【摘要】：研究背景軟骨肉瘤是軟骨來源的惡性骨腫瘤,其發(fā)病率僅次于骨肉瘤而位于原發(fā)性惡性骨腫瘤的第二位。手術雖是軟骨肉瘤首選和有效的治療手段,但要求腫瘤徹底切除,否則極易復發(fā)。由于軟骨肉瘤對化療的敏感性欠佳,因此化療在臨床上的應用受到了極大的限制。如何提高軟骨肉瘤對化學治療的敏感性,一直是軟骨肉瘤治療領域的一個核心問題。微小RNA (microRNA, miRNA)是由19-25個核苷酸組成的小單鏈非編碼RNA,廣泛表達于高等真核生物基因組中。每一種miRNA都具有物種特異性。到目前為止,已發(fā)現(xiàn)一百多種miRNA在細胞的增殖、分化、遷移以及細胞周期、凋亡等生物過程中發(fā)揮重要作用。隨著研究的不斷深入,miRNA在腫瘤的發(fā)生、發(fā)展、治療等各個環(huán)節(jié)所起的重要作用已為大家公認。1miRNA通過與mRNA結合對機體進行調(diào)控,相同miRNA在不同組織中與不同的靶基因結合發(fā)揮不同的調(diào)節(jié)作用,都顯示了miRNA的調(diào)節(jié)機制是一個復雜的網(wǎng)絡結構。正常情況下,miRNA通過精準的網(wǎng)絡調(diào)節(jié)維持機體各細胞、組織中蛋白水平維持平衡,然而當其功能缺失或獲得,將打破原有平衡。有研究表明niRNA作為一種潛在的抑癌/促癌因子,在許多人類腫瘤組織中miRNA表達異常,因此在腫瘤的診斷、治療和預后中具有重要意義。隨著對miRNA與腫瘤發(fā)生發(fā)展相關性研究的不斷深入,越來越多的證據(jù)表明miRNA參與了調(diào)控腫瘤細胞對化療的敏感性,這對揭示腫瘤的耐藥機制有著重要意義。腫瘤化療耐藥是腫瘤患者預后差的主要原因之一。miRNA突變、異常表達或異常加工均會導致miRNA功能異常,并導致其靶蛋白表達異常。若靶蛋白與腫瘤細胞對藥物敏感性相關,則相關miRNA的異常將會導致腫瘤細胞對藥物敏感性的改變。miRNA參與化療耐藥的機制一般可分為：與凋亡相關的miRNA介導的化療耐藥,與藥物轉(zhuǎn)運相關miRNA介導的化療耐藥,與細胞修復相關miRNA介導的化療耐藥,以及與藥物作用靶點相關miRNA介導的化療耐藥等。目前,miRNA在腫瘤耐藥中的研究剛剛起步,目前研究大多是通過對比耐藥細胞株與親本敏感細胞株之間miRNA表達譜異常,尋找與藥物敏感性相關的關鍵miRNA,從而進一步對腫瘤細胞化療耐受機制進行深入研究和尋找新的藥物作用靶點。順鉑是抗實體腫瘤的一線用藥,為鉑的金屬絡合物,其作用類似于烷化劑,干擾DNA復制,為周期非特異性抗腫瘤藥物,具有抗癌譜廣、作用強、與多種抗腫瘤藥有協(xié)同作用且無交叉耐藥等特點,是目前聯(lián)合化療最常使用的藥物之一。本研究分為兩個部分,以順鉑為例分別探討microRNA-100 (miR-100)與microRNA-23b (miR-23b)恢復軟骨肉瘤細胞對順鉑敏感性的機制,為miRNAs成為軟骨肉瘤的治療策略提供理論基礎。第一部分：miR-100恢復軟骨肉瘤細胞對順鉑敏感性的實驗研究[研究目的]探討miR-100增加軟骨肉瘤細胞對順鉑敏感性的機制,為軟骨肉瘤基于miR-100的化療策略提供了理論基礎。[方法]1.采用實時定量PCR (quantitative real-time PCR, qRT-PCR)檢測miR-100在多種人軟骨肉瘤細胞系、正常軟骨細胞系以及患者軟骨肉瘤組織標本中的表達水平。2.