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. 2021 Jul 20;41(7):1022–1029. [Article in Chinese] doi: 10.12122/j.issn.1673-4254.2021.07.08

miR-let-7c-5p通过靶向HMGA2抑制膀胱癌细胞侵袭和迁移

miR-let-7c-5p inhibits invasion and migration of bladder cancer cells by targeting HMGA2

Yue YAO 1, Chong ZHANG 1, Yanjun XIONG 1, Bing HAN 1, Xingfu GAO 1, Sheng WANG 1,*
PMCID: PMC8329690  PMID: 34308851

Abstract

Objective

To investigate whether miR-let-7c-5p inhibits invasion and migration of bladder cancer cells by regulating HMGA2.

Methods

We used bioinformatics methods to determine the key genes of miR-let-7c-5p. RT-qPCR and Western blotting were used to detect the expressions of miR-let-7c-5p mRNA and HMGA2 protein in bladder cancer and adjacent tissues. With human normal bladder SV-HUC-1 cells as the control, we detected the expression levels of miR-let-7c-5p mRNA and HMGA2 protein in bladder cancer cell lines T24, UM-UC-3 and 5637 with RT-qPCR and Western blotting. We observed the effects of miR-let-7c-5p upregulation (by transfection with a miR-let-7c-5p mimic), miR-let-7c-5p downregulation (using a miR-let-7c-5p inhibitor), and knockdown of both HMGA2 and miR-let-7c-5p on invasion, migration and expressions of epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin, N-cadherin, vimentin, and Snail) in UM-UC-3 cells. Dual luciferase assay, RT-qPCR and Western blotting were used to verify the targeting relationship between miR-let-7c-5p and HMGA2.

Results

HMGA2 was identified as one of the target genes of miR-let-7c-5p. Compared with the adjacent tissues, bladder cancer tissues showed a significantly decreased expression of miR-let-7c-5p and an increased expression of HMGA2 protein (P < 0.05). In UM-UC-3 cells, the expression of miR-let-7c-5p was significantly reduced and that of HMGA2 was significantly increased as compared with those in SV-HUC-1 cells (P=0.001). Up-regulating miR-let-7c-5p expression significantly lowered the invasion and migration abilities of UM-UC-3 cells, and down-regulating miR-let-7c-5p expression obviously promoted the invasion and migration of UM-UC-3 cells (P < 0.05). Knockdown of both miR-let-7c-5p and HMGA2 expression significantly lowered the invasion and migration (P < 0.05) and inhibited the expressions of EMT-related proteins of UM-UC-3 cells (P < 0.05).

Conclusion

miR-let-7c-5p inhibits EMT of bladder cancer UM-UC-3 cells by targeting HMGA2, thereby inhibiting the cell invasion and migration.

Keywords: bladder cancer, miR-let-7c-5p, HMGA2, epithelial-mesenchymal transition, invasion and migration

膀胱癌是最常见的泌尿系恶性肿瘤，是全球男性中第6大最常见癌症之一[1]。在过去几十年中已提出并采用了各种治疗策略，根治性膀胱切除术被认为是肌层浸润性膀胱癌的标准治疗方法[2]，浸润性低的膀胱癌患者根治性膀胱切除术后5年生存率为62% [3]。目前，膀胱癌治疗方法效果不佳。因此，探索新的靶点靶向治疗膀胱癌至关重要。

let-7 miRNA家族在癌细胞生物学过程中起着重要作用，被广泛认为是抑癌的miRNA，参与了抑癌途径调控，并在包括膀胱癌在内的许多类型的肿瘤组织中被下调[4-9]。miR-let-7c-5p属于let-7家族，虽然已有报道表明miR-let-7c-5p在多种癌细胞中发挥抑癌作用[10-15]，但miR-let-7c-5p在膀胱癌进展中的确切作用和机制尚不清楚。我们前期研究发现miR-let-7c-5p在膀胱癌组织中低表达，本实验旨在研究miR-let-7c-5p表达水平对膀胱癌细胞侵袭和迁移的影响，并以高迁移率族蛋白A2（HMGA2）为线索分析miR-let-7c-5p抑制膀胱癌细胞侵袭和迁移的分子机制，为膀胱癌的临床诊疗提供理论基础和实验依据。

