miRNA-195在膀胱癌细胞中功能的研究

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陈婧, 孙奋勇, 马纪. miRNA-195在膀胱癌细胞中功能的研究. 检验医学,2014, 29(5): 540-544
CHEN Jing, SUN Fenyong, MA Ji. Research on the function of miRNA-195 on bladder cancer cell.Labratory Medicine, 2014, 29(5): 540-544 复制到剪切板

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Shanghai pudong new area of hongshan road 528

miRNA-195在膀胱癌细胞中功能的研究

陈婧¹, 孙奋勇¹, 马纪²

1. 同济大学附属上海市第十人民医院检验科,上海 200072

2. 同济大学附属第十人民医院中心实验室,上海 200072

作者简介：陈婧,女,989年生,学士,主要从事肿瘤及其分化的研究。

通讯作者：孙奋勇,电话：021-66306909。

摘要

目的

研究微小RNA(miRNA)-195对人膀胱癌细胞5637增殖、迁移及侵袭能力的影响。

方法

利用阳离子脂质体方法将miRNA-195转染至人膀胱癌细胞5637(miRNA-195组),同时以只转染病毒载体的5637细胞作为对照组。采用实时荧光定量聚合酶链反应(PCR)检测转染细胞中miRNA-195的表达量。采用噻唑蓝(MTT)法、细胞计数法和集落形成实验测定miRNA-195对5637细胞体外增殖的影响。采用划痕试验、Transwell实验、Matrigel 肿瘤细胞侵袭实验检测miRNA-195对 5637细胞迁移、侵袭能力的影响。

结果

实时荧光定量PCR检测结果表明转染后的5637细胞高表达miRNA-195。MTT法、细胞计数法及集落形成实验证明miRNA-195可以抑制5637细胞在体外的增殖,miRNA-195组和对照组比较差异有统计学意义(P<0.05)。划痕实验、Transwell实验以及Matrigel肿瘤细胞侵袭实验证明miRNA-195可以抑制5637细胞的迁移和侵袭能力,miRNA-195组和对照组比较差异有统计学意义(P<0.05)。

结论

miRNA-195能够抑制膀胱癌细胞5637在体外的增殖、迁移以及侵袭能力。

关键词: 微小RNA-195; 人膀胱癌细胞; 细胞增殖; 细胞迁移

中图分类号:Q74 文献标志码:A 文章编号:1673-8640(2014)05-0540-05

Research on the function of miRNA-195 on bladder cancer cell

CHEN Jing¹, SUN Fenyong², MA Ji³

1. Department of Clinical Laboratory, Shanghai Tenth People's Hospital, Tongji University, Shanghai 200072, China

2. Department of Central Laboratory, Shanghai Tenth People's Hospital, Tongji University, Shanghai 200072, China

Abstract

Objective

To investigate the influence of microRNA (miRNA)-195 on the proliferation, migration and invasion of human bladder cancer cell 5637.

Methods

Human bladder cancer cell 5637 was transfected with miRNA-195 by cationic liposomes as miRNA-195 group and transfected with the vector as control group. The expression of miRNA-195 was determined by real-time fluorescence quantitation polymerase chain reaction (PCR). Methyl thiazolyl tetrazolium(MTT) assay, cell counting assay and colony formation assay were used to detect the cell proliferation . Cell migration and invasion were determined by wound healling assay, Transwell assay and Matrigel tumour invasion assay.

Results

Real-time fluorescence quantitation PCR showed that there was high expression of miRNA-195 after transfection of cell 5637. MTT assay, cell counting assay and colony formation assay demonstrated that miRNA-195 can suppress cell 5637 proliferation in vitro, and there were statistical significance between miRNA-195 and control groups(P<0.05).Wound healing assay, Transwell assay and tumour invasion assay showed that miRNA-195 can suppress cell migration and invasion with statistical significance between the 2 groups(P<0.05).

Conclusions

MiRNA-195 has inhibitory ability on the proliferation, migration and invasion of bladder cancer cell 5637 cell.

