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      miR-15a/16在多发性骨髓瘤患者中的表达研究 Translated title: Dysfunction of miR-15a/16 in multiple myeloma

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          Abstract

          染色体异常和血管生成在多发性骨髓瘤(multiple myeloma, MM)发病中占有重要地位[1]。13q14缺失是MM患者最常见的染色体异常,miR-15a/16即定位于该染色体上。血管内皮生长因子A(vascular endothelial growth factor, VEGF-A)在MM的发病中具有诱导新生血管形成、诱导骨髓瘤细胞增殖和迁移、增加破骨细胞活性和调节免疫细胞活性等多种功能。在对骨肉瘤及神经胶质瘤的研究中也证实了miRNA对VEGF-A的调控作用[2]–[3],然而在MM中关于miRNA是否可以调节VEGF-A表达的研究仍较少。本研究中我们拟分析MM患者miR-15a/16、VEGF-A的表达情况;并通过细胞模型进一步研究miR-15a/16对VEGF-A的调控作用,从而为靶向miR-15a/16及抗血管生成治疗MM提供新的依据。 病例与方法 1.病例资料及细胞株来源:病例为2013年11月至2015年2月我院收治的45例初诊MM患者,男25例,女20例,中位年龄62岁,其中IgG型22例,IgA型10例,轻链型12例,不分泌型1例。所有患者均符合国际骨髓瘤工作组(IMWG)诊疗标准[4]。对照组为15名健康体检者,其中男9名,女6名,中位年龄56岁。本研究获得我院伦理委员会批准,所有患者及对照者均签署知情同意书。骨髓瘤细胞株U266细胞由本实验室冻存。 2.骨髓单个核细胞的收集:采集患者及对照者EDTA抗凝骨髓5 ml,采用淋巴细胞分离液密度梯度离心法分离单个核细胞和骨髓上清,冻存于−80 °C冰箱备用。 3.荧光定量PCR(qRT-PCR)法检测miR-15a/16、VEGF-A mRNA表达水平:采用TRIzol法提取单个核细胞总RNA,经琼脂糖凝胶电泳检验RNA质量后反转录成cDNA(TRIzol试剂盒和M-MLV反转录酶购自美国Invitrogen公司)。qRT-PCR试剂盒购自瑞士Roche公司。引物序列:GAPDH:上游:5′-CATCAAGAAGGTGGTGAAGCAG-3′,下游:5′-CAAAGGTGGAGGAGTGGGTG-3′;VEGF-A:上游:5′-CTACCTCCACCATGCCAAGT-3′,下游:5′-AGGGGCCATCCACAGTCTTC-3′;miR-15a:上游:5′-GCGGCGGTAGCAGCACATAATG-3′,下游:5′-ATCCAGTGCAGGGTCCGAGG-3′;miR-16:上游:5′-GCGGCGGTAGCAGCACGTAAAT-3′,下游:5′-ATCCAGTGCAGGGTCCGAGG-3′;U6:上游:5′-ATTGGAACGATACAGAGAAGATT-3′,下游:5′-GGAACGCTTCACGAATTTG-3′。引物由生工生物工程(上海)股份有限公司合成。反应条件:95 °C 10 min,95 °C 15 s,60 °C 1 min,40个循环。扩增完成后作熔解曲线验证产物的特异性,采用2−ΔΔCt法计算miR-15a/16、VEGF-A mRNA相对表达水平。 4.ELISA法检测VEGF-A表达水平:ELISA试剂盒购自上海富勒生物科技有限公司,按照说明书进行操作。 5.慢病毒感染U266细胞:U266细胞常规培养于含10%胎牛血清的RPMI 1640培养基中,置于37 °C、5%CO2培养箱常规传代培养。取对数生长期细胞用于实验。绿色荧光蛋白(GFP)标记的过表达miR-15a/16以及空载体(con)慢病毒由上海吉凯公司包装完成。