采用濃度梯度法對軟骨肉瘤CH-2879細胞系進行藥物篩選,獲得對順鉑耐受的CH-2879細胞(命名為：CH-2879/DDP細胞),qRT-PCR檢測并比較CH-2879細胞及CH-2879/DDP細胞的miR-100表達水平：MTT及克隆形成實驗檢測比較兩種細胞在順鉑作用下的增殖能力和克隆形成能力。3. miRNA數(shù)據(jù)庫(TargetScan, Pictar, and MicroRNA)預測miR-100的作用靶點。4.采用脂質(zhì)體2000法將pre-miR-100和anti-miR-100以及它們的陰性對照分別轉(zhuǎn)入CH-2879細胞,qRT-PCR驗證轉(zhuǎn)染后細胞miR-100的表達水平,蛋白質(zhì)印跡法(western blot)檢測提高或抑制miR-100后mTOR的表達水平以及]mTOR下游蛋白S6K和4EBP1的磷酸化水平。5.熒光素酶報告基因法(Luciferase reporter assay)進一步驗證miR-100作用于mTOR的3’端非翻譯區(qū)(3'untranslated region, UTR)。6.將CDDP. BEZ235以及CDDP+BEZ235分別處理CH-2879/DDP細胞及CH-2879細胞,觀察抑制mTOR通路,軟骨肉瘤細胞對順鉑的敏感性。7.將miR-100及其對照分別轉(zhuǎn)染CH-2879細胞及CH-2879/DDP細胞,觀察miR-100高表達與軟骨肉瘤細胞對順鉑敏感性的關系。8.外源性S6K過表達質(zhì)粒轉(zhuǎn)染事先已經(jīng)轉(zhuǎn)染過miR-100的軟骨肉瘤細胞,觀察mTOR通路活性恢復后,軟骨肉瘤細胞對順鉑耐受性的變化。[結果]1.miR-100在人軟骨肉瘤細胞和患者軟骨肉瘤標本中的表達下調(diào)。2. CH-2879/DDP細胞中miR-100表達下調(diào),提示該基因與軟骨肉瘤化療耐受有關。3. mTOR及其所在信號通路是目前腫瘤病理機制研究的熱點,正如我們預測的一樣,mTOR是miR-100的直接靶點之一。miR-100的過／低表達能抑制／恢復mTOR的表達水平。4.miR-100的高表達增加軟骨肉瘤對順鉑的敏感性,進一步印證了該基因與軟骨肉瘤化療耐受有關。5. mTOR通路活性的恢復使miR-100過表達的軟骨肉瘤細胞表現(xiàn)出對順鉑的耐受,提示miR-100的過表達增加軟骨肉瘤細胞對順鉑的敏感性是通過對mTOR通路的抑制實現(xiàn)的。[結論]與正常軟骨細胞相比,miR-100在軟骨肉瘤細胞及軟骨肉瘤患者標本中的表達均下調(diào),提示miR-100是軟骨肉瘤的抑制基因；與原代軟骨肉瘤細胞相比,miR-100在CH-2879/DDP細胞中表達下調(diào),這不僅說明miR-100具有腫瘤抑制功能,而且與軟骨肉瘤對順鉑的化療敏感性有關。nTOR在多種惡性腫瘤中活化,可以成為治療癌癥的一個有前景的治療靶點。在本研究中,我們確定了mTOR是miR-100直接靶目標,并首次報道了miR-100過表達通過抑制mTOR通路增加軟骨肉瘤細胞對順鉑的敏感性,為軟骨肉瘤基于miR-100的治療策略提供了理論基礎。第二部分：miR-23b恢復軟骨肉瘤細胞對順鉑敏感性的實驗研究[研究目的]探討miR-23b增加軟骨肉瘤細胞對順鉑敏感性的機制,為軟骨肉瘤基于miR-23b的化療策略提供了理論基礎。[方法]1.采用qRT-PCR檢測miR-23b在多種人軟骨肉瘤細胞系、正常軟骨細胞系以及患者軟骨肉瘤組織標本中的表達水平。2.