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. 2020 Dec 20;40(12):1720–1725. [Article in Chinese] doi: 10.12122/j.issn.1673-4254.2020.12.04

ALKBH5通过抑制上皮-间质转化降低人滋养细胞的迁移和侵袭能力

ALKBH5 suppresses migration and invasion of human trophoblast cells by inhibiting epithelial-mesenchymal transition

Jianping HE 1,2, Xiaojuan LI 2,3,2, Mengxin LÜ 1, Jue WANG 4, Jian TANG 1, Shengjun LUO 1, Yuan QIAN 1,2,4,5,6,7,*
PMCID: PMC7835694  PMID: 33380386

Abstract

Objective

To investigate the effects of ALKBH5 on migration, invasion and epithelial-mesenchymal transition (EMT) of human trophoblast cells.

Methods

The expression plasmid of ALKBH5 or a negative control plasmid (ALKBH5-NC) was transfected in human trophoblast HTR-8 /SVneo cells, and the expressions of ALKBH5 mRNA and protein were detected by qRT-PCR and Western blotting. Transwell assay was used to assess the changes in migration and invasion abilities of the trophoblast cells after the transfection. Western blotting was performed to detect the expressions of EMT-related proteins in the cells including vimentin, fibronectin, E-cadherin, N-cadherin, MMP9 and MMP2.

Results

ALKBH5 mRNA and protein expressions were significantly higher in ALKBH5 group than in the control group (P < 0.05). Over-expression of ALKBH5 significantly attenuated migration and invasion abilities of HTR-8/Svneo cells (P < 0.05). Compared with the control cells, the cells overexpressing ALKBH5 showed an up-regulated expression of E-cadherin and down-regulated expressions of vimentin, fibronectin, N-cadherin, MMP9 and MMP2 (P < 0.05).

Conclusion

ALKBH5 is involved in the pathogenesis of preeclampsia by inhibiting EMT of trophoblast cells and hence reducing their migration and invasion abilities.

Keywords: ALKBH5, preeclampsia, epithelial-mesenchymal transition, migration, invasion

子痫前期（PE）是妊娠中晚期常见疾病，主要表现为血压升高、蛋白尿、血小板减少等，重症者可引起全身多系统损害甚至导致母儿死亡[1]。研究认为子痫前期病理过程一旦开始就很难被逆转和缓解，治疗子痫前期比预防子痫前期更加困难[2]。目前研究表明多参数联合预测子痫前期效果虽高于单一指标预测，但特异性有限未能广泛用于临床[3]，故发掘有效的预测标志物对于子痫前期的预防至关重要[4]。

目前研究认为子痫前期病理过程可分为两个阶段，其中第一阶段滋养细胞功能障碍、胎盘血管重铸不佳是引起后续病理进展的主要原因[5-6]。胎盘形成过程中滋养细胞正常分化至关重要，此过程与蜕膜微环境及各种调控因子密切相关[7]。滋养细胞功能与血管生成因子、免疫调节因子、EMT相关蛋白等多种因子密切相关[8-10]，母胎界面微环境中的各种因子的表达不仅受细胞内mRNA水平的影响还可受转录后调控方式的影响[11-12]。目前已有研究发现习惯性流产患者绒毛组织中N6-腺嘌呤（m6A）水平显著降低，而去甲基酶ALKBH5水平显著升高，且m6A修饰对于滋养细胞的侵袭功能至关重要[13]。但m6A转录后调控方式是否参与子痫前期发病机制尚不明确。

ALKBH同源蛋白5（ALKBH5）于2013年作为第二个哺乳动物m6A去甲基酶由何川等人首次报导，通过对mRNA的m6A去甲基化来调控mRNA出核、合成、剪切[14-15]。目前研究报导ALKBH5可通过调控m6A影响精子凋亡及减数分裂中期的精母细胞，从而参与精子形成与发育等重要生理过程[14, 16]。同时还有研究表明ALKBH5可通过调控mRNA降解参与多种疾病的发生。在乳腺癌干细胞中ALKBH5可增加NANOG mRNA的稳定性提高癌细胞在缺氧微环境中的生存能力[17]。在胶质母细胞瘤中ALKBH5可提高FOXM1表达水平促进恶性胶质瘤干细胞的增殖[18]。表明ALKBH5在调控细胞生命活动及基因表达方面发挥重要作用，但ALKBH5是否参与子痫前期的发生有待进一步探索。本研究将ALKBH5过表达及抑制载体转染人滋养细胞HTR-8/SVneo后，检测滋养细胞迁移、侵袭功能变化，及EMT相关蛋白的表达情况。以探讨ALKBH5与子痫前期发病机制的关系，ALKBH5通过何种机制参与子痫前期的发生，并为探索子痫前期新的预测标志物及潜在药物靶点提供依据。