感染前1 d U266细胞换液,每孔接种4×105个细胞于96孔板中,每孔加终浓度为1 × 108 TU/ml的病毒10 µl, 10 mg/L聚凝胺10 µl,用RPMI 1640培养基补至总体积为100 µl,混匀后置于37 °C、5% CO2培养箱中培养。3 d后将细胞扩大培养,采用流式细胞术分选出GFP阳性细胞用于后续实验。 6.CCK-8法检测细胞增殖:实验分组:①U266细胞组;②U266-miR-15a组:转染过表达miR-15a慢病毒载体U266细胞组;③U266-miR-16组:转染过表达miR-16慢病毒载体U266细胞组;④U266-con组:转染空载体U266细胞组。分别取对数生长期细胞,每孔接种4×104个细胞于96孔板中,每孔终体积100 µl,置于37 °C、5%CO2培养箱中培养。于22、46、70 h时加CCK-8溶液(试剂盒购自日本同仁化学研究所),每孔5 µl,轻微振荡混匀,37 °C孵育2 h后采用自动酶标仪测定波长450 nm处的吸光度(A)值。结果以(A 处理组−A 空白组)/(A 未处理组−A 空白组)表示。每组设3复孔,实验重复3次。 7.流式细胞术检测细胞凋亡:实验分组同上。Annexin Ⅴ/7-AAD凋亡试剂盒购自美国eBioscience公司。每管取1×106个细胞,按试剂盒说明书进行孵育、洗涤后上流式细胞仪检测。每组设3复孔,实验重复3次。 8.Western blot法检测凋亡相关蛋白:实验分组同上。参照文献[5]方法进行操作,检测过表达miR-15a/16后抗凋亡蛋白Bcl-2的变化。 9.统计学处理:采用SPSS16.0软件对数据进行统计学分析。数据以 x ±s表示,两组间比较采用t检验或非参数检验。以P<0.05为差异有统计学意义。 结果 1.miR-15a/16、VEGF-A mRNA在MM患者中的表达:结果显示miR-15a/16在MM患者中的表达水平均较健康对照组低(P值分别为0.018和0.035),两组VEGF-A mRNA水平比较差异无统计学意义(P=0.387),MM患者骨髓上清中VEGF-A含量显著高于健康对照组(P=0.000)(表1)。 表1 多发性骨髓瘤患者中miR-15a/16、VEGF-A mRNA的表达( x ±s) 组别 例数 miR-15a miR-16 VEGF-A mRNA VEGF-A(ng/L) 多发性骨髓瘤患者组 45 0.34±0.27 0.86±0.80 1.62±3.59 239.93±126.38 健康对照组 15 0.85±0.62 1.27±0.66 3.94±6.42 104.93±60.74 P值 0.018 0.035 0.387 0.000 注:VEGF:血管内皮生长因子 2.慢病毒感染U266细胞:病毒感染U266细胞72 h后,荧光显微镜下均可见到绿色荧光,显示慢病毒成功感染细胞。采用流式细胞术分选GFP阳性细胞,其阳性率达98%以上(图1)。 图1 荧光显微镜下观察转染过表达miR-15a慢病毒载体U266细胞(U266-miR-15a组) 3.过表达miR-15a/16对U266细胞VEGF-A表达水平、细胞凋亡及细胞增殖的影响:与U266-con组比较,过表达miR-15a/16后,U266细胞VEGF-A mRNA表达水平无明显变化(P值均>0.05),但细胞培养上清液中VEGF-A表达水平明显降低(P值均<0.05),细胞凋亡率增加(P值均<0.05)(表2);抗凋亡蛋白Bcl-2表达下降(图2)。与U266-con组相比,U266-miR-15a、U266-miR-16组细胞增殖受到抑制(P值均< 0.05),U266-con组与U266组比较差异无统计学意义(P> 0.05)(表2)。 