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. 2020 Dec 20;40(12):1712–1719. [Article in Chinese] doi: 10.12122/j.issn.1673-4254.2020.12.03

MiR-4443通过抑制PEBP1的表达促进乳腺癌细胞的迁移和侵袭

MiR-4443 promotes migration and invasion of breast cancer cells by inhibiting PEBP1 expression

Jinyan WANG 1,2,2, Jinqiu WANG 3,2, Quan GU 4,2, Yan1 YANG 1, Yajun MA 1, Jing ZHU 1, Quanan ZHANG 1,*
PMCID: PMC7835697  PMID: 33380387

Abstract

Objective

To investigate the effect of miR-4443 expression on migration and invasion of breast cancer.

Methods

We examined the expression of miR-4443 in breast carcinoma in situ and paired adjacent tissues from 3 breast cancer patients with high-throughput sequencing and verified the results using TCGA database. We also detected miR-4443 expressions using real-time quantitative PCR (RT-qPCR) in low invasive and highly invasive breast cancer cells (MCF-7 and MDA-MB-231 cells, respectively). The changes in apoptosis, migration and invasion of MCF-7 and MDA-MB-231 cells after transfection with miR-4443 mimics, mimics-NC, miR-4443 inhibitor or inhibitor-NC were analyzed using flow cytometry, wound healing assay and Transwell invasion assay. The target gene of miR-4443 was predicted by bioinformatics software and validated by a dual luciferase reporter gene system. RT-qPCR and Western blotting were performed to detect the expression of recombinant human phosphatidyl ethanolamine binding protein 1 (PEBP1) in the transfected cells.

Results

The expression of miR-4443 was significantly higher in the breast cancer tissues than in the adjacent tissues (P < 0.01), and was significantly up-regulated in MDA-MB-231 cells as compared with MCF-7 cells (P < 0.01). Transfection with miR-4443 mimics or inhibitors did not obviously affect apoptosis rate of the breast cancer cells (P>0.05), but significantly enhanced or weakened the migration and invasion abilities of the cells, respectively (P < 0.01). Bioinformatic analysis identified PEBP1 as the target gene of miR-4443 with a close correlation with metastasis of breast cancer (P < 0.01), and the result was confirmed by double luciferase reporter gene assay. The mRNA and protein expression of PEBP1 were significantly lower in MDA-MB-231 cells than in MCF-7 cells (P < 0.01), and miR-4443 over-expression or knockdown significantly down-regulated or up-regulated PEBP1 expressions in the cells, respectively (P < 0.01).

Conclusion

MiR-4443 promotes the migration and invasion of breast cancer cells by inhibiting the expression of PEBP1, suggesting the possibility of suppressing miR-4443 expression as a potential therapeutic strategy for breast cancer.

Keywords: microRNAs, phosphatidylethanolamine binding protein 1, neoplasm metastasis, breast neoplasms

乳腺癌不仅是女性最常见的肿瘤，也是全球女性癌症死亡的首要原因[1]，近年来乳腺癌的发病率和死亡率正在迅速攀升[2-3]。虽然早期乳腺癌在手术和化疗后预后良好，但约90%的乳腺癌患者的死亡是由于原发肿瘤的复发和远处转移[4]。因此，识别新的与预后和治疗相关的生物标志物已成为一个紧迫的问题，这可能会有助于提高患者的生存率。

