MicroRNAs增加软骨肉瘤细胞对顺铂敏感性的实验研究
发布时间：2018-08-15 16:14
【摘要】：研究背景软骨肉瘤是软骨来源的恶性骨肿瘤,其发病率仅次于骨肉瘤而位于原发性恶性骨肿瘤的第二位。手术虽是软骨肉瘤首选和有效的治疗手段,但要求肿瘤彻底切除,否则极易复发。由于软骨肉瘤对化疗的敏感性欠佳,因此化疗在临床上的应用受到了极大的限制。如何提高软骨肉瘤对化学治疗的敏感性,一直是软骨肉瘤治疗领域的一个核心问题。微小RNA (microRNA, miRNA)是由19-25个核苷酸组成的小单链非编码RNA,广泛表达于高等真核生物基因组中。每一种miRNA都具有物种特异性。到目前为止,已发现一百多种miRNA在细胞的增殖、分化、迁移以及细胞周期、凋亡等生物过程中发挥重要作用。随着研究的不断深入,miRNA在肿瘤的发生、发展、治疗等各个环节所起的重要作用已为大家公认。1miRNA通过与mRNA结合对机体进行调控,相同miRNA在不同组织中与不同的靶基因结合发挥不同的调节作用,都显示了miRNA的调节机制是一个复杂的网络结构。正常情况下,miRNA通过精准的网络调节维持机体各细胞、组织中蛋白水平维持平衡,然而当其功能缺失或获得,将打破原有平衡。有研究表明niRNA作为一种潜在的抑癌/促癌因子,在许多人类肿瘤组织中miRNA表达异常,因此在肿瘤的诊断、治疗和预后中具有重要意义。随着对miRNA与肿瘤发生发展相关性研究的不断深入,越来越多的证据表明miRNA参与了调控肿瘤细胞对化疗的敏感性,这对揭示肿瘤的耐药机制有着重要意义。肿瘤化疗耐药是肿瘤患者预后差的主要原因之一。miRNA突变、异常表达或异常加工均会导致miRNA功能异常,并导致其靶蛋白表达异常。若靶蛋白与肿瘤细胞对药物敏感性相关,则相关miRNA的异常将会导致肿瘤细胞对药物敏感性的改变。miRNA参与化疗耐药的机制一般可分为：与凋亡相关的miRNA介导的化疗耐药,与药物转运相关miRNA介导的化疗耐药,与细胞修复相关miRNA介导的化疗耐药,以及与药物作用靶点相关miRNA介导的化疗耐药等。目前,miRNA在肿瘤耐药中的研究刚刚起步,目前研究大多是通过对比耐药细胞株与亲本敏感细胞株之间miRNA表达谱异常,寻找与药物敏感性相关的关键miRNA,从而进一步对肿瘤细胞化疗耐受机制进行深入研究和寻找新的药物作用靶点。顺铂是抗实体肿瘤的一线用药,为铂的金属络合物,其作用类似于烷化剂,干扰DNA复制,为周期非特异性抗肿瘤药物,具有抗癌谱广、作用强、与多种抗肿瘤药有协同作用且无交叉耐药等特点,是目前联合化疗最常使用的药物之一。本研究分为两个部分,以顺铂为例分别探讨microRNA-100 (miR-100)与microRNA-23b (miR-23b)恢复软骨肉瘤细胞对顺铂敏感性的机制,为miRNAs成为软骨肉瘤的治疗策略提供理论基础。第一部分：miR-100恢复软骨肉瘤细胞对顺铂敏感性的实验研究[研究目的]探讨miR-100增加软骨肉瘤细胞对顺铂敏感性的机制,为软骨肉瘤基于miR-100的化疗策略提供了理论基础。[方法]1.采用实时定量PCR (quantitative real-time PCR, qRT-PCR)检测miR-100在多种人软骨肉瘤细胞系、正常软骨细胞系以及患者软骨肉瘤组织标本中的表达水平。2.采用浓度梯度法对软骨肉瘤CH-2879细胞系进行药物筛选,获得对顺铂耐受的CH-2879细胞(命名为：CH-2879/DDP细胞),qRT-PCR检测并比较CH-2879细胞及CH-2879/DDP细胞的miR-100表达水平：MTT及克隆形成实验检测比较两种细胞在顺铂作用下的增殖能力和克隆形成能力。3. miRNA数据库(TargetScan, Pictar, and MicroRNA)预测miR-100的作用靶点。4.采用脂质体2000法将pre-miR-100和anti-miR-100以及它们的阴性对照分别转入CH-2879细胞,qRT-PCR验证转染后细胞miR-100的表达水平,蛋白质印迹法(western blot)检测提高或抑制miR-100后mTOR的表达水平以及]mTOR下游蛋白S6K和4EBP1的磷酸化水平。5.荧光素酶报告基因法(Luciferase reporter assay)进一步验证miR-100作用于mTOR的3’端非翻译区(3'untranslated region, UTR)。6.将CDDP. BEZ235以及CDDP+BEZ235分别处理CH-2879/DDP细胞及CH-2879细胞,观察抑制mTOR通路,软骨肉瘤细胞对顺铂的敏感性。7.将miR-100及其对照分别转染CH-2879细胞及CH-2879/DDP细胞,观察miR-100高表达与软骨肉瘤细胞对顺铂敏感性的关系。8.外源性S6K过表达质粒转染事先已经转染过miR-100的软骨肉瘤细胞,观察mTOR通路活性恢复后,软骨肉瘤细胞对顺铂耐受性的变化。[结果]1.miR-100在人软骨肉瘤细胞和患者软骨肉瘤标本中的表达下调。2. CH-2879/DDP细胞中miR-100表达下调,提示该基因与软骨肉瘤化疗耐受有关。3. mTOR及其所在信号通路是目前肿瘤病理机制研究的热点,正如我们预测的一样,mTOR是miR-100的直接靶点之一。miR-100的过／低表达能抑制／恢复mTOR的表达水平。4.miR-100的高表达增加软骨肉瘤对顺铂的敏感性,进一步印证了该基因与软骨肉瘤化疗耐受有关。5. mTOR通路活性的恢复使miR-100过表达的软骨肉瘤细胞表现出对顺铂的耐受,提示miR-100的过表达增加软骨肉瘤细胞对顺铂的敏感性是通过对mTOR通路的抑制实现的。[结论]与正常软骨细胞相比,miR-100在软骨肉瘤细胞及软骨肉瘤患者标本中的表达均下调,提示miR-100是软骨肉瘤的抑制基因；与原代软骨肉瘤细胞相比,miR-100在CH-2879/DDP细胞中表达下调,这不仅说明miR-100具有肿瘤抑制功能,而且与软骨肉瘤对顺铂的化疗敏感性有关。nTOR在多种恶性肿瘤中活化,可以成为治疗癌症的一个有前景的治疗靶点。在本研究中,我们确定了mTOR是miR-100直接靶目标,并首次报道了miR-100过表达通过抑制mTOR通路增加软骨肉瘤细胞对顺铂的敏感性,为软骨肉瘤基于miR-100的治疗策略提供了理论基础。第二部分：miR-23b恢复软骨肉瘤细胞对顺铂敏感性的实验研究[研究目的]探讨miR-23b增加软骨肉瘤细胞对顺铂敏感性的机制,为软骨肉瘤基于miR-23b的化疗策略提供了理论基础。[方法]1.采用qRT-PCR检测miR-23b在多种人软骨肉瘤细胞系、正常软骨细胞系以及患者软骨肉瘤组织标本中的表达水平。2.采用脂质体2000法将pre-miRs和control-miRs转染SW1353细胞；并在转染后不同时间点检测细胞的增殖情况。3.在常规细胞培养条件下,CH-2879细胞与CH-2879/DDP细胞分别接受顺铂刺激48小时,利用显微镜观察细胞形态的改变以及利用qRT-PCR检测miR-23b的表达。4.根据以往报道我们预测并验证Src是miR-23b的直接靶目标。5.通过采用不同浓度的顺铂分别作用于CH-2879细胞,48小时后收集细胞行western blot分析,验证miR-23b过表达抑制Src-Akt通路的作用。6.