表2 过表达miR-15a/16后对U266细胞VEGF-A表达水平、细胞凋亡及增殖的影响( x ±s) 组别 VEGF-A mRNA VEGF-A(ng/L) 细胞凋亡率(%) 不同时间点细胞增殖(A值比值) 24 h 48 h 72 h U266细胞组 1.04±0.26 384.01±5.89 1.51±0.81 0.35±0.08 1.12±0.16 1.08±0.03 U266-con组 1.00 379.08±4.70 2.08±1.25 0.30±0.05 0.93±0.09 1.09±0.15 U266-miR-15a组 1.11±0.27 347.62±6.94a 2.98±0.95a 0.22±0.03a 0.76±0.15a 0.99±0.06a U266-miR-16组 1.16±0.49 346.44±5.72a 2.90±1.33a 0.19±0.03a 0.62±0.09a 0.99±0.07a 注:U266-con组:转染空载体U266细胞组;U266-miR-15a组:转染过表达miR-15a慢病毒载体U266细胞组;U266-miR-16组:转染过表达miR-16慢病毒载体U266细胞组;与U266-con组比较,a P<0.05。每组设3个复孔,实验重复3次 图2 Western blot法检查过表达miR-15a/16对U266细胞抗凋亡蛋白Bcl-2表达水平的影响 1:U266细胞组;2:转染空载体U266细胞组;3:转染过表达miR-15a慢病毒载体U266细胞组;4:转染过表达miR-16慢病毒载体U266细胞组 讨论 越来越多的研究表明miRNA在实体肿瘤以及包括MM在内的血液系统肿瘤中均存在表达失调[6]–[7]。miRNA通过靶向原癌基因或抑癌基因的表达调节肿瘤的发生、发展,可以作为疾病诊断、进展及预后的新型标志[8]–[9]。近来有研究表明miRNA表达还与肿瘤细胞的耐药相关[10]–[11],使其成为肿瘤治疗的新靶点。 miR-15a/16是第一个被证明参与肿瘤发病的miRNA,定位于13q14上。研究证实在多种实体肿瘤、淋巴瘤、白血病及约50%的MM患者中都存在该染色体的缺失或miR-15a/16低表达[12]–[13],但关于miR-15a/16对MM细胞株生物学行为的影响及其机制的报道并不多。在本研究中我们的结果显示,与健康对照组相比,MM患者miR-15a/16的表达水平降低,与Roccaro等[14]的研究结果一致,且我们的前期研究结果表明随着MM进展miR-15a/16的表达水平呈进行性下降[15]。同时,我们选取低表达miR-15a/16的骨髓瘤细胞株U266细胞为研究对象,应用慢病毒转染技术使U266细胞过表达miR-15a/16,探讨其对骨髓瘤细胞生长的影响。实验结果显示过表达miR-15a/16后,细胞增殖速度降低、凋亡增加,证实了miR-15a/16在MM发病中的抑癌基因作用。 Bcl-2是一种重要的抗凋亡基因,它通过抑制细胞膜超极化、调节caspase信号通路等机制促进细胞生存,抑制细胞凋亡。在本研究中我们发现过表达miR-15a/16后,U266细胞中Bcl-2的表达明显下调,说明miR-15a/16可能是通过抑制Bcl-2基因的表达实现促肿瘤细胞凋亡作用的。关于miR-15a/16在MM中是否参与其他信号通路调节及其机制还在进一步研究中。 血管新生在MM的发生、发展中发挥重要作用,而VEGF-A是目前已知最强的促血管生成因子[16]。MM细胞既可以直接分泌VEGF-A,也可以诱导骨髓基质细胞分泌VEGF-A[17],进而促进血管内皮细胞增殖,诱导肿瘤新生血管生成,为MM细胞提供更适宜的生存环境,促进MM进展。在本研究中我们的结果显示MM患者VEGF-A的表达水平显著高于健康对照者,过表达miR-15a/16后,U266细胞VEGF-A分泌水平下降,而VEGF-A mRNA水平并无变化,这与Sun等[18]的研究结果一致,说明miR-15a/16对VEGF-A的调控作用发生在转录后水平。miR-15a/16通过下调VEGF-A的表达减少新生血管形成,制造不利于MM细胞生存的环境,至少在一定程度上可以延缓MM的发展过程。但本研究只是体外实验,关于其体内效应还在进一步研究中。 综上,通过研究我们验证了miR-15a/16在MM患者中存在低表达,更进一步证实了miR-15a/16不仅可以直接抑制MM细胞的生长,促进其凋亡,还可以通过降低骨髓微环境中VEGF-A的分泌水平,减少血管生成,间接调控MM进展,发挥抑癌基因作用。