微小RNA（miRNAs）通过与信使RNA（mRNA）的3'非翻译区（3'UTR）结合，在转录和转录后水平上调控基因表达[5]。近十年来，越来越多的证据表明，miRNAs参与调控细胞凋亡、增殖、血管生成、耐药、迁移和侵袭等多种生物代谢过程[6]。许多miRNAs已被证明可以调节肿瘤的侵袭和转移[7-8]，如miR-29a[9]，miR-34a[10]和miR-105[11]。已有研究表明miR-4443在转移性和浆液性卵巢癌组织中的表达下调，并且低表达的miR-4443有助于肿瘤的转移和发生[12]。miR-4443可通过靶向肌醇多磷酸4-磷酸酶I型基因（INPP4A），激活JAK2/ STAT3信号通路，从而促进非小细胞肺癌对表柔比星的耐药[13]。同样，在转移性结肠癌中miR-4443可抑制肿瘤的侵袭转移[14]。miR-4443和长链非编码RNA的相互作用与各种肿瘤的发生发展密切相关，包括骨肉瘤[15]，头颈部鳞状细胞癌[16]，肝癌[17]和胶质母细胞瘤[18]等。但是，目前关于miR-4443在乳腺癌中的作用机制的报导较为少见，2016年有研究首次报导miR-4443可能诱导乳腺癌的化疗耐药[19]。本研究团队后续进一步探索miR-4443与乳腺癌肝转移的关系，发现高侵袭性乳腺癌细胞分泌外泌体miR-4443传递到原发灶及肝转移灶的基质细胞，抑制组织抑制金属蛋白酶2（TIMP2）的表达，并进一步激活基质金属蛋白酶（MMPs），从而破坏了抗转移的天然屏障，因此乳腺癌来源的外泌体miR- 4443诱导的微环境中TIMP2的丢失极大地促进了乳腺癌的肝转移[20]。然而乳腺癌发生肝转移几率远低于肺、脑、骨等器官的转移[21-22]。研究发现肿瘤中异常表达的miRNAs与肿瘤的器官特异性转移密切相关，比如肿瘤分泌的miR- 940通过靶向骨转移微环境中的ARHGAP1基因诱导成骨[23]，外泌体miRNAs诱导的微环境中PTEN的缺失可特异性促进脑转移灶的生长[24]。因此，本研究使用高侵袭性的乳腺癌MDA-MB-231细胞株和低侵袭性的乳腺癌MCF-7细胞株来探索miR-4443在乳腺癌凋亡、迁移、侵袭中的调节作用，探索miR-4443参与乳腺癌侵袭转移的潜在靶点，为后续进一步研究miR-4443与乳腺癌器官特异性转移相关的信号通路提供研究基础。

1. 资料和方法

1.1. 样本收集和高通量鉴定

收集江苏省肿瘤医院收治的3例乳腺癌患者的乳腺癌组织和配对的癌旁组织，肿瘤组织和癌旁组织均经组织病理学证实。所有患者均提供书面同意书，经南京医科大学肿瘤研究所伦理委员会批准（批准号：2016- 230）。手术时立即将组织冷冻在液氮中，并在-80 ℃保存。RNA的提取在RT-qPCR方法中进行描述。然后对总RNA进行高通量测序鉴定。

1.2. 癌症基因组图谱数据集

从癌症基因组图谱数据库（TCGA，2017年11月）的门户下载了1066个乳腺癌组织和104个正常乳腺组织的miRNA数据[25]。

1.3. 细胞培养

本研究使用的人乳腺癌细胞株MCF-7和MDAMB-231购自中国科学院细胞库。所有细胞株在含10%胎牛血清(FBS；赛默飞世尔科技有限公司)的DMEM培养基（江苏凯基生物技术股份有限公司)中培养，在37 ℃和5% CO2的温箱中孵育。

1.4. 实时定量PCR

如前所述，使用茎环法检测miR-4443的表达[26]并且采用U6小核RNA（snRNA）作为内参。在磷脂酰乙醇胺结合蛋白1（PEBP1）的检测中，以β-actin为内参。本研究所用引物见表 1。

1.

miR-4443, PEBP1, U6 and β-actin的引物序列

Primer sequences of miR-4443, PEBP1, U6 and β-actin

Genes Primer sequence
RT: Reverse transcription.
MiR-4443
  RT primer 5'-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACAAAACCC-3'
  Forward 5'-GGAAGGTTGGAGGCGTGG-3'
  Reserve 5'-CAGTGCGTGTCGTGGAGT-3'
U6
  RT primer 5'-AACGCTTCACGAATTTGCGT-3'
  Forward 5'-CTCGCTTCGGCAGCACA-3'
  Reserve 5'-AACGCTTCACGAATTTGCGT-3'
PEBP1
  Forward 5'-CGCCCACCCAGGTTAAGAAT-3'
  Reserve 5'-GGTCTGTCAGGACCAAGGTG-3'
β-actin
  Forward 5'-CACCTTCTACAATGAGCTGCGTGTG-3'
  Reserve 5'-ATAGCACAGCCTGGATAGCAACGTAC-3'
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当细胞融合度达80%~90%时收集细胞。使用RNA提取试剂盒（天根生化科技有限公司）提取总RNA，使用逆转录试剂盒（赛默飞世尔科技有限公司）进行逆转录。采用Nanodrop 2000分光光度法（赛默飞世尔科技有限公司）在260、280 nm处（260/280 nm，1.8-2.0）的紫外吸收度和甲醛变性凝胶电泳对RNA的浓度和质量进行评价。