1. 材料和方法

1.1. 实验材料、试剂

人正常膀胱细胞SV-HUC-1，膀胱癌细胞系UMUC-3，5637购自上海细胞库，膀胱癌细胞系T24由蚌医一附院郭圆圆课题组惠赠，DMEM培养基、胰蛋白酶（Hyclone）、胎牛血清（FBS）（浙江天杭生物科技），miRlet-7c-5pmimic和inhibitor试剂盒，HMGA2 mimic和inhibitor试剂盒，Lipofectamin2000（吉玛基因），Mateigel基质胶和双荧光素酶报告系统检测试剂盒（碧云天），RNA提取试剂盒，逆转录试剂盒，荧光定量PCR试剂盒，miR-let-7c-5p、HMGA2引物均（Thermo），HMGA2、E-cadherin、N-cadherin、vimentin、Snail、β-actin及兔/鼠二抗均（上海艾博抗）。

1.2. 细胞培养

人正常膀胱细胞SV-HUC-1和膀胱癌细胞系T24，UM-UC-3，5637细胞培养在含10%胎牛血清、100 mg/L链霉素、100 U/mL青霉素的DMEM培养基中，放置于37 ℃、5% CO2饱和湿度的细胞恒温培养箱中日常培养。

1.3. 标本来源

蚌埠医学院第一附属医院收集癌旁组织（距膀胱癌右后壁5 cm处）及膀胱癌组织例，所有患者术前均未接受过靶向、放化疗或内分泌等辅助治疗，所有患者均签署知情同意书，实验方案由蚌埠医学院第一附属医院伦理委员会审核批准。

1.4. 靶基因检测及验证

利用靶基因预测软件miRTarbasemiRTarBase（<a href="http://mirtarbase.mbc.nctu.edu.tw/php/index.php" target="_blank">http://mirtarbase.mbc.nctu.edu.tw/php/index.php</a>），miRWalk（<a href="http://mirwalk.umm.uni-heidelberg.de" target="_blank">http://mirwalk.umm.uni-heidelberg.de</a>），TargetScan（<a href="http://www.targetscan.org/vert_72/" target="_blank">http://www.targetscan.org/vert_72/</a>）预测miR-let-7c-5p的靶基因。

1.5. 细胞转染分组

取UM-UC-3细胞株接种于六孔板，待细胞融合度为整个六孔板的60%严格按照说明书，对UM-UC-3细胞中miR-let-7c-5p上调和下调，上调实验设模拟物阴性对照组（mimic NC）、miR-let-7c-5p模拟物组（mimicmiRlet-7c-5p），下调实验设阴性对照组（inhibitor NC）及miR-let-7c-5p抑制剂组（si-miR-let-7c-5p）；同时对UMUC-3细胞中miR-let-7c-5p和HMGA2下调，实验按照如下分组：si-miR-let-7c-5p+NC组、si-miR-let-7c-5p+ si-HMGA2组。在37 ℃、5% CO2细胞培养箱中转染8 h，换成10%胎牛血清的DMEM培养基继续培养24 h。

1.6. 双荧光素酶报告实验

取对数生长期的UM-UC-3细胞接种于6孔板中，实验分为HMGA2-WT + miRNA阴性对照组（NCWT）、HMGA2-WT+miR-let-7c-5p模拟物组（HMGA2-WT）、HMGA2-Mut+miRNA阴性对照组（NC-Mut）、HMGA2-Mut + miR-let-7c-5p模拟物组（HMGA2-Mut），将构建成功的pGL3-HMGA2-3'UTR-Wt和pGL3-HMGA2-3'UTR-Mut质粒转染至UM-UC-3细胞，转染24 h后应用双荧光素酶报告系统检测试剂盒检测HMGA2的荧光强度，具体操作按试剂盒说明书要求进行。分析检测结果：A活性倍数=（R/F）样品/（ R/F）对照，萤火虫荧光素酶用F表示，海肾荧光素酶用R表示。最后以萤火虫荧光素酶与海肾荧光素酶荧光活性的相对活性比值作为报告基因活性值（海肾荧光素酶荧光值作为内参）。

1.7. 总RNA提取及实时荧光定量PCR检测mRNA的相对表达量

使用TRizol试剂提取SV-HUC-1和膀胱癌细胞系T24，UM-UC-3，5637、各转染组细胞以及膀胱癌组织和癌旁组织中总RNA，使用cDNA反转录试剂盒合成互补DNA模板；进行实时定量PCR以检测细胞中mRNA的表达水平。U6作为内源对照F: 5'-CGCTTCGGCAGC ACATATAC-3' R: 5'-CAGGGGCCATGCTAATCTT-3'，miR-let-7c-5p F：5'-GCCAACGUUCGAUUUCUACC UCA-3'R: 5'-UUGGUAUGUUGGAUGAUGGAGU-3'，HMGA2 F: 5'-TCCCTCTAAAGCAGCTCAAAA-3' R: 5'-ACTTGTTGTGGCCATTTCCT-3'。RT-qPCR条件：95 ℃持续3 min，94℃持续15 s，60℃持续55 s，72℃持续30 s，进行34个循环，通过2-ΔΔCt方法计算mRNA相对表达量，实验重复3次。