采用脂质体2000法将pre-miR-negative或pre-miR-23b分别转入CH-2879细胞,qRT-PCR验证转染后细胞1miR-23b的表达水平,western blot检测提高或抑制miR-23b后总的Src和磷酸化Src的水平。7.采用脂质体2000法,将含有野生型(wt)和突变型(mut) Src 3'UTR转染细胞,转染后利用荧光素酶报告基因法进一步验证miR-23b作用于Src3'UTR区。8.以contral-miR或pre-miR-23b分别转染CH-2879细胞、SW1353细胞和CH-2879/DDP细胞,用不同浓度的顺铂处理细胞后MTT法测定细胞活性。9.将：niR-23b转染CH-2879细胞及CH-2879/DDP细胞,观察miR-23b过表达恢复软骨肉瘤细胞对顺铂敏感性的作用。10.外源性Src过表达质粒转染事先已经转染过niR-23b的软骨肉瘤细胞,观察Src-Akt通路活性恢复后,软骨肉瘤细胞对顺铂耐受性的变化。[结果]1. miR-23b在多种软骨肉瘤细胞系及软骨肉瘤患者标本组织中的表达下调。2. CH-2879/DDP细胞中miR-23b表达下调,提示该基因也与软骨肉瘤对顺铂的化疗耐受有关。3.Src激酶参与了多种肿瘤的增殖、迁移和入侵,CH-2879/DDP细胞的Src-Akt通路活性的上调4.软骨肉瘤细胞中Src是miR-23b的直接靶目标5. miR-23b的过度表达通过直接抑制Src-Akt通路恢复了软骨肉瘤细胞对顺铂的敏感性。6.Src-Akt通路活性的恢复增加了miR-23b过表达软骨肉瘤细胞对顺铂的耐药性。[结论]同miR-100一样,miR-23b在软骨肉瘤细胞中的表达下调,提示它是一种肿瘤抑制基因,与软骨肉瘤对顺铂的化疗敏感性有关。我们经实验确认,Src是miR-23b的一个直接靶向目标,Akt是一种用于调节新陈代谢、细胞周期进展和细胞存活率的细胞质丝氨酸和苏氨酸激酶,miR-23b可以通过对Src-Akt通路的负向调节提高软骨肉瘤细胞对顺铂的敏感性,为软骨肉瘤基于miR-23b的治疗策略提供了理论依据。
[Abstract]:Background Chondrosarcoma is a malignant bone tumor of cartilage origin. The incidence of osteosarcoma is second only to osteosarcoma. Surgery is the first choice and effective treatment for chondrosarcoma, but it requires complete resection of the tumor, otherwise it is easy to recur. Clinical applications have been greatly limited. How to improve the sensitivity of chondrosarcoma to chemotherapy has always been a core issue in the field of chondrosarcoma treatment. Up to now, more than 100 kinds of microRNAs have been found to play an important role in cell proliferation, differentiation, migration, cell cycle, apoptosis and other biological processes. The same microRNAs bind to different target genes in different tissues and play different regulatory roles, indicating that the regulatory mechanism of microRNAs is a complex network structure. Studies have shown that niRNA, as a potential tumor suppressor/promoter, is abnormal in the expression of microRNAs in many human tumor tissues, so it is important in the diagnosis, treatment and prognosis of cancer. MicroRNAs are involved in the regulation of chemosensitivity of tumor cells, which is important for revealing the mechanism of tumor resistance. Chemosensitivity is one of the main causes of poor prognosis in cancer patients. Mutations in microRNAs, abnormal expression or abnormal processing can lead to abnormal function of microRNAs and abnormal expression of target proteins. The mechanisms of microRNAs involved in chemosensitivity include apoptosis-related microRNAs-mediated chemosensitivity, drug transport-related microRNAs-mediated chemosensitivity, and cell repair-related microRNAs-mediated chemosensitivity. At present, the study of microRNAs in tumor resistance has just begun. At present, most of the research is to find the key microRNAs related to drug sensitivity by comparing the abnormal expression profiles of microRNAs between drug-resistant cell lines and parental sensitive cell lines. Cisplatin is a first-line antitumor drug, a metal complex of platinum. It acts as an alkylating agent, interferes with DNA replication, and is a periodically nonspecific antitumor drug. It has a broad spectrum of anticancer drugs, has a strong effect, and has synergistic effect with a variety of