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Journal of Southern Medical University logoLink to Journal of Southern Medical University
. 2020 Jun 20;40(6):869–875. [Article in Chinese] doi: 10.12122/j.issn.1673-4254.2020.06.15

MiR-204通过靶向调控HNRNPA2B1抑制乳腺癌的侵袭和转移

MiR-204 inhibits invasion and metastasis of breast cancer cells by targeted regulation of HNRNPA2B1

张 丽萍 1, 白 俊 1, 胡 雅琼 1, 周 丹丹 1, 郑 荃 1, 尹 崇高 2, 牟 青杰 3, 李 洪利 4,*
PMCID: PMC7321266  PMID: 32895200

Abstract

目的

探讨miR-204通过靶向调控HNRNPA2B1对乳腺癌侵袭和转移的影响。

方法

生物信息学数据库查询miR-204在乳腺癌患者中的表达情况和miR-204对乳腺癌患者生存率的影响；RT-qRCR检测miR-204在乳腺癌细胞系中的表达情况；利用GV369-miR-204过表达慢病毒上调MDA-MB-231细胞中的miR-204；Transwell实验检测miR-204对MDA-MB-231细胞侵袭和迁移能力的影响；生物信息学方法确定miR-204的关键基因（Hub基因）；双荧光素酶实验检测miR-204和HNRNPA2B1的靶向关系；Western blot实验检测过表达miR-204后HNRNPA2B1的表达情况；Transwell实验检测MDA-MB-231/miR-204+ NC、MDA-MB-231/miR-204+HNRNPA2B1细胞的侵袭和迁移能力的变化。

结果

miR-204在乳腺癌组织中表达降低（P < 0.05），低表达水平的miR-204与患者不良预后相关（P < 0.05）；miR-204在乳腺癌细胞系MDA-MB-231细胞系中表达量低于正常乳腺上皮细胞MCF-10A的表达（P < 0.0001）。通过过表达慢病毒上调MDA-MB-231细胞中miR-204的表达，结果显示上调miR-204可抑制乳腺癌细胞的侵袭和迁移能力（P < 0.05）；HNRNPA2B1为miR-204的Hub基因，starbase网站发现miR-204-5p和HNRNPA2B1表达呈负相关且HNRNPA2B1在乳腺癌患者中表达升高（P < 0.05）；生存分析证明高表达HNRNPA2B1的乳腺癌患者预后不良（P < 0.05）；双荧光素酶实验证明miR-204和HNRNPA2B1可靶向结合（P < 0.05）。Western blot和Transwell实验证明，miR-204通过靶向调控HNRNPA2B1抑制乳腺癌的侵袭和迁移，且差异具有统计学意义（P < 0.05）。

结论

miR-204在乳腺癌组织和细胞中表达降低，且miR-204可通过靶向调控HNRNPA2B1抑制乳腺癌的侵袭和转移。

Keywords: 乳腺癌, miR-204, 肿瘤侵袭和迁移, HNRNPA2B1

乳腺癌是一种起源于乳腺组织的恶性肿瘤，是全世界妇女因癌症死亡的最常见原因[1]。侵袭转移是肿瘤细胞重要的生物学行为，与预后不良密切相关，严重威胁着转移患者的健康和生命[2]。因此，深入分析乳腺癌转移机制对改善乳腺癌患者的生存率和预后具有重要意义。

我们对基因如何驱动相应蛋白质改变从而影响表型改变的认识仍有欠缺，因此研究肿瘤组织与正常组织的差异基因从而指导我们更全面的认识肿瘤的发病机制具有重要意义。功能基因组学的进展表明，人类基因组是主动转录的。然而，绝大多数转录本是非编码RNA包括microRNAs（miRNAs）和长链非编码RNA（LncRNA）。MiRNA是一类内源性、进化保守的小非编码RNA分子，可调节靶基因的转录后处理，导致靶mRNA的降解及其翻译抑制[3]。多项研究表明[4-5]，miRNA参与细胞上皮间质转化等多种细胞和生理过程，且其异常表达与多种人类肿瘤的进展和转移有关。miRNA发挥作用主要通过与目标mRNA的3'-UTR区结合，导致目标mRNA的降解，从而调控靶蛋白的表达，从而影响人肿瘤的进展。另外，多项研究表明[6-8]，与正常组织相比，miR-204在多种人类肿瘤组织中下调，并被认为在多种类型的癌症中具有抑癌作用。核内不均一核糖核蛋白（hnRNPs）参与了包括增殖和凋亡在内的关键肿瘤进展过程，HNRNPA2和HNRNPB1是由HNRNPA2B1基因翻译而来的两个亚型[9]，有研究证明HNRNPA2B1在肿瘤发生过程中参与多种重要基因的表达，HNRNPA2B1通过激活Lin28B表达促进卵巢癌的恶性表型并增强其稳定性[10]。

miR-204在乳腺癌中的具体作用仍有待研究，因此，本研究将探讨miR-204是否可以通过靶向调控HNRNPA2B1影响乳腺癌的侵袭和转移，为抑制乳腺癌细胞转移和治疗乳腺癌提供新的靶点。

