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miR-21介导的和厚朴酚在骨肉瘤细胞中的作用及机制研究
中文摘要

骨肉瘤(Osteosarcoma,OS)也叫成骨肉瘤,是较常见的发生在20岁以下的青少年或儿童的一种恶性骨肿瘤,在小儿骨恶性肿瘤中最多见,约为小儿肿瘤的5%。骨肉瘤是骨恶性肿瘤中最多见的一种,是从间质细胞系发展而来,肿瘤迅速生长是由于肿瘤经软骨阶段直接或间接形成肿瘤骨样组织和骨组织。下肢负重骨在外界因素(如病毒)的作用下,使细胞突变,可能与骨肉瘤形成有关。有研究资料显示,骨肉瘤患者五年的生存率约为68%,但是一旦发生转移,其5年生存率则锐减至20%左右。因此,抗骨肉瘤化疗药物的开发成为迫切需要和研究的热点。 中国地域广阔,自然环境复杂多变,中药资源极为丰富,为从中药中提取和开发抗肿瘤有效成分提供了丰富的来源。厚朴为木兰科植物,是一味具有广泛药理作用的祖国传统草本中药。和厚朴酚(honokiol, HNK)是中药厚朴的主要活性成分之一,具有中度抗肿瘤活性的生物学作用,其抗癌机制越来越受到关注。有研究报道,HNK可以抑制肺鱗癌细胞的增殖,诱导其细胞凋亡。HNK还可以通过抑制上皮间质转化而抑制乳腺癌细胞MCF-7和MDA.MB-231的侵袭和转移,从而产生抗乳腺癌作用。然而其在骨肉瘤中的作用及机制目前尚不明确。 MicroRNA(miRNA)是一种有21-25个核苷酸组成的单链小分子RNA,广泛存在于真核生物中,是一组不编码蛋白质的短序列RNA。近期多项研究发现,若干miRNAs在骨肉瘤组织或细胞中的表达水平有不同程度上调或下调,这一现象初步揭示了骨肉瘤的发生与miRNA表达之间存在相关性。Vanita Vanas等人研究发现在骨肉瘤细胞及人成骨细胞中MiR-21表达上调,并证实miR-21能够促进骨肉瘤细胞的增殖,侵袭与转移,诱导细胞凋亡。然而,miRNAs是否参与HNK的抗骨肉瘤作用仍是未知的。本课题初步研究HNK在抗骨肉瘤中的作用,并且对其作用机制进行了初步探讨,为后续试验奠定了理论和技术的基础,为HNK应用于骨肉瘤的临床治疗可能性提供理论基础和实验依据。 第一部分 和厚朴酚对骨肉瘤细胞増殖和凋亡的影响 目的 观察不同浓度HNK对骨肉瘤细胞增殖和凋亡的影响。 方法 1.应用MTT法检测不同浓度HNK处理后骨肉瘤细胞存活率。采用MTT比色法,取不同浓度HNK处理24h后的骨肉瘤细胞,检测HNK对骨肉瘤细胞Saos-2和MG-63的增殖抑制率的影响; 2.应用双染色法和流式细胞技术,检测不同作用时间和不同浓度HNK对骨肉瘤细胞Saos-2和MG-63细胞凋亡的影响; 3.Western Blot方法检测HNK处理后骨肉瘤细胞凋亡相关蛋白的表达水平。 结果 1.HNK抑制人骨肉瘤细胞增殖。Saos-2细胞系和MG-63细胞系用不同浓度HNK处理24小时后,用MTT法检测细胞活性,结果显示, HNK呈剂量依赖性显着抑制人骨肉瘤细胞增殖。HNK在Saos-2细胞系中的半抑制浓度(The half maximal inhibitory concentration, IC50)为37.85μM,在MG-63细胞系中为38.24μM,两组无明显差异。 2.HNK促进人骨肉瘤细胞系的凋亡。采用双染色法和流式细胞技术研究了 HNK在人骨肉瘤细胞系中的促凋亡活性。结果表明,和对照组比较,HNK显著促进人骨肉瘤细胞凋亡,差别有统计学差异(P< 0.01)。并且,HNK促进骨肉瘤细胞的凋亡呈现剂量依赖性。在人骨肉瘤细胞系Saos-2和MG-63中,HNK显示出基本相同的凋亡诱导作用。 3.Western Blot方法检测HNK处理后骨肉瘤细胞凋亡相关蛋白的表达水平,结果显示,用不同浓度HNK处理人骨肉瘤细胞系后,凋亡相关蛋白cleaved-caspase-3、cleaved-PARP和Bax水平显著上调,而Bcl-2水平显著下调。并且,HNK的这种上调和下调作用呈现剂量依赖性。 结论 1.HNK在体外对人骨肉瘤细胞具有明显的抑制增殖作用。 2.HNK促进人骨肉瘤细胞凋亡。 3.Western Blot方法检测结果提示,HNK可能是通过影响细胞间凋亡信号通路而导致人骨肉瘤细胞的凋亡。 关键词 骨肉瘤;和厚朴酚;miR-21;PTEN;PI3K/AKT通路 第二部分 和厚朴酚处理人骨肉瘤细胞前后miRNA表达谱检测 目的 检测HNK处理前后骨肉瘤细胞中miRNAs表达情况,寻找存在明显表达差异的miRNA,探寻受HNK调控的miRNAs并予以鉴定。 方法 1.运用生物信息学方法,选择微阵列数据显示的HNK处理前后表达差异显著的6种miRNA (miR-188-5p,miR-202和miR-623显著上调的;miR-21,miR-532-5p 和 miR-628-3p 显著下调)进行 RT-PCR验证; 2.RT-PCR验证HNK处理Saos-2和MG-63细胞前后差异化表达最显著的miR-21,并对其表达量进行检测; 3.HNK介导的miR-21在人骨肉瘤细胞系中的表达和调控。采用脂质体转染法转染miRNA进入细胞,根据miRNA序列设计和经化学修饰的核苷酸片段特定的miRNA mimic上调细胞中的miR-21水平,运用MTT法和流式细胞技术,观察HNK对骨肉瘤的抑制作用效果的变化。 结果 1.应用RT-PCR技术验证HNK处理前后人骨肉瘤细胞系MG-63和Saos-2中差异化表达最显著的6种miRNA,结果显示,miR-202和miR-623显著上调,miR-21显著下调,差别有统计学意义(P<0.01)。而miR-188-5p,miR-532-5p和miR-628-3p与载体组相比无显著差异; 2.HNK处理人骨肉瘤细胞系MG-63和Saos-2前后miR-21差异化表达显著,并呈剂量依赖性; 3.HNK+miR-21组与HNK组相比,Saos-2和MG-63细胞的凋亡明显减少,miR-21的过表达逆转了 HNK对人骨肉瘤细胞的抑制作用。 结论 HNK处理人骨肉瘤细胞系MG-63和Saos-2前后miR-21差异化表达显著,并呈剂量依赖性。miR-21的过表达逆转了 HNK对人骨肉瘤细胞的抑制作用,HNK可能通过miR-21介导进而影响细胞间凋亡信号通路而导致人骨肉瘤细胞的凋亡。 关键词:骨肉瘤;和厚朴酚;miR-21; PTEN; PI3K7AKT通路 第三部分 miR-21介导HNK在骨肉瘤细胞中作用及机制研究 目的 分析miR-21在HNK抗骨肉瘤机制中的作用,寻找其作用的靶基因,阐明其对靶基因的调节作用,探讨HNK介导的miR-21在骨肉瘤细胞中的作用机制,寻找骨肉瘤治疗的新靶点。 方法 1.miR-21靶基因的生物信息学方法预测。进入microRNA靶基因预测网站TargetScan,输入后提交预测; 2.miR-21靶基因的验证。应用双荧光素酶活性检测,构建相应靶基因的荧光素酶报告基因质粒,检测荧光素酶活性,判断miR-21的靶基因; 3.