antitumor drugs without cross-resistance. This study is divided into two parts. Take microRNA-100 and microRNA-23b as examples to explore the mechanism of restoring the sensitivity of chondrosarcoma cells to cisplatin, and to provide theoretical basis for microRNAs to become a therapeutic strategy for chondrosarcoma. Part I: MicroRNA-100 restores cartilage. [Objective] To explore the mechanism of increasing the sensitivity of chondrosarcoma cells to cisplatin by microarray-100, and to provide a theoretical basis for the chemotherapy strategy of chondrosarcoma based on microarray-100. [Methods] 1. Real-time quantitative PCR (qRT-PCR) was used to detect microarray-100 in multiple human chondrosarcoma cells. The expression levels of CH-2879 cells (named CH-2879/DDP cells) and CH-2879 cells and CH-2879/DDP cells were detected by qRT-PCR. The expression levels of microarray-100 in CH-2879 cells and CH-2879/DDP cells were compared. Ping: MTT assay and cloning assay were used to compare the proliferation and cloning ability of the two cells under cisplatin. 3. The Target Scan (Pictar, and MicroRNA) database predicted the target of microRNA-100. 4. Pre-microRNA-100, anti-microRNA-100 and their negative control were transferred into CH-2879 cells by liposome 2000, respectively. PCR was used to verify the expression of microRNAs-100, Western blot was used to detect the expression of mTOR after microRNAs-100, and phosphorylation of downstream mTOR proteins S6K and 4EBP1.5 Luciferase reporter assay was used to further verify the effect of microRNAs-100 on the 3'untranslated region of mTOR. The sensitivity of chondrosarcoma cells to cisplatin and inhibition of mTOR pathway were observed. 7. Mi-100 and its control were transfected into CH-2879 cells and CH-2879/DDP cells respectively. The high expression of Mi-100 and the sensitivity of chondrosarcoma cells to cisplatin were observed. Relationship. 8. Exogenous S6K overexpression plasmid was transfected into chondrosarcoma cells which had previously been transfected with Mi-100 to observe the changes of cisplatin tolerance of chondrosarcoma cells after the activity of mTOR pathway was restored. [Results] 1. The expression of Mi-100 in human chondrosarcoma cells and chondrosarcoma samples was down-regulated. 2. The expression of Mi-100 in CH-2879/DDP cells was down-regulated. It is suggested that this gene is related to chemotherapeutic tolerance of chondrosarcoma. 3. mTOR and its signaling pathway are the hotspots in the study of tumor pathology. As we predicted, mTOR is one of the direct targets of microRNA-100. Over/under-expression of microRNA-100 can inhibit/restore the expression of mTOR. 4. Over-expression of microRNA-100 can increase the cis-cis of chondrosarcoma. The sensitivity to platinum further confirmed that the gene was related to chemotherapeutic tolerance of chondrosarcoma. 