1. 材料和方法

1.1. 材料与仪器

MCF-10A，MDA-MB-231，293T细胞（ATCC），胎牛血清和RPMI 1640培养基（HyClone）。MiRNA上下游引物由生工生物工程有限公司设计合成。8-μm孔径Transwell小室（BD Biosciences）。Lipofectamine 2000（Invitrogen）。包含HNRNPA2B1 3'-UTR序列的荧光素酶报告基因质粒和含有HNRNPA2B1 3'-UTR突变序列的荧光素酶报告基因质粒均由吉凯公司构建并鉴定。HNRNPA2B1抗体为Rabbit Anti- HNRNPA2B1 antibody（ab31645），β-actin抗体为Rabbit Anti-beta Actin antibody（ab8227），抗体均购自Abcam公司。

1.2. 生物信息学数据库

GEO数据库：从基因表达谱GEO数据库（<a href="https://www.ncbi.nlm.nih.gov" target="_blank">https://www.ncbi.nlm.nih.gov</a>）中下载两组乳腺癌患者资料。GSE44124数据集为50例乳腺肿瘤组织与30个正常组织miRNA表达谱。GSE38167数据集为31例原发性三阴性乳腺癌（TNBC）患者组织（IDC），13例淋巴结转移（LN）患者组织，23例正常乳腺组织（NAT）的miRNA表达谱。Kaplan- Meier Plotter（Kmplot）网站：Kmplot（<a href="https://kmplot.com/analysis/" target="_blank">https://kmplot.com/analysis/</a>）网站能够评估21种癌症类型中54000基因对生存率的影响。最大的数据集包括乳腺癌（6234例）、卵巢癌（2190例）、肺癌（3452例）和胃癌（1440例）。miRNA子系统包括20种不同癌症类型的11 000样本。该系统包括基因芯片和RNA-seq数据源，数据库包括GEO、EGA和TCGA数据库<sup>[<xref ref-type="bibr" rid="b11">11</xref>]</sup>。

1.3. 细胞培养与分组

细胞培养参考本课题先前已发表文献[12]，将MDA-MB-231细胞分组：（1）con组，转入空载慢病毒；（2）miR-204组，转入miR-204过表达慢病毒；（3）miR- 204 + NC组，同时转入miR-204过表达慢病毒和HNRNPA2B1过表达对照质粒；（4）miR-204 + HNRNPA2B1组，同时转入miR-204过表达慢病毒和HNRNPA2B1过表达质粒。

1.4. RT-qPCR实验

RNA提取及逆转录过程参考本课题先前已发表文献[13]。PCR条件为95 ℃ 5 s、63 ℃ 30 s、72 ℃ 30 s进行35个循环。以U6作为内参。MiR-204-5p的上游引物为CGCGTTCCCTTTGTCATCCT，下游引物为AGTGCAGGGTCCGAGGTATT，茎环结构GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGGCAT。

1.5. Transwell实验

Transwell实验是在Transwell小室中进行的，如文献[3]所述。迁移实验：取对数生长期的MDA-MB-231细胞（2×105）悬浮于200 μL无血清的RPMI 1640培养基中，将细胞悬浮液加至无基质胶Transwell上室中，下室为500 μL胎牛血清。侵袭实验：取对数生长期的MDA-MB-231细胞（2×105）悬浮于200 μL无血清的RPMI 1640培养基中，将细胞悬浮液加至有基质胶Transwell上室中，下室为500 μL胎牛血清。固定染色方法：将细胞培养24 h，用棉签将未通过气孔的细胞除去。用4%多聚甲醛进行细胞固定，姬姆萨（Sigma-Aldrich, St. Louis, USA）染色。显微镜下随机选取5个视野拍照计数，取穿至下室细胞的平均数为实验结果，实验重复3次。