1. 材料和方法

1.1. 细胞株及主要试剂

人胎盘滋养细胞株HTR-8/SVneo购自BioVector质粒载体菌种细胞基因保藏中心，RPMI 1640培养基、胎牛血清（BI）。LipofectamineTM 2000（Invitrogen）、TRIzol(Sigma)、RIPA细胞裂解液（索莱宝），Transwell小室（Costar）、Material基质胶（Corning），ALKBH5抗体、Fibronectin抗体、N-cadherin抗体、E-cadherin抗体、MMP9抗体、MMP2抗体、Vimentin抗体、GAPDH抗体（abcam），Mouse二抗（Sigma）、Rabbit二抗（Sigma）、PVDF膜（Millipore）。qRT-PCR试剂盒（上海吉玛）。ALKBH5过表达、抑制及阴性对照质粒均由上海吉玛制药技术有限公司合成。

1.2. 方法

1.2.1. 实验分组及细胞转染

实验共分为4组：未转染Control组、阴性对照NC组、ALKBH5组及shALKBH5组。取P3-P7代对数生长期细胞，常规胰酶消化后制备成单细胞悬液，计数后按3×105/孔接种到6孔板中，待细胞密度到达70%~80%时按照LipofectamineTM 2000说明书进行转染，5 h后换液，48 h收集细胞，并用qRT-PCR、WB检测细胞中ALKBH5 mRNA及蛋白表达水平验证过表达效率。

1.2.2. 实时荧光定量PCR（qRT-PCR）法检测ALKBH5表达情况

转染后细胞培养48 h用1mL TRIzol收集细胞，提取RNA，通过qRT-PCR实验检测ALKBH5表达情况。ALKBH5上游引物5'-TCAAGCCTATTCGGG TGTCG-3'，下游引物5'-AGCAGCATATCCACTGAG CA-3'；GAPDH上游引物5'-CATGTTCCAATATGATT CCACCCA-3'，下游引物5'-CCTGGAAGATGGTGAT GGGATT-3'。反应条件：95 ℃ 10 min、95 ℃ 15 s、60 ℃表s，合计45个循环。每个实验重复3次。以GAPDH为内参采用2-△△CT法进行数据的相对定量分析。

1.2.3. 蛋白免疫印迹实验（WB）

转染后细胞培养48 h用RIPA细胞裂解液收集细胞，提取蛋白。定量后配制上样体系，100 ℃煮沸10 min，10% SDS-PAGE凝胶分离蛋白，转移至PVDF膜上，5%脱脂牛奶4 ℃封闭过夜后加入一抗（ALKBH5抗体、Vimentin抗体、Fibronectin抗体、N-cadherin抗体、E-cadherin抗体、MMP9抗体、MMP2抗体、GAPDH抗体，稀释比例均为1:1000）4 ℃过夜，TBST洗膜后加入二抗（1:10 000），室温孵育2 h。ECL化学发光试剂显影，化学发光成像系统拍照。

1.2.4. Transwell实验

迁移实验中，细胞处理48 h后，0.25%胰酶消化收集细胞，1×PBS洗2次，用无血清1640重悬细胞并计数，将5×104细胞种到小室上室；侵袭实验中，Transwell小室上层提前加入100 μL无血清1640与基质胶混合液并于37 ℃放置1 h使其成胶，将105细胞种到小室上室中。向Transwell小室下室中加入500 μL含10%胎牛血清的RPMI 1640培养基，37 ℃培养箱培养24 h后，用棉签擦拭掉上室中的细胞，1×PBS洗小室3次，甲醇固定20 min，置0.1%结晶紫溶液中染色10 min后，倒置显微镜下随机选取5个视野，200倍光学显微镜下拍照并计数穿膜细胞数，相同独立实验重复3次。

1.3. 统计学分析

采用SPSS 19.0进行统计学分析，计量数据以均数±标准差表示，两组间比较采用独立样本t检验，多组间比较采用单因素方差分析，组间多重比较采用LSD-t检验，P < 0.05为差异有统计学意义。

2. 结果

2.1. 转染ALKBH5后滋养细胞ALKBH5高表达及抑制效果显著

qRT-PCR结果表明：ALKBH5组ALKBH5 mRNA表达水平较Control组及NC组均升高（P < 0.05）；shALKBH5组ALKBH5 mRNA表达水平较Control组及NC组均降低（P < 0.05，图 1）。WB结果表明：ALKBH5组ALKBH5蛋白表达水平较Control组及NC组均上调（P < 0.05）；shALKBH5组ALKBH5 蛋白表达水平较Control组及NC组均下调（P < 0.05，图 2）。

1.