采用染料法（SYBR Green PCR Master Mix，罗氏公司）在LightCycler®480实时定量PCR系统（罗氏公司）上进行RT-qPCR分析。每个基因的Ct值由内参进行标准化控制，并使用2-ΔΔCt方法计算基因表达水平。每项实验均采用阴性对照（无核酸酶水或无逆转录提取的RNA）。所有样本均独立重复3次，数据以均数±标准差表示。

1.5. 细胞转染

人源的miR-4443模拟物（mimics）及模拟物的阴性对照（mimics-NC），人源的miR-4443抑制物及抑制剂的阴性对照（inhibitors-NC）是由锐博生物科技有限公司合成。转染方法按照既往描述的进行[27]。将miR-4443模拟物及模拟物的阴性对照加入至含1×106乳腺癌细胞的100 μL无血清无抗生素的培养基中，使模拟物的最终浓度控制在50 nmol/L，抑制物的最终浓度控制在100 nmol/L。然后将混合物放入至脉冲比色杯中，使用Super Electroporator NEPA 21 Type II（日本NEPAGENE）电转（poring pulse: pulse voltage, 125 V; pulse length, 5 ms; pulse interval, 50 ms; pulse number, 2）。空白对照组是不含模拟物、抑制物或其阴性对照的接受电转染的细胞。细胞接受电转染后以5×105细胞/孔的密度接种于6孔板中，用2 mL预热的不含抗生素的完全培养基中培养24 h。

1.6. 凋亡实验

电转染后的MCF-7细胞以5×105细胞/孔的密度接种于6孔板中，用2 mL预热的不含抗生素的完全培养基培养24 h。使用不含EDTA的胰酶（Gibco）消化细胞，PBS清洗2遍后，使用5 μL的APC Annexin V和5 μL的7-AAD（美国BD）暗室中孵育15 min。最后，将400 μL的1×结合缓冲液加入到每个流式管中并使用流式细胞分析仪（美国BD）进行分析。

1.7. 划痕实验

将转染后的乳腺癌细胞以5×105细胞/孔的密度接种于6孔板中，使用2 mL完全培养液在37 ℃温箱中孵育24 h。当细胞融合达到90%时，使用200 μL的枪头在培养板上划出笔直均一的划痕，磷酸缓冲盐水（PBS）冲洗2次，使悬浮细胞脱落。然后，在无血清的培养基中培养细胞。转染48 h后验证MDA-MB-231细胞的迁移程度，转染72 h观察MCF-7细胞的迁移程度，并在显微镜（日本奥林巴斯）下观察和拍照。

1.8. Transwell侵袭实验

使用无抗生素培养基以1:9的浓度稀释基质（美国康宁），稀释后的基质均匀铺于Transwell小室底部（小室平均分布有8.0 μm的孔隙，美国康宁）。37 ℃孵化2~ 3 h后小室底部形成基质膜，移除上清液后，将2×104乳腺癌细胞置于小室上层不含血清的培养基中孵育。小室的下层添加500 μL含25%胎牛血清的完全培养基。MDA-MB-231细胞和MCF-7细胞分别在37.0 ℃孵育24 h和48 h后用棉签拭子擦除小室上层未侵袭的细胞，4%福尔马林固定膜下未侵袭的细胞，0.05%结晶紫染色。最后，将穿透细胞在3个随机选取的区域进行人工计数，并在倒置显微镜下拍照。

1.9. miR-4443靶基因的预测

使用TargetScan（<a href="http://www.targetscan.org" target="_blank">http://www.targetscan.org</a>）<sup>[<xref ref-type="bibr" rid="b28">28</xref>]</sup>预测miR-4443的靶基因。为探索与转移相关的靶基因中，我们将预测基因和“转移”一词在GenClip 2.0（<a href="http://ci.smu.edu.cn/GenCLiP2" target="_blank">http://ci.smu.edu.cn/GenCLiP2</a>；2016年4月5日）<sup>[<xref ref-type="bibr" rid="b29">29</xref>-<xref ref-type="bibr" rid="b30">30</xref>]</sup>中进行搜索，其中“转移”和至少一个基因需同时出现在一个句子中。

1.10. 双荧光素酶报告基因实验

使用pmiR-RB-REPORTTM vector（锐博生物科技有限公司）构建含有PEBP1野生型3'UTR（p-Luc-WT）或PEBP1突变型3'UTR（p-Luc-MT）的质粒。然后将构建的质粒（p-Luc-WT或p-Luc-MT）和miR-4443 mimics（或mimics-NC）共转染MCF-7细胞。使用双荧光素酶测定系统测定荧光素酶活性，并使用海肾荧光进行标化。