1.8. Transwell检测上调和下调miR-let-7c-5p后UMUC-3细胞侵袭迁移能力

侵袭实验：细胞分组同1.4，每孔加入60 μL Materigel胶均匀覆盖Transwell小室底部，培养60 min使之呈半凝固态。分别取转染后的各组细胞，消化离心后，用无血清培养基重悬计数，每孔均含1×105细胞。于24孔板即外室中加入600 μL含10% FBS的DMEM培养基，每组均设3个复孔。于37 ℃、5% CO2、饱和湿度下培养48 h；取出小室，PBS清洗2次，4%多聚甲醛固定15 min，用1%结晶紫染液室温下染色10 min；PBS漂去多余染料，使用电子显微镜在200倍放大倍率下对粘附在孔下部膜上的细胞进行计数。侵袭抑制率（%）=[1-mimic miR-let-7c-5p（或si-miR-let-7c-5p）组侵袭细胞个数/NC组细胞侵袭个数]×100%，迁移实验：除无需铺基质胶外步骤同侵袭实验，迁移抑制率（%）=[1-mimic miR-let-7c-5p（或si-miR-let-7c-5p）组迁移细胞个数/NC组细胞迁移个数]×100%，实验重复3次。

1.9. Western blot法检测蛋白表达水平

提取组织和各组细胞蛋白，冰上裂解30 mim，12 000 r/min离心30 min，吸取上清液，每组样取60 μg蛋白进行SDS-PAGE电泳；蛋白转至聚偏氟乙烯（PVDF）膜；快速封闭液封闭15 min；4 ℃孵育一抗（HMGA2、E-cadherin、N-cadherin、vimentin、Snail、β-actin）过夜；PBS清洗3次，二抗室温下孵育2 h；ECL试剂盒暗室发光显影，凝胶成像系统获取图像。

1.10. 统计分析方法

实验数据采用SPSS21.0统计软件进行统计分析，实验数据均以均数±标准差表示，多组比较采用单因素方差分析及两因素方差分析，组间两两比较采用LSDt检验，P < 0.05认为差异具有统计学意义。

2. 结果

2.1. 生物信息学软件Target Scan预测miR-let-7c-5p与HMGA2靶向关系

miR-let-7c-5p在miRTarbase、Targetscan、miRWalk 3个在线软件中分别存在1019、726、2170个潜在靶基因，经过绘制Venny图并取其交集共有4个潜在的靶基因，HMGA2为其中之一（图 1）。

1.

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生物信息学软件预测miR-let-7c-5p与HMGA2靶向关系

Prediction of the targeting relationship between miR-let-7c-5p and HMGA2 using bioinformatics software.

2.2. 膀胱癌和癌旁组织中miR-let-7c-5p和HMGA2的相对表达量

RT-qPCR结果显示，与对照组相比，癌旁组织相比miR-let-7c-5pmRNA在膀胱癌组织中表达降低（P= 0.003，图 2A），Western blot结果显示膀胱癌组织中HMGA2蛋白水平表达增加（P=0.001，图 2B、C）。

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膀胱癌和癌旁组织中miR-let-7c-5p和HMGA2的相对表达量

Relative expression of miR-let-7c-5p and HMGA2 in bladder cancer and adjacent tissues. A: RT-qPCR was used to detect the relative expression of miR-le-7c-5p in bladder cancer and adjacent tissue; B: Western blotting for detecting the expression of HMGA2 protein (n=3, *P < 0.05, **P < 0.01 vs Normal group).

2.3. 膀胱癌细胞株中miR-let-7c-5p和HMGA2相对表达量

以人正常膀胱细胞SV-HUC-1作为对照，检测膀胱癌细胞系T24、UM-UC-3、5637细胞中miR-let-7c-5p mRNA相对表达量，qRT-PCR结果显示，与SV-HUC-1相比，miR-let-7c-5p mRNA在UM-UC-3细胞中表达显著降低（P=0.003，图 3A），HMGA2 mRNA在UM-UC-3细胞中表达显增加（P=0.001，图 3B），同时Western blot结果表明，HMGA2在UM-UC-3细胞中显著增加（P= 0.003，图 3C、D）。

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膀胱癌细胞株中miR-let-7c-5p和HMGA2相对表达量

Expression of miR-let-7c-5p and HMGA2 in bladder cancer cell lines. A: RT-qPCR for detecting the expression of miR-let-7c-5p. B: RT-qPCR for detecting HMGA2 mRNA expression. C, D: Western blotting for detecting HMGA2 protein expression (n=3, *P < 0.05, **P < 0.01 vs SV-HUC-1 group).

