤转移,五年生存率未有明显提高,这预示仍需新的治疗方法来改善患者愈后,发现新的基因对骨肉瘤的影响似乎是一种有效的手段。长链非编码RNA(Long non-coding RNA,lncRNA)是长度大于200个核苷酸的非编码RNA。做为遗传学研究热点,许多研究表明,lncRNA在剂量补偿效应、表观遗传调控、细胞周期调控和细胞分化调控等众多生命活动中发挥重要作用。lncRNA表达量的变化或其功能异常与导致人类疾病的发生密切相关,包括癌症、退行性神经疾病等在内的许多对人类健康危害严重的重大疾病,具体表现为lncRNA在序列和空间结构的异常、表达水平的异常、与结合蛋白相互作用的异常等。lncRNA ANRIL是细胞周期激酶抑制因子4b(INK4b)位点的反义非编码RNA(antisense non-coding RNA in the INK4 Locus),其表达与INK4a的表观遗传学沉默相关。INK4b-ARF-INK4a位点对细胞周期、衰老和调亡的调控中具有重要的作用。然而,关于lncRNA ANRIL对骨肉瘤细胞生物学行为的影响尚未见报道。因此,探讨lncRNA ANRIL对人骨肉瘤细胞生物学功能的影响,从而为骨肉瘤的临床治疗提供新的思路和策略。本实验通过收集骨肉瘤癌组织及癌旁组织的标本,比较lncRNA ANRIL在癌组织及癌旁组织表达的差异。将小干扰RNA(siRNA)分子转染进入骨肉瘤细胞来抑制lncRNA ANRIL基因的表达,观察抑制lncRNA ANRIL的表达对骨肉瘤细胞增殖、凋亡及侵袭迁移能力的影响,为lncRNA ANRIL在骨肉瘤的防治中提供实验依据。方法1.在郑州大学第一附属医院骨科手术室收集25例配对骨肉瘤患者的癌组织及癌旁组织标本,保存于-80°C冰箱。使用荧光实时定量PCR(real-time quantitative PCR,RT-q PCR)方法检测25例配对骨肉瘤癌组织及癌旁组织标本中lncRNA ANRIL的表达水平。2.试剂公司人工合成lncRNA ANRIL的抑制物:siRNA-ANRIL。实验分为三组,实验组(siRNA-ANRIL):将siRNA-ANRIL转染入骨肉瘤细胞MG63中,阴性对照组(Nagetive control,NC):转染lncRNA ANRIL Nagetive control,空白组(Blank):仅转染脂质体。转染后,将细胞至于CO2培养箱中,用于后续实验。3.转染48h后,用RT-q PCR技术测量三组细胞内lncRNA ANRIL基因的表达量,观察siRNA-ANRIL对lncRNA ANRIL表达的抑制效果。4.利用CCK-8细胞计数试剂盒(Cell Counting Kit-8,CCK-8)在处理24h、48h、72h、96h后分别测量三组细胞的增殖能力,观察抑制lncRNA ANRIL表达对骨肉瘤细胞增殖的影响。5.用流式细胞技术和caspase-3酶活性检测试剂盒分别对三组细胞的凋亡进行测量,观察抑制lncRNA ANRIL表达后对骨肉瘤细胞凋亡的影响。6.Transwell侵袭实验,抑制lncRNA ANRIL表达后在12h时观察对骨肉瘤细胞侵袭能力的影响。7.划痕实验,对三组骨肉瘤细胞进行划痕处理,在处理12h后观察抑制lncRNA ANRIL表达后对骨肉瘤细胞迁移能力的影响。8.利用蛋白质印迹法(Western blot)分别检测转染后的三组骨肉瘤细胞中肿瘤转移相关基因1蛋白(metastasis-associaled gene 1,MTA1),上皮细胞钙黏蛋白(E-cadherin,E-cad),B淋巴细胞瘤-2蛋白(bcl-2)的表达量。结果1.与癌旁组织相比,骨肉瘤癌组织中lncRNA ANRIL基因的表达水平明显增高,其差异具有统计学意义(P0.05)。2.RT-q PCR结果显示,转染后siRNA-ANRIL组lncRNA ANRIL基因的表达水平明显低于阴性对照组和空白组,其差异具有统计学意义(P0.05),阴性对照组和空白组lncRNA ANRIL表达量无明显差异(P0.05)。3.CCK-8结果显示,siRNA-ANRIL组的骨肉瘤细胞在OD450处的吸光度值明显下降,且随时间延长,吸光度值与阴性对照组和空白组相比差异越明显,其差异具有统计学意义(P0.05),阴性对照组和空白组的吸光度值相近,且随时间变化不明显,差异无统计学意义(P0.05)。4.流式细胞技术和caspase-3酶活性检测试剂盒结果均表明,与阴性对照组和空白组相比,siRNA-ANRIL组的骨肉瘤细胞的凋亡率和Caspase-3酶活性均明显升高,其差异具有统计学意义(P0.05),阴性对照组和空白组骨肉瘤细胞的凋亡率和Caspase-3酶活性无明显差异(P0.05)。5.Transwell侵袭实验结果显示,siRNA-ANRIL组骨肉瘤细胞穿过基底膜的数量明显低于阴性对照组和空白组,其差异具有统计学意义(P0.05),而阴性对照组和空白组骨肉瘤细胞穿过基底膜的数量差异无统计学意义(P0.05)。6.对三组细胞进行划痕处理12h以后的结果提示,siRNA-ANRIL组骨肉瘤细胞的迁移能力与阴性对照组和空白组相比明显降低,阴性对照组和空白组骨肉瘤细胞的迁移能力无明显差异。7.Western blot结果显示,siRNA-ANRIL组中MTA1表达量降低,E-cad表达量升高,bcl-2蛋白表达量降低,与阴性对照组和空白组相比较,其差异具有统计学意义(P0.05),而阴性对照组和空白组MTA1,E-cad和bcl-2蛋白的表达量相近,差异无统计学意义(P0.05)。结论1.lncRNA ANRIL在骨肉瘤癌组织的表达高于癌旁组织,且可通过使用siRNA-ANRIL来抑制骨肉瘤细胞lncRNA ANRIL的表达。2.抑制lncRNA ANRIL基因的表达能够促进骨肉瘤细胞的凋亡,且能够抑制骨肉瘤细胞的增殖、侵袭和迁移能力。
[Abstract]:Background and objective osteosarcoma is a common malignant tumor in bone surgery. It originates in bone marrow mesenchymal stem cells (MSCs) and develops young children well. Operation and radiotherapy combined with chemotherapy are the basic methods for the treatment of this disease. With the improvement of surgical methods and the use of new chemotherapeutic drugs, the prognosis of this disease has been improved significantly. However, about 40% of the patients still have tumor metastasis after treatment, and the five year survival rate has not been significantly improved. This indicates that a new treatment is still needed to improve the patient's recovery. It is found that the effect of the new gene on osteosarcoma seems to be an effective means. Long chain non coded RNA (Long non-coding RNA, lncRNA) is a non coding length greater than 200 nucleotides. RNA. is a hot topic in genetics. Many studies have shown that lncRNA plays an important role in many life