5. Restoration of the activity of mTOR pathway made the over-expressed chondrosarcoma cells show tolerance to cisplatin, suggesting that the overexpression of microRNA-100 increased the sensitivity of chondrosarcoma cells to cisplatin by inhibiting the mTOR pathway. Compared with normal chondrocytes, the expression of miR-100 was down-regulated in chondrosarcoma cells and chondrosarcoma specimens, suggesting that it was an inhibitor gene of chondrosarcoma; compared with primary chondrosarcoma cells, the expression of miR-100 was down-regulated in CH-2879/DDP cells, which not only showed that the expression of microRNA-100 had tumor-suppressing function, but also showed that it had the effect of cisplatin on chondrosarcoma cells. In this study, we identified mTOR as a direct target for microarray-100, and reported for the first time that microarray-100 overexpression increases the sensitivity of chondrosarcoma cells to cisplatin by inhibiting the mTOR pathway. Part II: Experimental study on the effect of microRNAs-23b on cisplatin sensitivity of chondrosarcoma cells [Objective] To explore the mechanism of microRNAs-23b increasing the sensitivity of chondrosarcoma cells to cisplatin, and to provide a theoretical basis for the chemotherapy strategy of chondrosarcoma based on microRNAs-23b. [Methods]1. QRT-PCR was used to detect the sensitivity of chondrosarcoma cells to cisplatin. Expression of microRNAs-23b in chondrosarcoma cell lines, normal chondrocyte lines and chondrosarcoma tissue specimens from patients. 2. Pre-microRNAs and control-microRNAs were transfected into SW1353 cells by liposome 2000 method. Cell proliferation was detected at different time points after transfection. 3. CH-2879 cells and CH-2 cells were cultured under normal conditions. 879 / DDP cells were stimulated by cisplatin for 48 hours. Cell morphological changes were observed under microscope and the expression of microarray-23b was detected by qRT-PCR. 4. According to previous reports, we predicted and verified that Src was the direct target of microarray-23b. 5. Different concentrations of cisplatin were used to treat CH-2879 cells. After 48 hours, the cells were collected and Wester was performed. Pre-microRNA-negative or pre-microRNA-23b were transfected into CH-2879 cells by liposome 2000. The expression of 1microRNA-23b was verified by qRT-PCR. The total Src and phosphorylated Src levels were increased or inhibited by Western blot. Liposome 2000 was used. METHODS: Transfected cells containing wild type (wt) and mutant type (mut) Src 3'UTR were transfected and further validated by luciferase reporter gene assay. 8. Contrl-Mi or pre-Mi-23b were used to transfect CH-2879 cells, SW1353 cells and CH-2879/DDP cells respectively. MTT assay was used to detect the effect of microwave-23b on Src 3'UTR region. Activity: NiR-23b was transfected into CH-2879 cells and CH-2879/DDP cells to observe the effect of microarray-23b overexpression on restoring the sensitivity of chondrosarcoma cells to cisplatin. [Results] 1. The expression of Mi-23b was down-regulated in chondrosarcoma cell lines and samples from patients with chondrosarcoma. 2. The down-regulated expression of Mi-23b in CH-2879/DDP cells suggested that the gene was also involved in the chemotherapeutic tolerance of chondrosarcoma to cisplatin. 