Keyword: Micro RNA-195; Human bladder cancer cell; Cell proliferation; Cell migration

Show Figures

膀胱癌是世界上发病率排名第九的肿瘤,是我国泌尿系统最常见的肿瘤,其中90%是移行上皮癌。已经明确膀胱癌的发生、发展与吸烟、环境污染等因素有关^{[ 1, 2]},但其分子机制尚不明确。研究结果显示在膀胱癌发生时,微小RNA(micro RNA,miRNA)的表达发生异常改变,并且在膀胱癌的发展中起重要作用^{[ 3]}。

miRNA是一类非编码小RNA,在进化中高度保守。越来越多的证据表明,miRNA在转录后水平调节基因表达,并在细胞增殖和肿瘤形成中发挥重要作用。抑制肿瘤的miRNA下调或者促进肿瘤的miRNA上调都会引起肿瘤形成^{[ 4]}。有研究证实miRNA-23b、miRNA-409-3p在膀胱癌细胞中表达下调^{[ 5, 6]}。

miRNA-195可抑制人胶质瘤细胞、胃癌细胞的增殖和转移^{[ 7, 8]}。为探索miRNA-195在膀胱癌细胞中的作用,我们将miRNA-195转染人膀胱癌细胞5637,以观察其对5637细胞增殖、迁移以及侵袭能力的影响,为进一步明确miRNA-195与肿瘤的关系提供实验依据。

材料和方法

一、材料

人膀胱癌细胞5637购自美国ATCC公司;miRNA-195由上海吉玛制药技术有限公司合成;细胞RNA抽提试剂盒购自QIAGEN公司;RNA酶抑制剂、逆转录酶、cellTiter96AQ单溶液细胞增殖检测试剂盒购自Promega公司;Trizol购自Invitrogen公司;RPMI 1640培养基购自Gibco公司;胰蛋白酶购自Sigma公司;胎牛血清购自HyClone公司。

二、方法

1.阳离子脂质体法转染细胞 5637细胞在37 ℃、5%CO₂孵箱中用含10%胎牛血清(fetal bovine serum,FBS)的RPMI 1640培养基培养。在转染前一天将5637细胞以1×10⁴/cm²的密度接种于24孔板上,培养基为含10%FBS的RPMI 1640培养基。转染当天,当细胞汇合度为50%~60%时,吸去旧的培养基,每孔加入300 μL无血清新鲜的RPMI 1640清洗细胞。以200 μL/孔加入配制好的含有阳离子脂质体、miRNA的转染培养基,在37 ℃、5%CO₂培养箱中保温4~6 h,吸去转染培养基,换成含10%FBS的完全培养基。

2.实时荧光定量聚合酶链反应(polymerase chain reaction,PCR)检测转染后5637细胞miRNA-195的表达量用Trizol提取膀胱癌细胞5637的总mRNA,逆转录成cDNA,用实时荧光定量PCR进行检测。miRNA-195引物:5'-ACACTCCAGCTGGGTAGCAGCACAGAAATATT-3 ',5'- CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAG-GCCAATAT -3';内参基因U6引物:5'- CTCGCTTCGGCAGCACA-3',5'-AACGCTTCACGAATTTGCGT-3'。实时荧光定量PCR反应条件:94 ℃ 5 min、94 ℃15 s、60 ℃ 15 s、72 ℃ 20 s,40 个循环,72 ℃保温10 min。每个样品设置3个复孔;2%琼脂糖凝胶电泳检测目的条带特异性。用2^-ΔΔCt 法计算差异表达的相对水平。

3. 噻唑蓝[即3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐,3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide,MTT]法检测miRNA-195对5637细胞增殖的影响将转染miRNA-195的5637细胞(miRNA-195组)以及只转染病毒载体的5637细胞(对照组)以每孔1×10⁴个细胞的密度接种于96孔板,每组设置5个复孔。第2天用Lipofectamine^TM2000转染50 nmol/mL miRNA至细胞内,转染后4~6 h,换液。第3天起收集各个时间点(24、48、72 h)的细胞,加入cellTiter96AQ单溶液细胞增殖检测试剂(100 μL培养液加入10 μL检测液),孵育4 h后酶标仪读取吸光度( A_{490 nm})值。实验重复3次。

4.细胞计数法检测细胞生长速度转染miRNA-195至5637细胞,取对数生长期的对照组及miRNA-195组细胞,按5×10⁵/mL接种于6孔板内,每组设置3个复孔。于不同时间点(1、2、3 d)收集细胞,显微镜下计数,记录对照组及miRNA-195组细胞数量。实验重复3次。

5.集落形成实验检测细胞增殖活性取处于对数生长期的对照组及miRNA-195组细胞,以1 000/mL密度接种于12孔板,水平位左右前后晃动培养板,尽量使孔内细胞分布均匀。培养7 d后吸去各孔培养基,磷酸盐缓冲液(phosphate buffered saline,PBS)或生理盐水漂洗2次,各孔加0.5 mL结晶紫染色液(应充分覆盖孔底),20 min后将6孔板置于自来水下冲洗后晾干,计算集落数。实验重复3次。