转染miR-21模拟物及抑制物后通过RT-PCR和Western Blot技术检测靶基因的mRNA水平和蛋白表达水平的变化,分析miR-21对靶基因的调节作用; 4.IE基因及下游通路蛋白的检测。应用Western Blot方法检测HNK处理后人骨肉瘤细胞系Saos-2和MG-63后PI3K/AKT/mTOR通路的激活情况。 结果 1.用生物信息学方法分析并预测启动子区可能的转录因子结合位点,预测PTEN可能为miR-21作用的靶基因,其3’UTR区域与miR-21有8个碱基互补配对; 2.成功构建候选靶基因PTEN的荧光素酶报告基因质粒,转染miR-21 mimics 的 PTEN 3'-URT wt 组荧光信号显著降低;miR-21inhibitor及PTEN 3'-URT wt共转染组荧光显著增高,同时miR-21不抑制含有miR-21结合位点突变的PTEN的3MJTR的报告载体的荧光素酶活性,PTEN 3'-URT mut组的各组荧光无显著差异; 3.过表达miR-21的骨肉瘤细胞中,PTEN的mRNA明显下调,蛋白表达水平明显降低,而应用miR-21抑制物可上调PTEN的蛋白表达。miR-21通过抑制PTEN表达发挥作用; 4.miR-21通过与其3'-UTR结合而抑制PTEN的表达; 5.HNK可通过剂量依赖方式上调PTEN在骨肉瘤细胞中表达; 6.HNK 处理后 Saos-2 和 MG-63 细胞中 AKT,mTOR 和 p70S6K的表达显著下调,但转染miR-21 mimics后AKT,mTOR和p70S6K的表达显著上调,miR-21可逆转HNK的效应。HNK能够阻断人类骨肉瘤细胞中的PI3K/AKT信号传导途径,但是其可以被miR-21过表达逆转。 结论 HNK通过调节miR-21的表达影响人骨肉瘤细胞中PTEN/PI3K /AKT/mTOR信号通路,从而调控人骨肉瘤细胞凋亡。 关键词 骨肉瘤;和厚朴酚;miR-21; PTEN; PI3K/AKT/mTOR通路

英文摘要

Honokiol (HNK) is a small biphenolic compound, which exerts anti-neoplastic effects in various types of cancer. However, the mechanism underlying the anti-tumor effects of HNK in osteosarcoma (OS) cells is not yet fully understood. Emerging evidence has indicated that micro RNAs (miRNAs/miRs) serve key roles in numerous pathological processes, including cancer. It has previously been reported that Chinese medicinal herbs harbor anticancer properties via modulating mi RNA expression. Therefore, the present study aimed to determine whether HNK could suppress OS cell growth by regulating mi-RNA expression. The MTT assay and flow cytometric analysis were used to evaluate the cell proliferation and apoptosis in human OS cells after treatment with HNK, respectively. The results demonstrated that HNK inhibits proliferation and induces apoptosis of human OS cells in a dose-dependent manner. Furthermore, HNK-induced apoptosis was characterized by up-regulation of proapoptotic proteins, including cleaved-caspase-3, cleaved-poly (ADP-ribose) polymerase and B-cell lymphoma 2 (Bcl-2)-associated X protein, and down-regulation of the anti-apoptotic protein Bcl-2. RT-qPCR verified that HNK was able to induce aberrant expression of miRNAs in human OS cells, and miR-21 was one of the mi RNAs that was most significantly down-regulated. To further investigate miR-21 function, the present study validated that HNK reduces miR-21 levels in a dose-dependent manner. In addition, restoration of miR-21 expression abrogated the suppressive effects of HNK on OS cells. Luciferase assay and Western Blot analysis identified that miR-21 inhibits the expression of phosphatase and tensin homolog (PTEN) by directly targeting its 3'-UTR. Notably, HNK was able to suppress the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway; however, it was reactivated by miR-21 overexpression. Taken together, these data indicated that HNK may inhibit proliferation and induce apoptosis of human OS cells by modulating the miR-21/PTEN/PI3K/AKT signaling pathway. Therefore, miR-21 may be