3. Src kinase was involved in the proliferation, migration and invasion of various tumors, and Src-Akt pathway in CH-2879/DDP cells. Upregulation of pathway activity 4. Src is the direct target of microRNAs-23b in chondrosarcoma cells 5. Overexpression of microRNAs-23b restores the sensitivity of chondrosarcoma cells to cisplatin by directly inhibiting the Src-Akt pathway. 6. Recovery of Src-Akt pathway activity increases the resistance of microRNAs-23b overexpression chondrosarcoma cells to cisplatin. The down-regulation of R-23b expression in chondrosarcoma cells suggests that it is a tumor suppressor gene and is related to the chemosensitivity of chondrosarcoma to cisplatin. We have confirmed that Src is a direct target of microRNA23b, and Akt is a cytoplasmic serine and threonine used to regulate metabolism, cell cycle progression and cell survival. Acid kinase, microRNA23b, can enhance the sensitivity of chondrosarcoma cells to cisplatin by negatively regulating the Src-Akt pathway, providing a theoretical basis for the treatment strategy of chondrosarcoma based on microRNA23b.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R738
本文编号：2184733
[Abstract]:Background Chondrosarcoma is a malignant bone tumor of cartilage origin. The incidence of osteosarcoma is second only to osteosarcoma. Surgery is the first choice and effective treatment for chondrosarcoma, but it requires complete resection of the tumor, otherwise it is easy to recur. Clinical applications have been greatly limited. How to improve the sensitivity of chondrosarcoma to chemotherapy has always been a core issue in the field of chondrosarcoma treatment. Up to now, more than 100 kinds of microRNAs have been found to play an important role in cell proliferation, differentiation, migration, cell cycle, apoptosis and other biological processes. The same microRNAs bind to different target genes in different tissues and play different regulatory roles, indicating that the regulatory mechanism of microRNAs is a complex network structure. Studies have shown that niRNA, as a potential tumor suppressor/promoter, is abnormal in the expression of microRNAs in many human tumor tissues, so it is important in the diagnosis, treatment and prognosis of cancer. MicroRNAs are involved in the regulation of chemosensitivity of tumor cells, which is important for revealing the mechanism of tumor resistance. Chemosensitivity is one of the main causes of poor prognosis in cancer patients. Mutations in microRNAs, abnormal expression or abnormal processing can lead to abnormal function of microRNAs and abnormal expression of target proteins. The mechanisms of microRNAs involved in chemosensitivity include apoptosis-related microRNAs-mediated chemosensitivity, drug transport-related microRNAs-mediated chemosensitivity, and cell repair-related microRNAs-mediated chemosensitivity. At present, the study of microRNAs in tumor resistance has just begun. At present, most of the research is to find the key microRNAs related to drug sensitivity by comparing the abnormal expression profiles of microRNAs between drug-resistant cell lines and parental sensitive cell lines. Cisplatin is a first-line antitumor drug, a metal complex