1.6. 双荧光素酶实验

293T细胞购自ATCC，细胞培养方式按照ATCC建议。构建pGL3-HNRNPA2B1-3-UTR-MUT和pGL3- HNRNPA2B1-3-UTR-WT质粒，将293T细胞培养于24孔板中，100 ng的pGL3-HNRNPA2B1-3-UTR-MUT和pGL3-HNRNPA2B1-3-UTR-WT使用Lipofectamine 2000（Invitrogen，12566014）和miRNA对照及过表达载体分别共转染293T细胞。48 h后获得细胞，用双荧光素酶检测系统（Promega）测定荧光素酶活性。

1.7. Western blot

Western blot检测HNRNPA2B1水平，将转染后的细胞提取蛋白。上样，电泳，转膜，封闭，滴加一抗4 ℃过夜，滴加二抗，TBST洗PVDF膜，曝光。β-actin作为内参。

1.8. 靶基因预测和Hub基因筛选

利用miRTarbase（<a href="http://mirtarbase.mbc.nctu.edu.tw/php/index.php" target="_blank">http://mirtarbase.mbc.nctu.edu. tw/php/index.php</a>）网站、miRWalk（<a href="http://mirwalk.umm.uni-heidelberg.de/" target="_blank">http://mirwalk.umm.uni-heidelberg.de/</a>）网站、Targetscan（<a href="http://www.targetscan.org/vert_72/" target="_blank">http://www.targetscan.org/vert_72/</a>）网站对miR-204进行靶基因预测。利用STRING v11（<a href="https://string-db.org/" target="_blank">https://string-db.org/</a>）网站进行PPI分析，STRING v11可构建蛋白质-蛋白质关联网络，支持蛋白质相关信号通路的富集关系，蛋白质或者基因的分子功能，生物过程和细胞组成等的分析<sup>[<xref ref-type="bibr" rid="b14">14</xref>]</sup>。Starbase网站：starBase v2.0（<a href="http://starbase.sysu.edu.cn/index.php" target="_blank">http://starbase.sysu.edu.cn/index.php</a>），可以从大规模CLIP-Seq数据中解码miRNA- ceRNA、miRNA-ncRNA和蛋白-RNA相互作用网络<sup>[<xref ref-type="bibr" rid="b15">15</xref>]</sup>。

1.9. 统计学分析

采用SPSS 22.0进行统计学处理，实验数据用均数±标准差表示，两组间定量资料采用独立样本t检验，多组间比较采用单因素方差分析，以P < 0.05认为差异具有统计学意义。

2. 结果

2.1. MiR-204在乳腺癌患者组织中的表达情况

通过GEO数据库发现miR-204在GSE44124和GSE38167数据集中低表达，通过GEO2R分析发现，miR-204在GSE44124数据集中的表达降低的miRNA中P值最小（图 1A），miR-204在乳腺癌患者组织中的表达较正常乳腺组织降低（图 1B），在GSE38167数据集中，miR-204在浸润性导管癌和有淋巴结转移癌症患者中miR-204的表达水平同样降低（图 1C）。Kmplot数据库对6234例乳腺癌患者的300个月的随访分析结果显示，低表达水平的miR-204与患者不良预后相关，且差异具有显著性（P < 0.05，图 1D）。

1.

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MiR-204在乳腺癌患者组织中的表达量和miR-204与乳腺癌患者预后的相关性

Expression of miR-204 in breast cancer tissues and its association with the patients' prognosis. A: Volcano plot of GSE44124 showing miRNA expression in breast cancer tissues and normal tissues; B: Expression of miR-204 in GSE44124; C: Expression of miR-204 in GSE38167 (NAT: Normal adjacent tissues, IDC: Invasive ductal carcinoma tissues, LN: Breast cancer tissue with lymph node metastasis); (D) Survival analysis of patients with different expression levels of miR-204 in breast cancer tissues.

2.2. miR-204在三阴性乳腺癌细胞系MDA-MB-231中的表达

与正常乳腺细胞MCF-10A中miR-204的表达量（1.01±0.10）相比，miR-204在三阴性乳腺癌细胞系MDA-MB-231细胞中表达量（0.40±0.07）低，且差异具有统计学意义（图 2A）。miR-204在MDA-MB-231中的转染效率，相对于MDA-MB-231/con组细胞的miR- 204的表达量（1.01±0.09），miR-204在MDA-MB-231/ miR-204组的表达量（2.58±0.16）升高（图 2B）。

2.

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miR-204在各组细胞中的表达量

Expression of miR-204 in different cells. A: The expression of miR-204 in MCF-10A and MDA-MB-231 cells; B: The transfection efficiency of miR-204 in MDA-MB-231 cells.