considered a potential therapeutic target for the treatment of osteosarcoma with HNK. Osteosarcoma (OS) is the most frequent primary malignant bone tumor, which is commonly diagnosed in children and young adolescents, with a male predominance(1). OS is highly aggressive and primarily metastasizes to the lung(2). Surgical tumor resection and multi-agent chemotherapy are the main current therapeutic strategies used to treat OS. It has previously been reported that chemotherapy may increase the 5-year survival rate for localized disease by >50% compared with surgery alone. Conversely, patients diagnosed with metastases exhibit a poor prognosis, with a 5-year survival rate of 20-30% following surgical resection and/or radiotherapy(3,4). Furthermore, currently approved agents exhibit severe side effects(3,4); therefore, the development of a novel agent with increased efficiency and reduced toxicity in OS treatment is required. Honokiol (HNK) is a biphenolic compound extracted from the magnolia tree, which has been used to treat anxiety, thrombotic stroke and gastrointestinal symptoms in traditional Chinese and Japanese medicine(5). HNK has long been known to exert antimicrobial(6), anti-inflammatory(7)and anti-angiogenic(8,9) effects. Increasing evidence has revealed that HNK exerts anti-neoplastic functions in various types of cancer, including anti-osarcoma(8), colorectal carcinoma(10), breast cancer(11) and gastric cancer(12). Furthermore, HNK may trigger apoptotic pathways that result in mitochondrial dysfunction(13), influence retinoblastoma function and E2F transcription factor 1 transcriptional activity(14), and suppress the phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway(15). However, the molecular mechanism underlying the anticancer effects of HNK on OS cells remains to be elucidated. MicroRNAs (miRNAs/mills) are a class of small (19-24 nucleotide) noncoding RNAs that mediate post-transcriptional regulation of target genes by suppressing translation or promoting RNA degradation. MiRNAs have crucial functions in various biological and pathological processes, including cellular proliferation, differentiation, apoptosis and carcinogenesis(16). In recent years, it has been demonstrated that some natural products are able to control tumor-suppressive and oncogenic miRNAs, including curcumin (diferuloylmethane), which inhibits hepatocellular cancer cell proliferation via modulating miRNA expression(17). Furthermore, previous studies have reported that Chinese medicinal herbs exert antitumor effects by modulating miRNA expression(18,19). Zhang et al demonstrated that HNK suppresses bladder tumor growth by inhibiting the enhancer of zeste homolog 2/miR-143 axis(20). Avtanski et al also revealed that HNK rescued leptin-induced tumor progression by suppressing the Wntl-metastasis associated 1-β-catenin signaling pathway in a miR-34a-dependent manner(11). Therefore, it may be hypothesized that HNK