of platinum. It acts as an alkylating agent, interferes with DNA replication, and is a periodically nonspecific antitumor drug. It has a broad spectrum of anticancer drugs, has a strong effect, and has synergistic effect with a variety of antitumor drugs without cross-resistance. This study is divided into two parts. Take microRNA-100 and microRNA-23b as examples to explore the mechanism of restoring the sensitivity of chondrosarcoma cells to cisplatin, and to provide theoretical basis for microRNAs to become a therapeutic strategy for chondrosarcoma. Part I: MicroRNA-100 restores cartilage. [Objective] To explore the mechanism of increasing the sensitivity of chondrosarcoma cells to cisplatin by microarray-100, and to provide a theoretical basis for the chemotherapy strategy of chondrosarcoma based on microarray-100. [Methods] 1. Real-time quantitative PCR (qRT-PCR) was used to detect microarray-100 in multiple human chondrosarcoma cells. The expression levels of CH-2879 cells (named CH-2879/DDP cells) and CH-2879 cells and CH-2879/DDP cells were detected by qRT-PCR. The expression levels of microarray-100 in CH-2879 cells and CH-2879/DDP cells were compared. Ping: MTT assay and cloning assay were used to compare the proliferation and cloning ability of the two cells under cisplatin. 3. The Target Scan (Pictar, and MicroRNA) database predicted the target of microRNA-100. 4. Pre-microRNA-100, anti-microRNA-100 and their negative control were transferred into CH-2879 cells by liposome 2000, respectively. PCR was used to verify the expression of microRNAs-100, Western blot was used to detect the expression of mTOR after microRNAs-100, and phosphorylation of downstream mTOR proteins S6K and 4EBP1.5 Luciferase reporter assay was used to further verify the effect of microRNAs-100 on the 3'untranslated region of mTOR. The sensitivity of chondrosarcoma cells to cisplatin and inhibition of mTOR pathway were observed. 7. Mi-100 and its control were transfected into CH-2879 cells and CH-2879/DDP cells respectively. The high expression of Mi-100 and the sensitivity of chondrosarcoma cells to cisplatin were observed. Relationship. 8. Exogenous S6K overexpression plasmid was transfected into chondrosarcoma cells which had previously been transfected with Mi-100 to observe the changes of cisplatin tolerance of chondrosarcoma cells after the activity of mTOR pathway was restored. [Results] 1. The expression of Mi-100 in human chondrosarcoma cells and chondrosarcoma samples was down-regulated. 2. The expression of Mi-100 in CH-2879/DDP cells was down-regulated. It is suggested that this gene is related to chemotherapeutic tolerance of chondrosarcoma. 3. mTOR and its signaling pathway are the hotspots in the study of tumor pathology. As we predicted, mTOR is one of the direct targets of microRNA-100. Over/under-expression of microRNA-100 can inhibit/restore the expression of mTOR. 4. Over-expression of microRNA-100 can increase the cis-cis of chondrosarcoma. The sensitivity to platinum further confirmed that the gene was related to chemotherapeutic tolerance of chondrosarcoma. 