1.11. Western blotting

采用RIPA缓冲液（Biouniquer Technology）在冰上混合细胞30 min，提取总蛋白后4 ℃下14 000×g离心15 min以去除细胞碎片。用Nanodrop 2000分光光度仪（赛默飞世尔科技有限公司）检测蛋白浓度和纯度。将样本与十二烷基硫酸钠聚丙烯酰胺凝胶（SDS-PAGE）缓冲液（碧云天生物技术有限公司）混合，煮沸5 min，冷却后将等量的蛋白质进行电泳，使蛋白质转移至聚偏二氟乙烯膜（PVDF；Sigma）。在含5%脱脂牛奶的0.05% Tween20/TBS（TBST）中封闭1 h后使用PEBP1（Abcam；1:1000）和β-actin（Abcam；1:6000）4 ℃下孵育过夜，然后使用二抗室温孵育1 h。二抗是山羊抗兔（1: 2000，Abcam）和山羊抗鼠（1:4000，Abcam）。用TBST洗涤3次后，用ECL试剂盒（碧云天生物技术有限公司）观察结合蛋白，通过暗室曝光获取图像。

1.12. 数据分析

采用R软件3.3.2进行统计分析。所有实验，包括侵袭实验、RT-qPCR、双荧光素酶报告基因实验、免疫印迹实验、凋亡实验，至少独立重复3次。定量数据以均数±标准差表示，采用非配对t检验比较实验组和对照组差异，P < 0.05为差异有统计学意义。

为了分析TCGA miRNA数据，首先对miRNA原始数据进行归一化，并在R软件中使用DESeq2[19]计算差异miRNA表达量。

2. 结果

2.1. 人乳腺癌组织中miR-4443的表达

在乳腺癌组织中，miR-4443的表达高于癌旁组织（P < 0.01，图 1A）。利用TCGA数据库进一步验证了miR-4443在体内的表达水平，结果显示与正常乳腺组织相比，miR-4443在乳腺癌组织中的表达水平更高（P < 0.01，图 1B）。

1.

1

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不同乳腺癌组织及细胞中miR-4443的表达水平

Quantification of miR-4443 expressions in different breast cancer tissues and cells. A: Quantification of miR-4443 in human breast cancer tissues and paired adjacent tissues through high-throughput sequencing (Clinical samples, n=3); B: Quantification of miR-4443 in breast cancer tissues and normal breast tissues (TCGA database); C: Quantification of miR-4443 in MDA-MB-231 and MCF-7 cell lines; D: Quantification of miR-4443 in MCF-7 cells transfected with miR-4443 mimics in comparison with blank control and mimics-NC; E: Quantification of miR-4443 in MDA-MB-231 cells transfected with miR-4443 inhibitors in comparison with blank control and inhibitors-NC. Data are presented as Mean±SD from 3 independent experiments. ***P < 0.001.

2.2. miR-4443在人乳腺癌细胞株中的表达

使用RT-qPCR检测miR-4443在高侵袭性乳腺癌细胞MDA-MB-231和低侵袭性乳腺癌细胞MCF-7中的表达，结果显示，miR-4443在MDA-MB-231细胞中的表达是MCF-7细胞的3倍左右（P < 0.01，图 1C）。与转染阴性对照的细胞相比，转染miR-4443 mimics的细胞中miR-4443的上调幅度超过500倍（P < 0.01，图 1D），转染miR-4443 inhibitors的MDA-MB-231细胞的miR- 4443表达水平是阴性对照的五分之二（P < 0.01，图 1E）。

2.3. miR-4443对乳腺癌细胞凋亡的影响

研究结果表明，转染miR-4443 mimics的MCF-7细胞的凋亡率与转染阴性对照和空白对照细胞的凋亡率差异无统计学意义（P>0.05，图 2A）；转染了miR- 4443 inhibitors的MDA-MB-231细胞的凋亡率与转染阴性对照和空白对照细胞的凋亡率差异无统计学意义（P>0.05，图 2B）。

2.

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流式细胞术检测不同miR-4443表达水平的MCF-7和MDA-MB-231细胞的凋亡率

Flow cytometric analysis of apoptosis of MCF-7 and MDA-MB-231 cells with miR-4443 overexpression or knockdown. A: The apoptotic rate of MCF-7 cells transfected with miR-4443 mimics was similar to that in cells transfected with mimics-NC and blank controls. B: The apoptotic rate of MDA-MB-231 cells transfected with miR-4443 inhibitors was similar to that in cells transfected with inhibitors-NC and blank controls. Bars represent Mean±SD from at least 3 independent experiments.