6.划痕实验检测miRNA-195对5637细胞迁移的影响用笔在6孔板背后均匀划横线横穿过孔,每间隔0.5~1.0 cm划一条线,每孔至少穿过5条线。取对数生长期的细胞,按5×10⁵/mL接种于6孔板内。待细胞基本长满时,用丝裂霉素处理1 h,抑制细胞的分裂。用枪头比着直尺,尽量垂至于背后的横线划痕。用PBS洗细胞3次,去除划下的细胞,加入无血清培养基,放37 ℃、5%CO₂培养箱培养,按0、24 h取样,拍照。实验重复3次。

7.Transwell实验检测细胞迁移取对数生长期的对照组及miRNA-195组细胞,胰酶消化后用含0.2%牛血清白蛋白(bovine serum albumin,BSA)的无血清培养基重悬,调整细胞密度至1×10⁶,取100 μL加入上室,下室加入600 μL完全培养基,37 ℃、5%CO₂培养24 h。观察膜及孔下细胞数,用甲醛室温固定15 min。吸出固定液,加入结晶紫染液,室温作用10 min,用双蒸水洗涤。用棉签擦去上室内的细胞,拍照计数。实验重复3次。

8. Matrigel侵袭实验检测细胞侵袭能力将Matrigel在4 ℃融化后向Transwell小室每孔加入40 μL,37 ℃干燥30 min。干燥后上室加入100 μL无血清1640培养基,下室加入600 μL无血清1640培养基,37 ℃再平衡12 h后待用。收集细胞,用含0.2%BSA的无血清1640培养基调整细胞密度至1×10⁶。取200 μL加入上室,即上室细胞数为2×10⁵/孔;下室加入600 μL完全培养基,37 ℃、5%CO₂培养24 h,观察膜及孔下细胞数。用甲醛室温固定15 min,吸出固定液,加入结晶紫染液,室温作用10 min,用双蒸水洗涤。用棉签擦去基质胶和上室内的细胞,拍照计数。实验重复3次。

三、统计学方法

采用SPSS 16.0软件进行统计分析。所有数据均用x- ±s表示。两组间比较采用 t检验。 P<0.05为差异有统计学意义。

结果

一、转染后5637细胞miRNA-195的表达量

提取NC、miRNA-195细胞的总RNA进行逆转录,采用实时荧光定量PCR检测miRNA-195。结果显示转染后的5637细胞(即miRNA-195组)高表达miRNA-195,与对照组比较差异有统计学意义( P<0.05)。见图1。

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	图1 转染后5637细胞miRNA-195的表达量

二、miRNA-195对5637细胞增殖能力的影响

与对照组比较,miRNA-195组细胞增殖能力明显降低( P<0.05),见图2。

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	图2 miRNA-195对5637细胞增殖能力的影响

三、细胞生长速度

与对照组比较,miRNA-195组细胞的增长速度明显减弱( P<0.05),见图3。

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	图3 对照组及miRNA-195组细胞的生长速度比较

四、miRNA-195对5637细胞增殖能力的影响

采用细胞克隆形成实验检测miRNA-195对膀胱癌细胞生长能力的影响。结果显示miRNA-195组的细胞克隆数明显少于对照组( P<0.05),见图4。说明转染miRNA-195后5637细胞的增殖能力减弱。

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	图4 miRNA-195对5637细胞增殖能力的影响

五、miRNA-195对5637细胞体外迁移能力的影响

1.划痕实验采用倒置显微镜观察拍摄对照组、miRNA-195注细胞的划痕愈合情况并统计划痕面积变化情况。结果显示同一时间点miRNA-195组细胞的划痕愈合较慢,并且划痕愈合面积明显小于对照组( P<0.05),见图5。说明miRNA-195组细胞迁移能力受到抑制。

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	图5 划痕实验检测细胞迁移

2. Transwell实验 miRNA-195组穿过Transwell小室的细胞少于对照组( P<0.05),见图6。说明转染miRNA-195的5637细胞迁移能力减弱。

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	图6 Transwell实验检测细胞迁移

七、miRNA-195对5637细胞侵袭能力的影响

Matrigel侵袭实验显示miRNA-195组穿过matrigel胶的5637细胞明显少于对照组,见图7。说明转染miRNA-195后5637细胞的侵袭能力减弱。

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	图7 miRNA-195对5637细胞侵袭能力的影响

讨论

miRNAs通过调节各种基因的表达,在真核细胞的生长和肿瘤形成中发挥重要作用。因此,研究各种miRNA的作用机制有重要意义。miRNA是非编码小RNA,在转录后水平调节基因的表达^{[ 9]}。在各种恶性肿瘤的发生、发展中,miRNA发挥着重要作用。泌尿系统的多种恶性肿瘤细胞miRNA出现异常表达,其中包括膀胱癌细胞。说明miRNA在多种膀胱癌的信号通路中发挥作用^{[ 10]}。虽然miRNA的表达特点已经基本清楚,但是miRNA在膀胱癌发生及代谢中表达异常的机制尚不明确。