activities such as dose compensation effect, epigenetic regulation, cell cycle regulation and cell differentiation regulation. The changes in the expression of.LncRNA or its function abnormality are closely related to the occurrence of human diseases, including cancer and degenerative neurological disease. Many serious diseases, such as lncRNA in sequence and space structure, abnormal expression level, abnormal interaction with binding proteins,.LncRNA ANRIL are antisense non coded RNA (antisense non-coding RNA in the INK4), and so on. CUS), its expression and epigenetic silencing of INK4a have an important role in the regulation of cell cycle, senescence and apoptosis. However, the effect of lncRNA ANRIL on the biological behavior of osteosarcoma cells has not yet been reported. Therefore, the effect of lncRNA ANRIL on the biological function of human osteosarcoma cells is discussed, thus the effect of lncRNA ANRIL on the biological function of human osteosarcoma cells is discussed. This experiment provides a new way of thinking and strategy for the clinical treatment of osteosarcoma. By collecting the specimens of osteosarcoma and para cancer tissue, the difference of expression of lncRNA ANRIL in cancer tissue and para cancer tissue was compared. Small interference RNA (siRNA) molecules were transfected into osteosarcoma cells to inhibit the expression of lncRNA ANRIL gene and to observe the inhibition of lncRNA ANRIL. The effects of expression on the proliferation, apoptosis and invasion and migration of osteosarcoma cells were provided for lncRNA ANRIL in the prevention and treatment of osteosarcoma. Methods 1. the specimens of 25 cases of paired osteosarcoma in the Department of orthopedics, the First Affiliated Hospital of Zhengzhou University, were collected and preserved in the -80 C refrigerator. The fluorescence real-time quantitative PCR (R) was used. The eal-time quantitative PCR, RT-q PCR) method was used to detect the expression of lncRNA ANRIL in 25 cases of paired osteosarcoma and paracarkoma tissue. The inhibitor of lncRNA ANRIL was synthesized by.2. reagent company. The siRNA-ANRIL. experiment was divided into three groups, and the experimental group (siRNA-ANRIL) was transfected into osteosarcoma cells and negative control group. Tive control, NC): transfection of lncRNA ANRIL Nagetive control, blank group (Blank): transfection only liposomes. After transfection, the cells were used in the CO2 incubator for the follow-up experimental.3. transfection 48h. The 8 cell counting Kit (Cell Counting Kit-8, CCK-8) measured the proliferation ability of three groups of cells after 24h, 48h, 72h and 96h, and observed the effect of the inhibition of lncRNA ANRIL expression on the proliferation of osteosarcoma cells.5. using flow cytometry and caspase-3 enzyme activity detection kit to measure the apoptosis of three groups of cells respectively. The effect of A ANRIL expression on osteosarcoma cell apoptosis,.6.Transwell invasion experiment, inhibition of lncRNA ANRIL expression on the invasion ability of osteosarcoma cells after 12h,.7. scratch test, three groups of osteosarcoma cells were scratched, and the migration ability of osteosarcoma cells after lncRNA ANRIL expression was observed after 12h treatment. The expression of tumor metastasis related gene 1 protein (metastasis-associaled gene 1, MTA1), epithelial calcic mucin (E-cadherin, E-cad) and B lymphocytic -2 protein (Bcl-2) expression in three