inhibits proliferation and induces apoptosis, via the modulation of miRNA expression, in human OS cells. The present study investigated the effects of HNK on OS tumor growth inhibition and explored the underlying molecular mechanisms. The results indicated that HNK may inhibit growth and promote apoptosis of human OS cells in a dose-dependent manner. Furthermore, the results verified that HNK induces aberrant expression of miRNAs in human OS cells, and miR-21 suppresses phosphatase and tensin homolog (PTEN) by directly targeting its 3'-untrans-lated region (3'-UTR). Notably, the results indicated that HNK blocks the PI3K/protein kinase B (AKT) signaling pathway by inhibiting miR-21 expression in human OS cells. Collectively, these results suggested that the molecular mechanism by which HNK induces apoptosis was modulated by the miR-21/PTEN/PI3K/AKT axis in human OS cells. PART Ⅰ THE EFFECT OF HONOKIOL ON PROLIFERATION AND APOPTOSIS OF OSTEOSARCOMA CELLS Objective To investigate the effect of honokiol on proliferation and apoptosis of osteosarcoma cells. Methods 1.To investigate the anti-proliferative effects of HNK on OS cells, Saos-2 and MG-63 cells were treated with various concentrations of HNK for 24h, and the MTT assay was used to investigate the anti-proliferative effects of HNK on OS cells. 2.Apoptosis analysis. To examine HNK-induced apoptosis of OS cells, the cells were analyzed by Annexin V-PI staining following treatment with HNK was used to detect cell apoptosis. 3.To further explore the apoptotic mechanism, the intracellular apoptotic signaling pathway was investigated in OS cells following treatment with various concentrations of HNK. Western Blot analysis was used to detect the expression of apoptosis-related proteins in osteosarcoma cells following HNK treatment. Results 1.HNK inhibits growth of human OS cells. The results of MTT assay indicated that treatment with 1-100μM HNK reduced cell viability of Saos-2 and MG-63 cells in a dose-dependent manner. The half maximal inhibitory concentration (IC50) values of HNK were 37.85μM in Saos-2 and 38.24μM in MG-63 cells. Similar IC50 values of HNK were detected in human Saos-2 and MG-63 OS cells. 2.HNK induces apoptosis of human OS cells. The results of Annexin V-PI staining demonstrated that the proportion of apoptotic cells was markedly increased following HNK treatment compared with in the control group (P<0.01). Furthermore, following treatment with 10 or 40μM HNK, the number of apoptotic cells increased in a dose-dependent manner. With regards to apoptotic induction, Saos-2 and MG-63 had similar results. 3.The results revealed that the protein expression levels of cleaved-caspase-3, cleaved-PARP and Bax were significantly up-regulated, and Bcl-2 was significantly down-regulated following HNK treatment. Furthermore, HNK regulated these protein expression levels in a dose-dependent manner in human OS cells. These data suggested that HNK may induce apoptosis of human OS cells by activating the intracellular apoptotic signaling pathway. Conclusion 1.HNK has obvious inhibitory effect on proliferation of human osteosarcoma cells in vitro. 