2.4. miR-4443对乳腺癌细胞迁移和侵袭的影响

划痕实验显示，转染miR-4443 inhibitors的MDAMB-231细胞与转染inhibitors-NC的对照组相比，其迁移能力减弱（P < 0.01，图 3A）；转染miR-4443 mimics的MCF-7细胞与转染miR-4443 mimics-NC的对照组相比，其迁移能力增强（P < 0.01，图 3B）。同样，transwell侵袭实验证实，转染miR-4443 inhibitors的MDA-MB-231细胞与转染inhibitors-NC的对照组相比，其侵袭能力减弱（P < 0.01，图 3C）；转染miR-4443 mimics的MCF-7细胞与转染miR-4443 mimics-NC的对照组相比，其侵袭能力增强（P < 0.01，图 3D）。

3.

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划痕实验及Transwell侵袭实验检测miR-4443对乳腺癌细胞株迁移和侵袭的影响

Effect of miR-4443 overexpression or knockdown on migration and invasion of breast cancer cell lines. A: Wound healing assay in MDA-MB-231 cells transfected with miR-4443 inhibitors in comparison with inhibitors-NC and blank control; B: Wound healing assay in MCF-7 cells transfected with miR-4443 mimics in comparison with mimics-NC and blank control; C: Cell invasion assay in MDA-MB-231 cells transfected with miR-4443 inhibitors in comparison with inhibitors-NC and blank control (×40). D: Cell invasion assay in MCF-7 cells transfected with miR-4443 mimics in comparison with mimics-NC and blank control (×40). *P < 0.05.

2.5. miR-4443靶基因的预测与验证

从TargetScan中获得了4465个miR-4443的潜在靶基因，通过在GenClip 2.0中同时搜索预测基因和“转移”这个词，显示PEBP1是最常见的与“转移”相关的靶基因（表 1）。MCF-7细胞中双荧光素酶报告基因检测结果显示，miR-4443对PEBP1野生型3'UTR报告基因的荧光素酶活性有抑制作用，而对PEBP1突变型3'UTR则无抑制作用（图 4A）。

4.

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通过RT-qPCR和Western blot检测MCF-7和MDA-MB-231细胞中PEBP1的表达水平

RT-qPCR and Western blot analysis of PEBP1 expressions in MCF-7 and MDA-MB-231 cells. A: Luciferase assay of MCF-7 cells co-transfected with miR-4443 mimics and plasmids (p-Luc-WT: Plasmids containing wild-type 3' UTR of PEBP1; p-Luc-MT: Plasmids containing mutant-type 3' UTR of PEBP1). B, C: RT-qPCR and Western blotting results of PEBP1 expression in MCF-7 cells transfected with miR-4443 mimics and mimics-NC. D: RT-qPCR and Western blotting results of PEBP1 expression in MDA-MB-231 cells transfected with miR-4443 inhibitors and inhibitors-NC. *P < 0.05.

2.6. PEBP1和miR-4443与乳腺癌细胞转移的关系

RT- qPCR和Western blot实验结果显示，MDAMB-231细胞中PEBP1的mRNA表达量约是MCF-7细胞的五分之一（P < 0.01，图 4B）。转染miR-4443 mimics的MCF-7细胞的PEBP1 mRNA及蛋白表达水平比转染mimics-NC的细胞低（P < 0.01，图 4C）；转染miR-4443 inhibitors的MDA-MB-231细胞的PEBP1 mRNA及蛋白表达水平比转染inhibitors-NC的细胞高（P < 0.01，图 4D）。

2.

通过在GenClip 2.0中共同搜索miR-4443的靶基因和“转移”得到前20位基因

Top 20 genes by co-searching GenClip 2.0 using target genes of miR-4443 and the term "metastasis"