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          Most cited references14

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          MiRNAs and cancer.

          Cancer is the result of a complex multistep process that involves the accumulation of sequential alterations of several genes, including those encoding microRNAs (miRNAs). miRNAs are a class of 17- to 27-nucleotide single-stranded RNA molecules that regulate gene expression posttranscriptionally. A large body of evidence implicates aberrant miRNA expression patterns in most, if not all, human malignancies. This article reviews our current knowledge about miRNAs, focusing on their involvement in cancer and their potential as diagnostic, prognostic, and therapeutic tools.
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            MicroRNA-221/222 confers breast cancer fulvestrant resistance by regulating multiple signaling pathways.

            Fulvestrant is a selective estrogen receptor downregulator (SERD) and highly effective antagonist to hormone-sensitive breast cancers following failure of previous tamoxifen or aromatase inhibitor therapies. However, after prolonged fulvestrant therapy, acquired resistance eventually occurs in the majority of breast cancer patients, due to poorly understood mechanisms. To examine a possible role(s) of aberrantly expressed microRNAs (miRNAs) in acquired fulvestrant resistance, we compared antiestrogen-resistant and -sensitive breast cancer cells, revealing the overexpression of miR-221/222 in the SERD-resistant cell lines. Fulvestrant treatment of estradiol (E2)- and fulvestrant-sensitive MCF7 cells resulted in increased expression of endogenous miR-221/222. Ectopic upregulation of miR-221/222 in estrogen receptor-α (ERα)-positive cell lines counteracted the effects of E2 depletion or fulvestrant-induced cell death, thus also conferring hormone-independent growth and fulvestrant resistance. In cells with acquired resistance to fulvestrant, miR-221/222 expression was essential for cell growth and cell cycle progression. To identify possible miR-221/222 targets, miR-221- or miR-222- induced alterations in global gene expression profiles and target gene expression at distinct time points were determined, revealing that miR-221/222 overexpression resulted in deregulation of multiple oncogenic signaling pathways previously associated with drug resistance. Activation of β-catenin by miR-221/222 contributed to estrogen-independent growth and fulvestrant resistance, whereas TGF-β-mediated growth inhibition was repressed by the two miRNAs. This first in-depth investigation into the role of miR-221/222 in acquired fulvestrant resistance, a clinically important problem, demonstrates that these two 'oncomirs' may represent promising therapeutic targets for treating hormone-independent, SERD-resistant breast cancer.
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              miR-15a and miR-16 affect the angiogenesis of multiple myeloma by targeting VEGF.

              Deregulated microRNAs (miRNAs) and their roles in cancer development have attracted much attention. Two miRNAs, miR-15a and miR-16, which act as putative tumor suppressor by targeting the oncogene BCL2, have been implicated in cell cycle, apoptosis and proliferation. In this study, we investigated the possible role of miR-15a/16 in the angiogenesis of multiple myeloma (MM). Using a stem-loop quantitative reverse transcription-PCR, we analyzed miR-15a/16 expressions in bone marrow samples from newly diagnosed MM patients and a panel of MM cell lines. miRNA transfection, western blotting analysis and assay of luciferase activity were used to examine whether vascular endothelial growth factor (VEGF) is the target of miR-15a/16. The functional roles of miR-15a/16 on tumorigenesis and angiogenesis were examined by in vitro angiogenesis models and in vivo tumor xenograft model. We showed that miR-15a and miR-16 were significantly underexpressed in primary MM cells as well as in MM cell lines. The aberrant expression of miR-15a/16 was detected especially in advanced stage MM. In human MM cell lines and normal plasma cells, expression of miR-15a/16 inversely correlated with the expression of VEGF-A. Western blotting combined with the luciferase reporter assay demonstrated that VEGF-A was a direct target of miR-15a/16. Ectopic overexpression of miR-15a/16 led to decreased pro-angiogenic activity of MM cells. Finally, infection of lentivirus-miR-15a or lentivirus-miR-16 resulted in significant inhibition of tumor growth and angiogenesis in nude mice. This study suggest that miR-15a/16 could play a role in the tumorigenesis of MM at least in part by modulation of angiogenesis through targeting VEGF-A.
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                Author and article information

                Journal
                Zhonghua Xue Ye Xue Za Zhi
                Zhonghua Xue Ye Xue Za Zhi
                CJH
                Chinese Journal of Hematology
                Editorial office of Chinese Journal of Hematology (No. 288, Nanjing road, Heping district, Tianjin )
                0253-2727
                2707-9740
                July 2016
                : 37
                : 7
                : 611-613
                Affiliations
                [1]221002 徐州医学院附属医院(张冰云、姚瑶、罗建萍、李护君、陆倩、闫志凌、徐开林、李振宇),诊断教学实验中心(李艳杰)
                Author notes
                通信作者:李振宇,Email: lizhenyumd@ 123456163.com
                Corresponding author: Li Zhenyu, Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China. Email: lizhenyumd@ 123456163.com
                Article
                cjh-37-07-611
                10.3760/cma.j.issn.0253-2727.2016.07.014
                7365005
                27535864
                40651c28-ff63-43a9-9bdd-262d4ce73194
                2016年版权归中华医学会所有Copyright © 2016 by Chinese Medical Association

                This work is licensed under a Creative Commons Attribution 3.0 License (CC-BY-NC). The Copyright own by Publisher. Without authorization, shall not reprint, except this publication article, shall not use this publication format design. Unless otherwise stated, all articles published in this journal do not represent the views of the Chinese Medical Association or the editorial board of this journal.

                Funding
                基金项目:国家自然科学基金(81570183);江苏省科技厅临床医学科技专项(SBL201330199)
                Categories
                短篇论著

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