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转染ALKBH5相关质粒后ALKBH5 mRNA表达情况

Expression levels of ALKBH5 mRNA in HTR-8/SVneo cells after transfection with ALKBH5 plasmid. *P < 0.05 vs control and NC groups.

2.

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Western blot检测转染ALKBH5相关质粒后ALKBH5蛋白表达情况

Western blotting for detecting ALKBH5 protein expression in HTR-8/SVneo cells after transfection with ALKBH5-overexpressing plasmid. *P < 0.05 vs control and NC groups.

2.2. 高表达ALKBH5抑制滋养细胞迁移、侵袭功能，抑制ALKBH5表达促进滋养细胞迁移、侵袭功能

Transwell迁移实验结果表明：ALKBH5组穿膜细胞数与Control组及NC组相比显著减少（P < 0.05）。shALKBH5组穿膜细胞数与Control组及NC组相比均有增加（P < 0.05）。Transwell侵袭实验结果表明：ALKBH5组穿膜细胞数与Control组及NC组相比显著减少（P < 0.05）。shALKBH5组穿膜细胞数与Control组及NC组相比均有增加（P < 0.05，图 3）。

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Transwell实验检测转染ALKBH5相关质粒后滋养细胞迁移、侵袭功能变化

Transwell assay for detecting changes in trophoblast cell migration and invasion after transfection with ALKBH5 plasmid (Original magnification: ×200). *P < 0.05 vs control and NC groups.

2.3. 高表达ALKBH5抑制滋养细胞EMT过程间质相关蛋白表达，抑制ALKBH5促进滋养细胞EMT过程间质相关蛋白表达

WB结果显示：ALKBH5高表达后滋养细胞EMT相关蛋白中间质标记物：Fibronectin、N- cadherin、MMP2蛋白表达水平与shALKBH5组相比表达下调，差异有统计学意义（P < 0.05），Vimentin、MMP9蛋白表达水平无明显变化；上皮标记物E-cadherin蛋白表达上调，差异有统计学意义（P < 0.05）。ALKBH5抑制表达后滋养细胞EMT相关蛋白中间质标记物：Fibronectin、N-cadherin、MMP2蛋白表达水平与ALKBH5组相比表达上调，差异有统计学意义（P < 0.05），Vimentin、MMP9表达水平无明显变化；上皮标记物E-cadherin表达下调，差异有统计学意义（P < 0.05，图 4）。

4.

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Western blot检测转染ALKBH5相关质粒后EMT相关蛋白表达情况

Western blotting for detecting expressions of EMT-related proteins in the trophoblast cells after transfection with ALKBH5 plasmid (*P < 0.05).

3. 讨论

胎盘组织细胞内的mRNA水平并不总是与它们各自的蛋白水平成正比，这种差异表明除转录调控方式外转录后调控方式也参与了胎盘相关调控机制[19]。m6ARNA甲基化修饰广泛存在于真核生物中，是一种动态、可逆的转录后调控方式，通过甲基转移酶、结合蛋白和去甲基酶相互作用构建一个动态平衡的基因表达网络，在mRNA的剪切、翻译、出核、降解等方面发挥着重要的调控作用[20-21]。Taniguchi等[11]的研究发现胎儿生长受限与子痫前期胎盘中mRNA的5'非编码区（5'-UTR）和终止密码附近m6A水平降低。表明胎盘中m6A水平与子痫前期的发生相关，m6A转录后调控方式可能参与胎盘组织中相关蛋白的表达调控，这与此次研究发现ALKBH5表达水平可影响滋养细胞中EMT相关蛋白表达相符。