2.HNK induces apoptosis of human OS cells. 3.The results of Western Blot suggested that HNK may induce apoptosis of human osteosarcoma cells by affecting the signal transduction pathway between cells. Key words Honokiol; Osteosarcoma; miR-21; PTEN; PBK/AKT/mTOR PART Ⅱ HNK INDUCES ABERRANT EXPRESSION OF MIRNAS IN HUMAN OS CELLS Objective The present study aimed to determine whether HNK induces aberrant expression of miRNAs in human OS cells. Methods 1.To determine whether HNK also induces aberrant expression of miRNAs in human OS cells, six miRNAs (miR-188-5p, miR-202 and miR-623 were the most significantly up-regulated; miR-21, miR-532-5p and miR-628-3p were the most significantly down-regulated) were selected based on the microarray data, and were verified by RT-qPCR. 2.To investigate the function of miR-21 in human OS cells. OS cells were treated with 10-100μM HNK for 24h and the expression levels of miR-21 were determined by RT-qPCR. 3.RT-qPCR was used to confirm that miR-21 expression was specifically up-regulated/knocked down following transfection with mimics/inhibitor. Subsequently, OS cell proliferation and apoptosis were assessed by MTT assay and flow cytometric analysis, respectively. Results 1.To determine whether HNK induces aberrant expression of miRNAs in human OS cells, six miRNAs (miR-188-5p, miR-202 and miR-623 were the most significantly up-regulated; miR-21, miR-532-5p and miR-628-3p were the most significantly down-regulated) were selected based on the microarray data, and were verified by RT-qPCR. The results indicated that miR-202 and miR-623 were markedly up-regulated, and miR-21 was significantly down-regulated in human Saos-2 OS cells following HNK treatment (P<0.01), whereas miR-188-5p, miR-532-5p and miR-628-3p were not significantly different compared with in the vehicle group. 2.To investigated the function of miR-21 in human OS cells. OS cells were treated with 10-100μM HNK for 24h and the expression levels of miR-21 were determined by RT-qPCR. The results demonstrated that HNK reduced miR-21 levels in a dose-dependent manner in human OS cells. 3.Overexpression of miR-21 rescues the suppressive effects of HNK on OS cells. The proportion of apoptotic cells was significantly decreased in the HNK + miR-21 group compared with in the HNK group in Saos-2 and MG-63 cells (P<0.01). These data indicated that HNK may exert suppressive effects on human OS cells via down-regulating miR-21. Conclusion HNK reduced miR-21 levels in a dose-dependent manner in human OS cells. Overexpression of miR-21 rescues the suppressive effects of HNK on OS cells. HNK may exert antitumor effects via modulating miR-21 expression in human OS cells. Key words Honokiol; Osteosarcoma; miR-21; PTEN; PI3K/AKT/mTOR