采用脂質(zhì)體2000法將pre-miRs和control-miRs轉(zhuǎn)染SW1353細胞；并在轉(zhuǎn)染后不同時間點檢測細胞的增殖情況。3.在常規(guī)細胞培養(yǎng)條件下,CH-2879細胞與CH-2879/DDP細胞分別接受順鉑刺激48小時,利用顯微鏡觀察細胞形態(tài)的改變以及利用qRT-PCR檢測miR-23b的表達。4.根據(jù)以往報道我們預測并驗證Src是miR-23b的直接靶目標。5.通過采用不同濃度的順鉑分別作用于CH-2879細胞,48小時后收集細胞行western blot分析,驗證miR-23b過表達抑制Src-Akt通路的作用。6.采用脂質(zhì)體2000法將pre-miR-negative或pre-miR-23b分別轉(zhuǎn)入CH-2879細胞,qRT-PCR驗證轉(zhuǎn)染后細胞1miR-23b的表達水平,western blot檢測提高或抑制miR-23b后總的Src和磷酸化Src的水平。7.采用脂質(zhì)體2000法,將含有野生型(wt)和突變型(mut) Src 3'UTR轉(zhuǎn)染細胞,轉(zhuǎn)染后利用熒光素酶報告基因法進一步驗證miR-23b作用于Src3'UTR區(qū)。8.以contral-miR或pre-miR-23b分別轉(zhuǎn)染CH-2879細胞、SW1353細胞和CH-2879/DDP細胞,用不同濃度的順鉑處理細胞后MTT法測定細胞活性。9.將：niR-23b轉(zhuǎn)染CH-2879細胞及CH-2879/DDP細胞,觀察miR-23b過表達恢復軟骨肉瘤細胞對順鉑敏感性的作用。10.外源性Src過表達質(zhì)粒轉(zhuǎn)染事先已經(jīng)轉(zhuǎn)染過niR-23b的軟骨肉瘤細胞,觀察Src-Akt通路活性恢復后,軟骨肉瘤細胞對順鉑耐受性的變化。[結果]1. miR-23b在多種軟骨肉瘤細胞系及軟骨肉瘤患者標本組織中的表達下調(diào)。2. CH-2879/DDP細胞中miR-23b表達下調(diào),提示該基因也與軟骨肉瘤對順鉑的化療耐受有關。3.Src激酶參與了多種腫瘤的增殖、遷移和入侵,CH-2879/DDP細胞的Src-Akt通路活性的上調(diào)4.軟骨肉瘤細胞中Src是miR-23b的直接靶目標5. miR-23b的過度表達通過直接抑制Src-Akt通路恢復了軟骨肉瘤細胞對順鉑的敏感性。6.Src-Akt通路活性的恢復增加了miR-23b過表達軟骨肉瘤細胞對順鉑的耐藥性。[結論]同miR-100一樣,miR-23b在軟骨肉瘤細胞中的表達下調(diào),提示它是一種腫瘤抑制基因,與軟骨肉瘤對順鉑的化療敏感性有關。我們經(jīng)實驗確認,Src是miR-23b的一個直接靶向目標,Akt是一種用于調(diào)節(jié)新陳代謝、細胞周期進展和細胞存活率的細胞質(zhì)絲氨酸和蘇氨酸激酶,miR-23b可以通過對Src-Akt通路的負向調(diào)節(jié)提高軟骨肉瘤細胞對順鉑的敏感性,為軟骨肉瘤基于miR-23b的治療策略提供了理論依據(jù)。
[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.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R738

【共引文獻】

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