5. Restoration of the activity of mTOR pathway made the over-expressed chondrosarcoma cells show tolerance to cisplatin, suggesting that the overexpression of microRNA-100 increased the sensitivity of chondrosarcoma cells to cisplatin by inhibiting the mTOR pathway. Compared with normal chondrocytes, the expression of miR-100 was down-regulated in chondrosarcoma cells and chondrosarcoma specimens, suggesting that it was an inhibitor gene of chondrosarcoma; compared with primary chondrosarcoma cells, the expression of miR-100 was down-regulated in CH-2879/DDP cells, which not only showed that the expression of microRNA-100 had tumor-suppressing function, but also showed that it had the effect of cisplatin on chondrosarcoma cells. In this study, we identified mTOR as a direct target for microarray-100, and reported for the first time that microarray-100 overexpression increases the sensitivity of chondrosarcoma cells to cisplatin by inhibiting the mTOR pathway. Part II: Experimental study on the effect of microRNAs-23b on cisplatin sensitivity of chondrosarcoma cells [Objective] To explore the mechanism of microRNAs-23b increasing the sensitivity of chondrosarcoma cells to cisplatin, and to provide a theoretical basis for the chemotherapy strategy of chondrosarcoma based on microRNAs-23b. [Methods]1. QRT-PCR was used to detect the sensitivity of chondrosarcoma cells to cisplatin. Expression of microRNAs-23b in chondrosarcoma cell lines, normal chondrocyte lines and chondrosarcoma tissue specimens from patients. 2. Pre-microRNAs and control-microRNAs were transfected into SW1353 cells by liposome 2000 method. Cell proliferation was detected at different time points after transfection. 3. CH-2879 cells and CH-2 cells were cultured under normal conditions. 879 / DDP cells were stimulated by cisplatin for 48 hours. Cell morphological changes were observed under microscope and the expression of microarray-23b was detected by qRT-PCR. 4. According to previous reports, we predicted and verified that Src was the direct target of microarray-23b. 5. Different concentrations of cisplatin were used to treat CH-2879 cells. After 48 hours, the cells were collected and Wester was performed. Pre-microRNA-negative or pre-microRNA-23b were transfected into CH-2879 cells by liposome 2000. The expression of 1microRNA-23b was verified by qRT-PCR. The total Src and phosphorylated Src levels were increased or inhibited by Western blot. Liposome 2000 was used. METHODS: Transfected cells containing wild type (wt) and mutant type (mut) Src 3'UTR were transfected and further validated by luciferase reporter gene assay. 8. Contrl-Mi or pre-Mi-23b were used to transfect CH-2879 cells, SW1353 cells and CH-2879/DDP cells respectively. MTT assay was used to detect the effect of microwave-23b on Src 3'UTR region. Activity: NiR-23b was transfected into CH-2879 cells and CH-2879/DDP cells to observe the effect of microarray-23b overexpression on restoring the sensitivity of chondrosarcoma cells to cisplatin. [Results] 1. The expression of Mi-23b was down-regulated in chondrosarcoma cell lines and samples from patients with chondrosarcoma. 