一些证据表明,miRNA-195能够抑制肝癌细胞对内皮细胞迁移和血管再生的促进能力,并且能直接抑制肝癌细胞的迁移、侵袭能力,从而对肝癌起到抑制作用^{[ 11]}。在骨肉瘤中,miRNA-195通过作用于脂肪酸合成酶(FASN)抑制肿瘤的迁移和侵袭^{[ 12]}。本研究将miRNA-195转染入膀胱癌5637细胞,研究其在膀胱癌中的作用。考虑到病毒载体本身可能对细胞产生影响,因此本研究以转染空载的膀胱癌5637细胞作为对照。MTT法和集落形成实验结果显示miRNA-195可以抑制膀胱癌细胞5637的增殖。划痕实验、Transwell实验结果显示miRNA-195抑制了膀胱癌细胞的转移和侵袭。因此,miRNA-195可以作为治疗膀胱癌的潜在靶点,也为研究miRNA-195在其他肿瘤中的作用提供实验基础。

目前,关于miRNA与膀胱癌的研究在不断深入。但这些miRNA到底是通过哪些具体的机制来促进或抑制膀胱癌的发生还不清楚。miRNA通过表达上调或下调,与某些原癌基因、抑癌基因、凋亡相关基因相互作用,或调解某些细胞因子,参与肿瘤的生成、发展甚至侵袭转移^{[ 13]}。有文献报道,转染了miRNA-30a-3p/miRNA133a/miRNA-199a的膀胱癌细胞株,其细胞角蛋白7(KRT7)的表达减少,从而抑制肿瘤细胞的生长^{[ 14]}。Huang等^{[ 15]}发现,miRNA-125b通过抑制细胞周期中E2F3-cyclinA2信号途径,可以使膀胱肿瘤细胞生长停滞在G期。Lu等^{[ 16]}的研究结果表明miRNA-221在膀胱癌细胞中发生表达沉默,能通过肿瘤肿瘤坏死因子相关凋亡诱导配体(TRAIL)途径促使肿瘤细胞发生凋亡。本研究结果表明miRNA-195对于膀胱癌细胞的增殖、转移及侵袭都有影响,但机制尚不清楚。下一步我们将利用生物信息学的方法对miRNA-195的靶基因进行预测和分析,研究其相关机制。

(收稿日期:2013-12-30)

(本文编辑:龚晓霖)

The authors have declared that no competing interests exist.

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2013

1.941

0.0

. 2013, 133(2):446-454

Smoking is associated with lower age, higher grade, higher stage, and larger size of malignant bladder tumors at diagnosis

Eline H. van Roekel 1,2 ,Kar K. Cheng 1,* ,Nicholas D. James 3 ,D. Michael A. Wallace 4 ,Lucinda J. Billingham 1,3 ,Paul G. Murray 3 ,Richard T. Bryan 3 andMaurice P. Zeegers 1,5

1Department of Public Health, Epidemiology and Biostatistics, School of Health and Population Sciences, University of Birmingham, Birmingham, United Kingdom 2Department of Epidemiology, GROW − School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands 3School of Cancer Sciences, University of Birmingham, Birmingham, United Kingdom 4School of Surgery, University of Western Australia, Fremantle, Australia 5Department of Complex Genetics, Cluster of Genetics and Cell Biology, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Center+, Maastricht, The Netherlands *Correspondence to: Kar K. Cheng, Department of Public Health, Epidemiology and Biostatistics, School of Health and Population Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom, Tel.: +44-121-4146757, Fax: +44-121-4146216, E-mail: <a href="mailto:k.k.cheng@bham.ac.uk" title="Link to email address" shape="rect">k.k.cheng@bham.ac.uk</a>

Smoking is a strong risk factor of bladder cancer (BC), but it is currently unclear whether smoking is also associated with severity of BC at diagnosis. We performed a large hospital-based case-comparison study, examining the relation between smoking and clinical characteristics of BC at diagnosis. A total of 1,544 cases from participating hospitals in the West Midlands were recruited between 19 December 2005 and 21 April 2011. Eligible cases were adult BC patients without a previous history of this disease. At time of diagnosis, semi-structured face-to-face interviews were conducted by trained research nurses to collect smoking information. Clinical characteristics were obtained from medical records. Linear mixed models were performed to calculate predicted means in clinical outcomes for a variety of smoking behaviors. A p < 0.05 was considered statistically significant. After adjustment for age and gender, current smokers were on average 4.0 years younger at diagnosis (95% CI: −5.9 to −2.0), had larger tumors (mean difference: 0.48 cm, 95% CI: 0.04−0.91), a higher T stage (mean difference: 0.25, 95% CI: 0.08−0.41), and a borderline significantly higher grade than never smokers (mean difference: 0.15, 95% CI: 0.00−0.30). Our results suggest that smoking could be associated with a more malignant phenotype of BC at diagnosis. More research is needed on the relation between smoking and prognosis, but our results could strengthen the message about the potential risks of smoking to these patients.