Gene1 Hit2 Total3 Hit/Total
1The listed genes are those found in the sentences identified by the term "metastasis"; 2The number is the count of articles mentioning the corre-sponding gene and "metastasis" in one sentence; 3The number is the count of articles mentioning the corresponding gene.
PEBP1 127 676 0.188
FLT4 148 1665 0.089
CDH1 1084 14418 0.075
MMP2 926 16803 0.055
CCR7 121 2631 0.046
PTK2 222 5136 0.043
HGF 338 8386 0.040
TIMP2 153 4306 0.036
CDH2 120 3457 0.035
CAV1 150 4325 0.035
HIF1A 268 8021 0.033
EGFR 832 36 142 0.023
PGR 278 13 480 0.021
CTSB 126 8147 0.015
TG 116 8779 0.013
AKT1 516 44 234 0.012
EGF 152 19 511 0.008
BCL2 249 40 622 0.006
MAPK1 291 53 520 0.005
MAPK3 107 23 983 0.004
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3. 讨论

本研究首次在乳腺癌细胞水平及组织标本水平验证miR-4443的表达，通过高通量测序技术分析发现miR-4443在乳腺癌组织中的表达远高于癌旁组织，并且TCGA数据库亦进一步确认miR-4443在乳腺癌组织中的表达水平更高；进一步在细胞水平验证发现，与低侵袭性乳腺癌细胞MCF-7相比，miR-4443在高侵袭性乳腺癌细胞MDA-MB-231中上调。综上结果表明，miR-4443可能是一种潜在的致癌miRNA，可促进乳腺癌的侵袭和转移。为证实这一观点，本研究通过划痕实验和Transwell侵袭实验证实了miR-4443的高表达促进乳腺癌细胞的迁移和侵袭能力。为进一步了解miR- 4443促进乳腺癌恶性进展的分子机制，本研究首次通过生物信息学分析和双荧光素酶报告基因实验确认PEBP1是miR-4443的靶点。近年来，PEBP1(又称RKIP，即Raf激酶抑制蛋白)被认为是一种新的、与临床相关的转移抑制基因[31-32]。PEBP1参与了raf-1介导的磷酸化和MEK的激活，调控多种重要的细胞过程，包括增殖、分化、存活和细胞死亡[33]。PEBP1在各种侵袭性肿瘤如乳腺癌、贲门腺癌中的表达通常是下调的[34-35]。此外，据报导PEBP1通过调控基质金属肽酶13、趋化因子配体5等来抑制乳腺癌的侵袭和转移[36-37]。基于上述研究结果，本研究进一步通过RT-qPCR和Western Blot证实miR-4443可以抑制乳腺癌细胞内源性PEBP1的表达，这可能与乳腺癌侵袭转移密切相关。既往本团队已发现乳腺癌来源的外泌体miR-4443诱导的微环境中TIMP2的丢失极大地促进了乳腺癌的肝转移[20]。然而，本研究尚未涉及miR-4443与乳腺癌侵袭转移关系的体内研究，后续应进一步体内探讨miR-4443是否与乳腺癌脑转移、骨转移、肺转移等特异性器官转移相关。并且乳腺癌中miR-4443上调的机制亦尚无报导，本研究首次在乳腺癌细胞及组织水平验证发现miR-4443表达上调，后续可进一步从m6A甲基化、转录因子、长链非编码RNA、circRNA等角度探讨miR-4443上调的具体机制。同时，还需进一步验证miR-4443的其他潜在靶基因，确认抑制乳腺癌恶性进展的关键通路。因此，了解miR-4443在乳腺癌恶性进展中的作用，不仅将增强对乳腺癌生物学知识的了解，还可能证实miR-4443作为乳腺癌治疗和预后指标的新靶点。

综上所述，本研究的重要发现是乳腺癌中上调的miR-4443通过抑制内源性PEBP1的表达来增加乳腺癌的侵袭转移能力，miR-4443也许是作为致癌性miRNA在乳腺癌的恶性进展中发挥作用。

Biographies

王金焱，硕士，住院医师，E-mail: wjy_doctor@163.com

王晋秋，硕士，主任医师，E-mail: jinqiuwang_doctor@163.com

顾诠，硕士，住院医师，E-mail: 13905152166@139.com

Funding Statement

国家自然科学基金（81602551）；江苏省自然科学基金（BK20161110），江苏省研究生科研与实践创新计划（SJCX17_0387）；南京医科大学科技发展基金一般项目（NMUB2019235）；江苏卫生健康职业学院校级科研项目（JKC201948）

Supported by National Natural Science Foundation of China (81602551)

Contributor Information

王 金焱 (Jinyan WANG), Email: wjy_doctor@163.com.

王 晋秋 (Jinqiu WANG), Email: jinqiuwang_doctor@163.com.

顾 诠 (Quan GU), Email: 13905152166@139.com.

张 全安 (Quanan ZHANG), Email: quananzhang_doctor@163.com.

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