2.4. miR-let-7c-5p与HMGA2的靶向关系

与对照组相比，上调UM-UC-3细胞中miR-let-7c-5p表达后HMGA2蛋白的表达显著降低（P=0.03，图 4A~ 4C），而下调miR-let-7c-5p表达后HMGA2蛋白的表达显著增加（P=0.005，图 4A~C）。上调和下调UM-UC-3细胞中HMGA2表达后检测miR-let-7c-5p mRNA相对表达量，当上调和下调UM-UC-3细胞中HMGA2表达后miR-let-7c-5p mRNA表达均无明显变化（图 4D、E）。同时双荧光素酶报告基因实验显示，与对照组相比HMGA2-WT（野生型）相对荧光素酶活性较NC-WT组明显降低，miR-let-7c-5p能够有效抑制野生型HMGA2荧光素酶活性（P=0.01，图 4F），HMGA2-Mut（突变型）组相对荧光素酶活性与NC-Mut组相比无明显变化（图 4F）。

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miR-let-7c-5p与HMGA2的靶向关系

Targeting relationship between miR-let-7c-5p and HMGA2 A: RT-qPCR method for detecting miRlet-7c-5p expression in UM-UC-3 cells with up-regulation and down-regulation of miR-let-7c-5p. B, C: Western blotting for HMGA2 protein expression in UM-UC-3 cells with up-regulation and downregulation of miR-let-7c-5p. D: RT-qPCR for detecting the expression of HMGA2 mRNA in UM-UC-3 cells with up-regulation and down-regulation of HMGA2. E: RT-qPCR for detecting miR-let-7c-5p expression after up-regulation and down-regulation of HMGA2 in UM-UC-3 cells. F: Quantitative analysis of the relative luciferase activity (n=3, *P < 0.05, **P < 0.01 vs mimic NC/inhibitor NC/NC group).

2.5. miR-let-7c-5p对UM-UC-3细胞侵袭迁移的影响及其机制

Transwell结果表明，当上调miR-let-7c-5p表达，UM-UC-3细胞侵袭和迁移能力均显著下降（P=0.02，P=0.03，图 5A、B），下调miR-let-7c-5p表达，UM-UC-3细胞侵袭和迁移能力显著增加（P=0.02，P=0.04，图 5C、D）。Western blot结果显示，与对照组相比，上调miR-let-7c-5p表达，E-cadherin表达增加，N-cadherin，vimentin，Snail蛋白表达下降（P=0.04，P=0.04，P=0.02，P=0.01，图 5F），下调miR-let-7c-5p表达，E-cadherin表达下降，N-cadherin，vimentin，Snail蛋白表达增加（P=0.02，P= 0.03，P=0.01，P=0.04，图 5F）。

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miR-let-7c-5p对UM-UC-3细胞侵袭迁移的影响

Effect of modulation of miR-let-7c-5p expression on invasion and migration of UM-UC-3 cells. A, C: Transwell assay for assessing changes in UM-UC-3 cell invasion and migration; B, D: Data anlysis of A and C; E: RT-qPCR for detecting miR-let-7c-5p expression in UM-UC-3 cells with HMGA2 up-regulation and down-regulation. F-J: Western blotting for expressions of EMTrelated proteins in UM-UC-3 cells with up-regulation and down-regulation of miR-let-7c-5p (n=3, scale bar=100 μm, *P < 0.05, **P < 0.01 vs mimic NC/inhibitor NC group).

2.6. miR-let-7c-5p靶向调控HMGA2表达抑制UMUC-3细胞侵袭和迁移

Transwell侵袭和迁移结果均表明，UM-UC-3侵袭和迁移能力均较单独下调miR-let-7c-5p降低（P= 0.003，P=0.03，图 6A、B），Western blot结果显示，与对照组相比，当同时下调miR-let-7c-5p和HMGA2表达，Ecadherin表达增加，N-cadherin，vimentin，Snail蛋白表达下降（P=0.01，P=0.03，P=0.02，P=0.02，图 6D），与对照组相比，差异具有统计学意义（P < 0.05）。

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miR-let-7c-5p靶向HMGA2抑制UM-UC-3细胞侵袭

miR-let-7c-5p inhibits invasion and migration of UM-UC-3 cells by targeting HMGA2. A: Transwell assay for assessing invasion of UM-UC-3 cells with down-regulation of both miR-let-7c-5p and HMGA2 expression. B: Transwell migration assay for assessing migration of UM-UC-3 cells with down-regulation of both miR-let-7c-5p and HMGA2. C: Data anlysis of A and B. D-H: Western blotting for detecting the expressions of EMT-related proteins (n=3, *P < 0.05, **P < 0.01 vs si-miR-let-7c-5p+NC group).