... 已经明确膀胱癌的发生、发展与吸烟、环境污染等因素有关^[1,2],但其分子机制尚不明确 ...

2012

0.0

. 2012, 30(2):null-null

Bladder cancer risk from occupational and environmental exposures

... 已经明确膀胱癌的发生、发展与吸烟、环境污染等因素有关^[1,2],但其分子机制尚不明确 ...

2011

10.476

0.0

. 2011, 59(5):671-681 DOI:10.1016/j.eururo.2011.01.044

MicroRNA in prostate, bladder, and kidney cancer: a systematic review

<ul><li id="aff0005">aInstitute for Cancer Studies and Academic Urology Unit, University of Sheffield, Sheffield, UK</li><li id="aff0010">bDepartment and Laboratory of Urology, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain</li><li id="aff0015">cDepartment of Urology, Skåne University Hospital, Malmö, Sweden</li><li id="aff0020">dDepartment of Urology, University of California, Davis, School of Medicine, Sacramento, CA, USA</li><li id="aff0025">eDepartment of Urology, Technical University of Dresden, Dresden, Germany</li><li id="aff0030">fNuffield Department of Surgery, University of Oxford, Oxford, UK</li><li id="aff0035">gInstitute for Molecular Medicine Finland (FIMM), Nordic EMBL Partnership in Molecular Medicine, University of Helsinki, Helsinki, Finland</li><li id="aff0040">hProstate Cancer Centre, University Medical Centre, Hamburg-Eppendorf, Hamburg, Germany</li><li id="aff0045">iInstitute of Medical Technology, University of Tampere and Tampere University Hospital, Tampere, Finland</li></ul>

Context

MicroRNAs (miRNA) are noncoding RNAs that post-transcriptionally regulate gene expression. Their altered expression and function have been observed in most urologic cancers. MiRNAs represent potential disease biomarkers and novel therapeutic targets.

Objective

To review and evaluate the evidence implicating miRNAs in the pathogenesis of prostate cancer (PCa), bladder cancer (BCa), and renal cancer.

Evidence acquisition

A systematic review was performed using PubMed and Embase to search for reports using strings for microRNA, non-coding RNA, cancer, prostate, bladder, and renal cancer. Identified manuscripts were retrieved and references searched. Selected studies were required to concentrate on the role of miRNA in these urologic cancers.

Evidence synthesis

We reviewed articles that focus on this topic. More than 40 miRNAs have been implicated in urologic cancer and many target common carcinogenic pathways. In particular, apoptosis avoidance, cell proliferation, epithelial-to-mesenchymal transition, angiogenic signalling, and the generation of androgen independence are targeted or facilitated by more than one miRNA. Little work has been done to evaluate the translational applications for this knowledge to date. Novel therapeutic strategies have been developed and are under investigation to selectively modulate miRNAs; such work would potentially enable personalised tumour therapy.

Conclusions

MiRNAs appear to be important modulators of urologic cancer. Their expression is frequently altered in these tumours, and many are functionally implicated in their pathogenesis. They require evaluation to determine the translational role and therapeutic potential for this knowledge.

... 研究结果显示在膀胱癌发生时,微小RNA(micro RNA,miRNA)的表达发生异常改变,并且在膀胱癌的发展中起重要作用^[3] ...

2011

4.25

0.0

... 抑制肿瘤的miRNA下调或者促进肿瘤的miRNA上调都会引起肿瘤形成^[4] ...