PART Ⅲ THE MOLECULAR MECHANISM OF HNK IN HUMAN OS CELLS MEDIATED BY MIR-21 Objective To explore the molecular mechanism of HNK in human OS cells mediated by miR-21. Methods 1.The potential binding site between PTEN and miR-21 was identified using Target Scan. 2.In the present study, luciferase-reporter plasmids containing wt or mut type 3'-UTR segments of PTEN were constructed. The reporters were cotransfected along-side miR-21 mimics/inhibitor or NC into Saos-2 cells, after which luciferase activity was measured. 3.To further confirm that PTEN levels are modulated by miR-21, the Saos-2 human OS cell line was transfected with miR-21 mimic/inhibitor or NC, and the protein expression levels of PTEN were determined using Western Blot analysis. 4.To further verify whether PTEN expression was modulated by HNK, Saos-2 cells were treated with 10-100μM HNK for 24h and PTEN expression was measured by Western Blotting. 5.To determine whether HNK-mediated miR-21 modulation regulates the PI3K/AKT signaling pathway in OS cells. Saos-2 and MG-63 cells were transfected with or without miR-21 mimics following treatment with or without HNK, and Western Blot analysis was used to determine the expression levels of p-AKT, p-mTOR and p-p70S6K, which are major components of the PI3K/AKT signaling pathway. Results 1.miR-21 inhibits PTEN by directly targeting PTEN 3'-UTR. 2.The results demonstrated that HNK enhanced PTEN expression in a dose-dependent manner in human OS cells. 3.The results demonstrated that miR-21 mimic significantly suppressed luciferase activity compared with mimic NC; however, the miR-21 inhibitor markedly enhanced luciferase activity compared with inhibitor NC in the presence of wt 3'-UTR (P<0.01). In addition, miR-21 did not affect luciferase activity of the reporter vector containing mut PTEN 3'-UTR. 4.To further confirm that PTEN levels are modulated by miR-21, the human OS cell line was transfected with miR-21 mimic/inhibitor or NC, and the protein expression levels of PTEN were determined using Western Blot analysis. To further verify whether PTEN expression was modulated by HNK, OS cells were treated with 10-100μM HNK for 24h and PTEN expression was measured by Western Blotting. The results indicated that miR-21 inhibited PTEN expression in OS cells compared with the NC. 5.The expression levels of AKT, mTOR and p70S6K were significantly down-regulated following HNK treatment compared with mock vehicle-treated cells or miR-21 transfection in Saos-2 and MG-63 cells; however, the expression levels of these proteins were significantly up-regulated in HNK-treated OS cells post-transfection with miR-21 mimics compared with transfection without miR-21 mimics (P<0.01). Conclusion HNK suppresses the PI3K/AKT signaling pathway by inhibiting miR-21 expression in human OS cells. Key words Honokiol; Osteosarcoma; miR-21; PTEN; PI3K/AKT/mTOR

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