2. The down-regulated expression of Mi-23b in CH-2879/DDP cells suggested that the gene was also involved in the chemotherapeutic tolerance of chondrosarcoma to cisplatin. 3. Src kinase was involved in the proliferation, migration and invasion of various tumors, and Src-Akt pathway in CH-2879/DDP cells. Upregulation of pathway activity 4. Src is the direct target of microRNAs-23b in chondrosarcoma cells 5. Overexpression of microRNAs-23b restores the sensitivity of chondrosarcoma cells to cisplatin by directly inhibiting the Src-Akt pathway. 6. Recovery of Src-Akt pathway activity increases the resistance of microRNAs-23b overexpression chondrosarcoma cells to cisplatin. The down-regulation of R-23b expression in chondrosarcoma cells suggests that it is a tumor suppressor gene and is related to the chemosensitivity of chondrosarcoma to cisplatin. We have confirmed that Src is a direct target of microRNA23b, and Akt is a cytoplasmic serine and threonine used to regulate metabolism, cell cycle progression and cell survival. Acid kinase, microRNA23b, can enhance the sensitivity of chondrosarcoma cells to cisplatin by negatively regulating the Src-Akt pathway, providing a theoretical basis for the treatment strategy of chondrosarcoma based on microRNA23b.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R738
【共引文献】
相关期刊论文 前10条
1 李俊;王建中;黄新生;白广平;童华;周雷;刘建平;;RNAi沉默miRNA-224-5p对喉鳞状细胞癌Hep-2细胞增殖、凋亡及侵袭能力的影响[J];中国耳鼻咽喉头颈外科;2013年09期
2 裴丽玲;李建生;任维华;;miRNAs与肝细胞癌发生研究进展[J];实用肝脏病杂志;2014年02期
3 丁琛琛;李琼;王阁;;miRNA-224通过HOXD10影响Hep3B细胞迁移[J];第三军医大学学报;2014年13期
4 邓振宇;冯宇鹏;何小科;;NF-κB信号通路参与miR-183抑制结肠癌细胞侵袭和迁移性研究[J];解剖学研究;2014年03期
5 曾静;唐瑞雪;何融泉;陈罡;;MicroRNAs在视网膜母细胞瘤的研究新进展[J];国际眼科杂志;2014年11期
6 赵晓蕾;蔡林;邓洲铭;;microRNA与骨肉瘤的研究进展[J];中国骨与关节杂志;2014年10期
7 王路;余伯龙;岑建华;彭新宇;刘友利;曾芳芳;刘雄;;鼻咽癌患者血浆miR-24异常表达的临床意义[J];南方医科大学学报;2015年05期
8 刘雅琼;;埃兹蛋白、CD44v6、E-cadherin及β-catenin在宫颈鳞状细胞癌中的表达及与侵袭转移的关系[J];广东医学;2015年09期
9 喻超;江建新;黎志鹏;肖杰;潘耀振;孙诚谊;;microRNA-100对人胰腺癌细胞增殖及皮下成瘤能力的影响[J];贵阳医学院学报;2015年08期
10 朱楠;荆珏华;;骨肉瘤相关microRNA研究概况[J];临床骨科杂志;2013年05期
相关博士学位论文 前10条
1 范磊;MicroRNA145对骨肉瘤细胞增殖和转移等生物学行为的影响及相关机制研究[D];华中科技大学;2013年
2 金一;MicroRNA-376c靶向调控TGFA抑制骨肉瘤细胞增殖与转移的研究[D];中南大学;2013年
3 闫康;NKD1参与介导miR-195对骨肉瘤转移的抑制作用[D];第四军医大学;2013年
4 文铮;CDK5磷酸化Raf激酶抑制蛋白在帕金森病中的作用研究[D];华中科技大学;2013年
5 潘伟波;骨肉瘤细胞MG63中内源性microRNA-27a对骨肉瘤恶性行为的影响及其调控靶基因的研究[D];浙江大学;2013年
6 姜茂竹;乳腺癌不同分子亚型差异表达microRNAs的综合分析及microRNA-9-5p功能研究[D];南方医科大学;2013年
7 赵广义;microRNA-221在骨肉瘤中作用及机制的研究[D];第四军医大学;2013年
8 沈克;Has-miR-139调控人结肠癌细胞浸润转移及多药耐药的分子机理研究[D];华东理工大学;2014年
9 郭继刚;microRNA在神经母细胞瘤转移及骨肉瘤发生过程中的研究[D];南京大学;2011年
10 霍威;miRNA调控TRAIL表达对前列腺癌细胞产生选择性细胞毒作用[D];吉林大学;2014年
相关硕士学位论文 前10条
1 樊智彬;microRNA-224在大肠癌中的表达及其功能研究[D];河北医科大学;2013年
2 蒋庆详;MicroRNA-429对膀胱癌细胞增殖侵袭的影响[D];南华大学;2013年
3 朱英;PAK5和Ezrin基因在骨肉瘤组织中的表达及其临床意义[D];苏州大学;2014年
4 朱楠;MicroRNA-199a-3p对人骨肉瘤细胞凋亡的影响[D];安徽医科大学;2014年
5 裴丽玲;四种miRNAs在肝细胞性肝癌中的表达及意义[D];安徽医科大学;2014年
6 陈雪;miR-34а对肺腺癌细胞辐射敏感性的影响及其作用机制的研究[D];中国疾病预防控制中心;2013年
7 何涵;miR-98促进肝癌发生发展机制初步研究[D];南方医科大学;2014年
8 陈鹏;P65、VEGF和MMP-9在骨肉瘤组织中的表达及临床病理意义[D];苏州大学;2013年
9 梁增辉;MiR-183在肝癌中的表达水平及其临床意义[D];天津医科大学;2014年
10 于开文;黄芩素对A549肺腺癌细胞的抗增殖作用及机制的研究[D];延边大学;2014年
，本文编号：2184733
本文链接：https://www.wllwen.com/yixuelunwen/zlx/2184733.html