2013

3.73

0.0

PLoS ONE. 2013, 8(7):e67686-null

MicroRNA-23b Functions as a Tumor Suppressor by Regulating Zeb1 in Bladder Cancer

Rajvir Dahiya 1 ,Soichiro Yamamura 1 ,Sharanjot Saini 1 ,Shahana Majid 1 ,Guoren Deng 1 ,Altaf A. Dar 2 ,Yuichiro Tanaka 1 ,Kirsten Greene 1 ,Inik Chang 1

1 Department of Urology, VA Medical Center and UCSF, San Francisco, California, United States of America 2 California Pacific Medical Center Research Institute, San Francisco, California, United States of America

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression by targeted repression of transcription and translation. In this study we show that miRNA-23b (miR-23b) acts as a tumor suppressor in bladder cancer. Quantitative real-time PCR analysis showed that miR-23b is significantly down-regulated in bladder cancer cell lines and tumor tissues compared to non-malignant cells and normal tissue samples. We also demonstrate that miR-23b expression has a potential to be diagnostic and prognostic biomarker in bladder cancer. High miR-23b expression is positively correlated with higher overall survival of bladder cancer patients as revealed by Kaplan-Meier analysis. ROC analysis showed that miR-23b expression can distinguish between normal and bladder cancer tissues. Further we elucidated the biological significance of miR-23b in bladder cancer. Over-expression of miR-23b in bladder cancer cells inhibited cell proliferation and impaired colony formation. Fluorescence activated cell sorting (FACS) analysis revealed that re-expression of miR-23b in bladder cancer cells induced G0/G1 cell cycle arrest and apoptosis while inhibiting cell migration and invasion. Luciferase reporter assays demonstrated that Zeb1, a crucial regulator of epithelial-to-mesenchymal transition (EMT), is a direct target of miR-23b in bladder cancer. These results show that loss of miR-23b confers a proliferative advantage and promotes bladder cancer cell migration and invasion. Furthermore, re-expression of miR-23b may be a beneficial therapeutic strategy for the treatment of human bladder cancer.

... 有研究证实miRNA-23b、miRNA-409-3p在膀胱癌细胞中表达下调^[5,6] ...

2013

2.21

0.0

. 2013, 36(1):62-68 DOI:10.1007/s10059-013-0044-7

MicroRNA-409-3p inhibits migration and invasion of bladder cancer cells via targeting c-Met

<li>144.Department of Urology, First Affiliated Hospital, Zhejiang University, Qingchun Road 79, Hangzhou, 310003, Zhejiang Province, China</li>

Abstract

There is increasing evidence suggesting that dysregulation of certain microRNAs (miRNAs) may contribute to tumor progression and metastasis. Previous studies have shown that miR-409-3p is dysregulated in some malignancies, but its role in bladder cancer is still unknown. Here, we find that miR-409-3p is down-regulated in human bladder cancer tissues and cell lines. Enforced expression of miR-409-3p in bladder cancer cells significantly reduced their migration and invasion without affecting cell viability. Bioinformatics analysis identified the pro-metastatic gene c-Met as a potential miR-409-3p target. Further studies indicated that miR-409-3p suppressed the expression of c-Met by binding to its 3′-untranslated region. Silencing of c-Met by small interfering RNAs phenocopied the effects of miR-409-3p overexpression, whereas restoration of c-Met in bladder cancer cells bladder cancer cells overexpressing miR-409-3p, partially reversed the suppressive effects of miR-409-3p. We further showed that MMP2 and MMP9 may be downstream effector proteins of miR-409-3p. These findings indicate that miR-409-3p could be a potential tumor suppressor in bladder cancer.

... 有研究证实miRNA-23b、miRNA-409-3p在膀胱癌细胞中表达下调^[5,6] ...

2013

3.73

0.0

PLoS ONE. 2013, 8(1):e54932-null

MicroRNA-195 Inhibits the Proliferation of Human Glioma Cells by Directly Targeting Cyclin D1 and Cyclin E1

Ba Yueyang 1,1 ,Wang Hui 1 ,Luo Lun 1 ,Li WenSheng 1 ,Lu Yuntao 2 ,Liang ChaoFeng 1 ,He HaiYong 1

1 Department of Neurosurgery, The Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong, China 2 Department of Neurosurgery, Nanfang Hospital, The First Affiliated Hospital of Southern Medical University, Guangzhou, Guangdong, China