groups of osteosarcoma cells after transfection were detected by Western blot (Western blot). Results 1. in the tissues of osteosarcoma carcinoma, LN.8. was compared with that of para cancerous tissue. The expression level of cRNA ANRIL gene was significantly higher, and the difference was statistically significant (P0.05).2.RT-q PCR results showed that the expression level of lncRNA ANRIL gene in the siRNA-ANRIL group was significantly lower than that of the negative control group and the blank group, and the difference was statistically significant (P0.05). There was no significant difference in the expression of lncRNA ANRIL in the negative control group and the blank group. P0.05.3.CCK-8 results showed that the absorbance value of osteosarcoma cells in group siRNA-ANRIL decreased obviously at OD450, and the difference of absorbance value was more obvious compared to negative control group and blank group with time, and the difference was statistically significant (P0.05). The absorbance value of negative control group and blank group was similar, and the change was not obvious with time. The difference was not statistically significant (P0.05).4. flow cytometry and caspase-3 enzyme activity detection kit showed that, compared with the negative control group and the blank group, the apoptosis rate and the Caspase-3 enzyme activity of the osteosarcoma cells in the siRNA-ANRIL group were significantly increased, and the difference was of the significance (P0.05), the negative control group and the blank group of osteosarcoma. The apoptosis rate and Caspase-3 enzyme activity of the cells were not significantly different (P0.05).5.Transwell invasion test results showed that the number of osteosarcoma cells passing through the basement membrane in siRNA-ANRIL group was significantly lower than that of the negative control group and the blank group, and the difference was statistically significant (P0.05), while the number of osteosarcoma cells in negative and blank groups passed through the basal membrane of the negative group and the blank group. The difference was not statistically significant (P0.05).6. after the scratch treatment of three groups of cells 12h results suggested that the migration of osteosarcoma cells in the siRNA-ANRIL group was significantly lower than that in the negative control group and the blank group. The migration ability of the osteosarcoma cells in the negative control group and the blank group was not significantly different from the.7.Western blot results, the siRNA-ANRIL group was shown in the siRNA-ANRIL group. The expression of MTA1 was reduced, the expression of E-cad increased and the expression of Bcl-2 protein decreased. Compared with the negative control group and the blank group, the difference was statistically significant (P0.05), while the expression of MTA1, E-cad and bcl-2 protein in the negative control group and the blank group was similar, and the difference was not statistically significant (P0.05). Conclusion 1.lncRNA ANRIL is in the osteosarcoma carcinoma tissue. The expression is higher than that of para cancerous tissue, and siRNA-ANRIL can be used to inhibit the expression of lncRNA ANRIL in osteosarcoma cells by.2. to inhibit the expression of lncRNA ANRIL gene, which can promote the apoptosis of osteosarcoma cells and inhibit the proliferation, invasion and migration of osteosarcoma cells.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R738.1

【参考文献】