2.3. miR-204抑制乳腺癌细胞的侵袭迁移能力

迁移实验显示，MDA-MB-231/miR-204组细胞穿至下室的数量（49.00±4.35）少于MDA-MB-231/con组细胞穿至下室的数量（131.00±7.00），过表达miR-204组侵袭能力降低（P < 0.05）；侵袭实验显示，MDA-MB-231/ miR-204组细胞穿过基质胶的细胞数目（40.67±1.53）少于MDA-MB-231/con（105.67±4.04）组细胞穿过基质胶的细胞数目（P < 0.05）（图 3A、B）。

3.

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各组MDA-MB-231细胞的侵袭能力

Overexpression of miR-204 inhibits the migration and invasion of breast cancer cells (Giemsa staining, scale bar=100 μm. Mean± SD, n=3, *P < 0.05 vs MDA-MB-231/con group).

2.4. HNRNPA2B1为miR-204的Hub基因

利用靶基因预测软件miRTarbase，miRWalk，TargetScan预测miR-204的靶基因，取交集得84个靶基因（图 4A）。利用此84个靶基因做PPI分析，利用Cytoscape软件的MCODE插件选取连接度超过4的靶基因MBNL1、KHDRBS1、TIAL1、HNRNPA2B1作为我们的Hub基因备选（图 4B）。Starbase网站和Kmplot数据库对HNRNPA2B1进行分析，结果显示，HNRNPA2B1在乳腺癌组织中表达和miR-204-5p的表达呈负相关，且差异具有显著性（图 4C）。与正常乳腺组织相比，HNRNPA2B1在乳腺癌中表达升高，且高表达水平的HNRNPA2B1与患者不良预后相关，且差异具有显著性（P < 0.05，图 4D、E）。

4.

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miR-204的Hub基因筛选

MBNL1, KHDRBS1, TIAL1, HNRNPA2B1 are Hub genes of miR-204. A: The venny plot of predicted genes; B: The Hub genes of targeted genes made by cytoscape; C: Co-Expression Analysis for hsa-miR-204-5p and HNRNPA2B1; D: The expression of HNRNPA2B1 in breast normal tissues and breast cancer tissues; E: The survival analysis of HNRNPA2B1 in breast cancer patients.

2.5. miR-204靶向调控HNRNPA2B1抑制乳腺癌的侵袭和迁移能力

HNRNPA2B1 mRNA的3'-UTR是miR-204的直接结合位点（P < 0.05，图 5A）。与对照组相比，过表达miR-204组细胞HNRNPA2B1表达水平的平均灰度值（0.50±0.05）比对照组HNRNPA2B1表达水平的平均灰度值（1.00±0.09）低，且差异有统计学意义（图 5B、C）。Transwell迁移实验结果显示，miR-204+HNRNPA2B1组细胞数（122.67±11.06）比miR-204+ NC组细胞穿至下室的细胞数（47.00±3.61）明显增多（P < 0.05）；侵袭实验显示，miR-204+HNRNPA2B1组穿过基质胶的细胞数（96.00+2.00）比miR-204+NC组细胞穿过基质胶的细胞数（34.67±2.08）明显增多（P < 0.05，图 5D）。

5.

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miR-204靶向调控HNRNPA2B1抑制乳腺癌的侵袭和迁移能力

miR-204 inhibits the migration and invasion ability of breast cancer by targeting the regulation of HNRNPA2B1. A: Luciferase activity of cells in different group was detected by luciferase experiment; B & C: The expression of HNRNPA2B1 in transfected cells. *P < 0.05 vs MDA-MB-231/con group; D: The migration and invasion ability in different transfected cells (Giemsa staining, scale bar= 100 μm, Mean±SD, n=3, *P < 0.05 vs miR-204+NC group).

3. 讨论

miRNA是一类21~23 nt的非编码小分子RNA，在多种肿瘤中异常表达，且在转移过程中发挥重要作用，如夏巍等发现miR-671-3p可靶向DEPTOR而抑制乳腺癌细胞的增殖与侵袭[19]；夏巍等[20]发现miR-152和miR-448靶向于Rictor可抑制结直肠癌细胞增殖。近来多项研究表明miRNA可通过不同方式调控肿瘤发生发展，其中一种重要的调控机制即miRNAs可影响mRNA的稳定性和转译，参与多细胞生物中基因表达的转录后调控，从而影响肿瘤的侵袭增殖能力[21-22]。因此，相较于mRNA，miRNA具有更大的潜力成为治疗乳腺癌的靶点[23-24]。