Glioma proliferation is a multistep process during which a sequence of genetic and epigenetic alterations randomly occur to affect the genes controlling cell proliferation, cell death and genetic stability. microRNAs are emerging as important epigenetic modulators of multiple target genes, leading to abnormal cellular signaling involving cellular proliferation in cancers.In the present study, we found that expression of miR-195 was markedly downregulated in glioma cell lines and human primary glioma tissues, compared to normal human astrocytes and matched non-tumor associated tissues. Upregulation of miR-195 dramatically reduced the proliferation of glioma cells. Flow cytometry analysis showed that ectopic expression of miR-195 significantly decreased the percentage of S phase cells and increased the percentage of G1/G0 phase cells. Overexpression of miR-195 dramatically reduced the anchorage-independent growth ability of glioma cells. Furthermore, overexpression of miR-195 downregulated the levels of phosphorylated retinoblastoma (pRb) and proliferating cell nuclear antigen (PCNA) in glioma cells. Conversely, inhibition of miR-195 promoted cell proliferation, increased the percentage of S phase cells, reduced the percentage of G1/G0 phase cells, enhanced anchorage-independent growth ability, upregulated the phosphorylation of pRb and PCNA in glioma cells. Moreover, we show that miR-195 inhibited glioma cell proliferation by downregulating expression of cyclin D1 and cyclin E1, via directly targeting the 3′-untranslated regions (3′-UTR) of cyclin D1 and cyclin E1 mRNA. Taken together, our results suggest that miR-195 plays an important role to inhibit the proliferation of glioma cells, and present a novel mechanism for direct miRNA-mediated suppression of cyclin D1 and cyclin E1 in glioma.

... miRNA-195可抑制人胶质瘤细胞、胃癌细胞的增殖和转移^[7,8] ...

2013

2.196

0.0

. 2013, 518(2):null-null

MicroRNA-195 and microRNA-378 mediate tumor growth suppression by epigenetical regulation in gastric cancer

... miRNA-195可抑制人胶质瘤细胞、胃癌细胞的增殖和转移^[7,8] ...

2012

0.742

0.0

. 2012, 188(2):null-null

Expression of miRNAs involved in angiogenesis, tumor cell proliferation, tumor suppressor inhibition, epithelial-mesenchymal transition and activation of metastasis in bladder cancer

... miRNA是非编码小RNA,在转录后水平调节基因的表达^[9] ...

2013

0.0

... 说明miRNA在多种膀胱癌的信号通路中发挥作用^[10] ...

2013

0.0

. 2013, 58(2):642-653

MicroRNA-195 Suppresses Angiogenesis and Metastasis of Hepatocellular Carcinoma by Inhibiting the Expression of VEGF, VAV2, and CDC42

Ruizhi Wang 1 ,Na Zhao 1 ,Siwen Li 1 ,Jian-Hong Fang 1 ,Mei-Xian Chen 2 ,Jine Yang 1 ,Wei-Hua Jia 3 ,Yunfei Yuan 2,* andShi-Mei Zhuang 1,*

1Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, People's Republic of China 2Department of Hepatobiliary Oncology, Sun Yat-sen University, Guangzhou, People's Republic of China 3Bank of Tumor Resources, Cancer Center, Sun Yat-sen University, Guangzhou, People's Republic of China *Address reprint requests to: Shi-Mei Zhuang, School of Life Sciences, Sun Yat-sen University, Xin Gang Xi Road 135#, Guangzhou 510275, People's Republic of China. E-mail: <a href="mailto:zhuangshimei@163.com" title="Link to email address" shape="rect">zhuangshimei@163.com</a> or <a href="mailto:lsszsm@mail.sysu.edu.cn" title="Link to email address" shape="rect">lsszsm@mail.sysu.edu.cn</a>; fax: +86-20-84113469. Yunfei Yuan, Department of Hepatobiliary Oncology, Cancer Center, Sun Yat-sen University, Dongfengdong Road 651#, Guangzhou 510060, People's Republic of China. E-mail: <a href="mailto:yuanyf@mail.sysu.edu.cn" title="Link to email address" shape="rect">yuanyf@mail.sysu.edu.cn</a>; fax: +86-20-87343392.

Hepatocellular carcinoma (HCC) is characterized by active angiogenesis and metastasis, which account for rapid recurrence and poor survival. There is frequent down-regulation of miR-195 expression in HCC tissues. In this study, the role of miR-195 in HCC angiogenesis and metastasis was investigated with in vitro capillary tube formation and transwell assays, in vivo orthotopic xenograft mouse models, and human HCC specimens. Reduction of miR-195 in HCC tissues was significantly associated with increased angiogenesis, metastasis, and worse recurrence-free survival. Both gain-of-function and loss-of-function studies of in vitro models revealed that miR-195 not only suppressed the ability of HCC cells to promote the migration and capillary tube formation of endothelial cells but also directly repressed the abilities of HCC cells to migrate and invade extracellular matrix gel. Based on mouse models, we found that the induced expression of miR-195 dramatically reduced microvessel densities in xenograft tumors and repressed both intrahepatic and pulmonary metastasis. Subsequent investigations disclosed that miR-195 directly inhibited the expression of the proangiogenic factor vascular endothelial growth factor (VEGF) and the prometastatic factors VAV2 and CDC42. Knockdown of these target molecules of miR-195 phenocopied the effects of miR-195 restoration, whereas overexpression of these targets antagonized the function of miR-195. Furthermore, we revealed that miR-195 down-regulation resulted in enhanced VEGF levels in the tumor microenvironment, which subsequently activated VEGF receptor 2 signaling in endothelial cells and thereby promoted angiogenesis. Additionally, miR-195 down-regulation led to increases in VAV2 and CDC42 expression, which stimulated VAV2/Rac1/CDC42 signaling and lamellipodia formation and thereby facilitated the metastasis of HCC cells. Conclusion: miR-195 deregulation contributes to angiogenesis and metastasis in HCC. The restoration of miR-195 expression may be a promising strategy for HCC therapy. (Hepatology 2013;58:642-653)