3. 讨论

越来越多的证据表明，miRNA在膀胱癌中异常表达，并通过影响细胞周期，增殖，侵袭和转移，耐药性和凋亡抑制作用来促进或抑制膀胱癌的发展和进程[16-18]，其中，miR-let-7c-5p在多种癌组织中表达降低并可能作为其生物标志物，其在多种癌细胞中发挥抑癌作用[19, 20]，如通过CTHRC1/AKT/ERK途径抑制食管鳞癌细胞增殖和迁移，促进细胞凋亡[21]，通过PI3K/Akt/FoxO信号通路抑制肝癌细胞增殖，迁移和侵袭，并诱导G1期阻滞导致抑制肝癌细胞生长[22]，同时能够抑制人类宫颈癌细胞的增殖和迁移[23]。结合最新研究发现尿液中let-7c簇的表达可以作为评估高级别非肌层浸润性膀胱癌患者疾病进展风险的无创工具[24]，猜测miR-let-7c-5p可能作为一个重要的肿瘤抑制因子参与膀胱癌的进程。前期实验研究发现，与膀胱癌癌旁组织相比，miR-let-7c-5p在膀胱癌组织中低表达，当上调miR-let-7c-5p表达，UM-UC-3细胞侵袭和迁移能力均显著下降，下调miRlet-7c-5p表达，UM-UC-3细胞侵袭和迁移能力均显著增加，为了研究miR-let-7c-5p抑制膀胱癌侵袭和迁移的机制，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-let-7c-5p结合的靶基因，三个网站预测的靶基因取交集得4个miR-let-7c-5p的靶基因，发现HMGA2可能为miR-let-7c-5p的潜在靶基因且HMGA表达与miR-let-7c-5p表达呈负相关。

HMGA2（又称HMGI-C）基因属于高迁移率族（HMGA）基因家族，是一种结构型转录因子，通常在许多恶性肿瘤中过表达，癌症患者中HMGA2的过度表达与较高的转移风险和不良预后相关[25-27]。我们研究发现，与相邻癌旁组织相比，HMGA2在膀胱癌组织中高表达。通过RT-qPCR、Western blot和双荧光素酶实验均证明HMGA2是miR-let-7c-5p的靶基因，此外，在非肌肉浸润性膀胱癌中，HMGA2的高表达与较高的复发率和进展率相关[28]。miR-let-7c-5p作为一个潜在的抑癌基因能否调控HMGA2影响膀胱癌细胞侵袭和迁移，当同时下调miR-let-7c-5p和HMGA2表达后，UM-UC-3细胞侵袭和迁移均能力被显著抑制。

沉默HMGA2能够抑制癌细胞迁移、侵袭和上皮- 间质转化（EMT）[29-31]。EMT是抑制上皮特征并上调间充质基因表达而促进癌细胞侵袭和转移的过程，是基本的细胞生物学过程，在胚胎发生、伤口愈合和肿瘤转移的早期阶段中发挥关键作用。同时，EMT也与膀胱癌的恶性表型密切相关[32]。为了研究miR-let-7c-5p抑制UM-UC-3细胞侵袭和迁移是否与EMT相关，Western blot结果显示，单独上调miR-let-7c-5p表达，E-cadherin表达增加，N-cadherin，vimentin，Snail蛋白表达下降，下调miR-let-7c-5p表达，E-cadherin表达下降，N-cadherin，vimentin，Snail蛋白表达增加，表明上调miR-let-7c-5p表达能够抑制UM-UC-3细胞EMT，下调miR-let-7c-5p表达促进UM-UC-3细胞EMT，当同时下调miR-let-7c-5p和HMGA2表达，UM-UC-3细胞EMT同样被显著抑制。

综上所述，通过细胞学实验证实，miR-let-7c-5p通过靶向HMGA2抑制EMT进而抑制UM-UC-3细胞侵袭和迁移，这些发现有可能为膀胱癌的诊断和治疗提供新的思路。

Biography

姚越，硕士，E-mail: 568243114@qq.com

Funding Statement

安徽省自然科学基金（1808085MH293）；蚌埠医学院2020科研创新计划项目（Byycx20030）

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