RNA结合蛋白（RBP）几乎参与了转录后调控层的所有步骤，决定了细胞中每个转录本的命运和功能，并确保细胞内环境的稳定。它们与其他蛋白质、编码RNA和非编码RNA建立高度动态的相互作用，形成称为核糖核蛋白复合物的功能单元[25-26]。作为一个RBP，HNRNPA2B1调节肿瘤发育相关RNA的剪接、稳定性和翻译[27-29]。先前研究揭示了HNRNPA2B1的下调可通过PI3K/AKT信号通路抑制宫颈癌细胞增殖、侵袭和细胞周期，从而引发细胞凋亡[30]。因此，HNRNPA2B1在癌症的发生、发展、基因表达和信号转导中起着直接的作用。

本研究通过GEO数据库中的乳腺癌患者相关数据集GSE44124、GSE38167发现miR-204在三阴性乳腺癌患者癌组织中表达降低，在转移性组织里miR-204表达同样降低；生存曲线分析显示低表达miR-204的患者具有更不良的预后。生物信息学数据显示miR-204可能作为抑癌基因在乳腺癌中发挥作用。RT-qPCR结果显示miR-204在乳腺癌细胞中低表达，过表达miR-204发现乳腺癌细胞的侵袭能力受抑制，以上结果证实miR-204在人乳腺癌发生发展过程中作为抑癌基因发挥作用。也有研究报道，miR-204在几种人类肿瘤中均有下调表达，miR-204能抑制膀胱癌细胞生长和转移，也能调节宫颈癌细胞的增殖，凋亡和自噬能力[31-32]，miR- 204-5p可靶向DSCAM-AS1抑制乳腺癌肿瘤生长[33]，miR-204也能可以靶向调控ZEB1抑制乳腺癌的上皮间质转化从而抑制乳腺癌的侵袭和转移[34]。结合我们的结果，miR-204可能作为一个重要的肿瘤抑制因子参与乳腺癌的进程。生物信息学在基础研究中发挥的作用越来越大，我们通过基因预测网站miRTarbase，miRWalk，Targetscan预测可能与miR-204结合的靶基因，三个网站预测的靶基因取交集得84个miR-204的靶基因。通过PPI分析和cytoscape软件的MCODE插件，我们选择这84个靶基因中蛋白与蛋白连接度大于4的作为我们的Hub基因，结果得MBNL1，KHDRBS1，TIAL1，HNRNPA2B1为miR-204的Hub基因。另外，我们发现HNRNPA2B1和miR-204-5p表达呈负相关，在乳腺癌组织中高表达且高表达HNRNPA2B1的乳腺癌患者预后不良，因此选取HNRNPA2B1进一步研究。在此基础上，为了进一步探讨所筛选出来的miR-204对HNRNPA2B1的调控是通过直接结合并作用于其3'- UTR之上，我们构建了野生型（WT）HNRNPA2B1和突变型（MUT）HNRNPA2B1载体，通过双荧光素酶实验证明miR-204可靶向结合HNRNPA2B1。Western blot结果证明，过表达miR-204后，HNRNPA2B1的表达量降低，证实miR- 204可调控HNRNPA2B1的表达，Transwell实验同样证实miR-204对乳腺癌细胞侵袭和迁移能力的抑制作用可被HNRNPA2B1逆转。这些结果表明miR-204可通过抑制HNRNPA2B1的表达从而抑制乳腺癌细胞侵袭转移，从而在乳腺癌中发挥抗肿瘤的作用。

综上所述，miR-204可通过靶向调节HNRNPA2B1抑制乳腺癌的侵袭和转移，有望作为乳腺癌早期诊断以及转移的参考指标，为乳腺癌的诊疗提供新思路。

Biography

张丽萍，在读硕士研究生，E-mail: zlp1317@163.com

Funding Statement

国家自然科学基金(81702932, 81402389, 81641111);山东省自然科学基金(ZR2019MH033);潍坊市科学技术发展计划(高校部分)(2018GX077);潍坊市科技局医学类项目(2019YX029);山东省研究生教育质量提升计划(SDYAL19155;SDYKC19157);山东省高等学校青创人才引育计划资助

Supported by National Natural Science Foundation of China (81702932, 81402389, 81641111)

Contributor Information

张 丽萍 (Liping ZHANG), Email: zlp1317@163.com.

李 洪利 (Hongli LI), Email: lihongli@wfmc.edu.cn.

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