... 一些证据表明,miRNA-195能够抑制肝癌细胞对内皮细胞迁移和血管再生的促进能力,并且能直接抑制肝癌细胞的迁移、侵袭能力,从而对肝癌起到抑制作用^[11] ...

2012

0.237

0.0

... 在骨肉瘤中,miRNA-195通过作用于脂肪酸合成酶(FASN)抑制肿瘤的迁移和侵袭^[12] ...

2011

0.0

... miRNA通过表达上调或下调,与某些原癌基因、抑癌基因、凋亡相关基因相互作用,或调解某些细胞因子,参与肿瘤的生成、发展甚至侵袭转移^[13] ...

2009

1.941

0.0

. 2009, 125(2):345-352

Identification of novel microRNA targets based on microRNA signatures in bladder cancer

Takahiro Ichimi 1 ,Hideki Enokida 1,†,* ,Yasushi Okuno 2 ,Ryo Kunimoto 2 ,Takeshi Chiyomaru 1 ,Ken Kawamoto 1 ,Kazuya Kawahara 3 ,Kazuki Toki 1 ,Kazumori Kawakami 1 ,Kenryu Nishiyama 1 ,Gozoh Tsujimoto 4 ,Masayuki Nakagawa 1 andNaohiko Seki 5

1Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan 2Department of Pharmacoinformatics, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan 3Kawahara Nephro-urology Clinic, Kagoshima, Japan 4Genomic Drug Discovery Science, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan 5Department of Functional Genomics, Graduate School of Medicine, Chiba University, Chiba, Japan *Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan

MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate protein-coding genes. To identify miRNAs that have a tumor suppressive function in bladder cancer (BC), 156 miRNAs were screened in 14 BCs, 5 normal bladder epithelium (NBE) samples and 3 BC cell lines. We identified a subset of 7 miRNAs (miR-145, miR-30a-3p, miR-133a, miR-133b, miR-195, miR-125b and miR-199a*) that were significantly downregulated in BCs. To confirm these results, 104 BCs and 31 NBEs were subjected to real-time RT-PCR-based experiments, and the expression levels of each miRNA were significantly downregulated in BCs (p < 0.0001 in all). Receiver-operating characteristic curve analysis revealed that the expression levels of these miRNAs had good sensitivity (>70%) and specificity (>75%) to distinguish BC from NBE. Our target search algorithm and gene-expression profiling in BCs (Kawakami et al., Oncol Rep 2006;16:521–31) revealed that Keratin7 (KRT7) mRNA was a common target of the downregulated miRNAs, and the mRNA expression levels of KRT7 were significantly higher in BCs than in NBEs (p = 0.0004). Spearman rank correlation analysis revealed significant inverse correlations between KRT7 mRNA expression and each downregulated miRNA (p < 0.0001 in all). Gain-of-function analysis revealed that KRT7 mRNA was significantly reduced by transfection of 3 miRNAs (miR-30-3p, miR-133a and miR-199a*) in the BC cell line (KK47). In addition, significant decreases in cell growth were observed after transfection of 3 miRNAs and si-KRT7 in KK47, suggesting that miR-30-3p, miR-133a and miR-199a* may have a tumor suppressive function through the mechanism underlying transcriptional repression of KRT7. © 2009 UICC

... 有文献报道,转染了miRNA-30a-3p/miRNA133a/miRNA-199a的膀胱癌细胞株,其细胞角蛋白7(KRT7)的表达减少,从而抑制肿瘤细胞的生长^[14] ...

2011

1.941

0.0

... Huang等^[15]发现,miRNA-125b通过抑制细胞周期中E2F3-cyclinA2信号途径,可以使膀胱肿瘤细胞生长停滞在G期 ...

2010

0.0

... Lu等^[16]的研究结果表明miRNA-221在膀胱癌细胞中发生表达沉默,能通过肿瘤肿瘤坏死因子相关凋亡诱导配体(TRAIL)途径促使肿瘤细胞发生凋亡 ...