去甲基酶ALKBH5可通过去甲基化作用降低靶基因的m6A水平，调控基因表达参与细胞生命活动及功能调节[22]。ALKBH5在肿瘤中研究较为广泛，目前研究认为ALKBH5在肿瘤细胞中既可发挥促癌作用亦可发挥抑癌作用。Zhang等[18]研究表明ALKBH5过表达可上调转录因子FOXM1表达促进胶质瘤母细胞瘤干细胞增殖。Zhang等[17]研究认为ALKBH5过表达可上调缺氧诱导因子NANOG的表达从而增强乳腺癌干细胞在缺氧微环境中的生存能力。但在在胰腺癌细胞中LKBH5过表达可下调lncRNA KCNK15-AS1的表达抑制胰腺癌细胞迁移和侵袭功能[23]。以上研究表明ALKBH5过表达后可通过降低下游靶基因如转录因子、插头蛋白、lncRNA等的m6A水平进而影响细胞的功能。滋养细胞浸润障碍引发的胎盘功能异常可能是子痫前期发病的重要机制，滋养细胞浸润子宫内膜的生物学行为与肿瘤发生过程相似[24]。ALKBH5对滋养细胞功能影响如何呢？Li等人研究发现ALKBH5可通过调控CYR61 mRNA的稳定性来抑制滋养细胞的侵袭功能[13]。此次研究在人滋养细胞HTR-8/Svneo中高表达ALKBH5后观察到滋养细胞迁移、侵袭功能降低，抑制ALKBH5后观察到滋养细胞迁移、侵袭功能增强这与Li等发现一致。

ALKBH5调控滋养细胞迁移、侵袭功能的具体机制目前尚不清楚，目前研究认为ALKBH5可通过EMT过程影响肿瘤细胞的迁移、侵袭功能。Jin等[25]研究表明ALKBH5过表达可抑制肺癌细胞EMT过程及细胞迁移、侵袭功能。He等[23]研究认为ALKBH5过表达可抑制胰腺癌细胞EMT过程。已有研究报导滋养细胞EMT过程可能参与子痫前期的发病机制。ZOU等[26]的研究认为白黎醇可通过促进细胞EMT过程增强滋养细胞侵袭能力同时通过介导Wnt/β-catenin信号通路参与子痫前期的发生。Shen等[27]研究认为circTNRC18负向调控miR-762表达影响滋养细胞EMT及迁移功能参与子痫前期的发生。可见滋养细胞EMT与子痫子痫前期的发生密切相关。

在胚胎发育与植入过程中，滋养外胚层通过EMT过程获得间充质特性的能力是胚胎植入的关键条件，在胚胎发育过程中上皮细胞失去极性和彼此的连接，产生具有侵袭性的间充质细胞，胚胎滋养细胞通过EMT转化获得侵入能力而进入母体蜕膜[28-29]。胚胎植入后滋养外胚层上皮细胞的一个子集，滋养干细胞转化为侵袭性的滋养细胞巨细胞，侵袭母体并形成胎盘[30]。此过程中绒毛外滋养细胞侵入母体蜕膜和子宫肌层使胎盘定植于子宫壁上，同时侵入到子宫螺旋动脉取代子宫内皮细胞发生螺旋动脉血管重铸[29]。以上研究表明胎盘植入、子宫螺旋动脉重铸与滋养细胞EMT过程密切相关，而胎盘植入过浅、子宫螺旋动脉重铸不良是目前子痫前期主要的病因学说。为进一步探究ALKBH5是否通过影EMT过程调控滋养细胞侵袭功能，此次研究检测高表达及抑制ALKBH5后滋养细胞迁移、侵袭功能变化，结果表明高表达ALKBH5后滋养细胞迁移、侵袭功能降低，抑制后ALKBH5表达后滋养细胞迁移、侵袭功能增强。通过下游通路探索发现ALKBH5高表达后上皮标记物E-cadherin蛋白表达水平增加，间充质标记物Fibronectin、N-cadherin、MMP2表达水平下降，提示高表达ALKBH5后滋养细胞EMT过程受到抑制。表明ALKBH5可影响滋养细胞EMT过程抑制其迁移、侵袭功能。

此次研究也存在一些不足之处，此次研究对下游机制研究主要集中于EMT过程对滋养细胞迁移、侵袭功能的影响，需发掘其它可能的通路是否参与了子痫前期的发病机制。此外体外细胞水平的结果尚需进一步动物体内实验的验证。

综上所述，ALKBH5与子痫前期的发病机制密切相关。ALKBH5可通过抑制EMT过程抑制滋养细胞的迁移、侵袭功能，从而影响子痫前期的病理过程。本实验为进一步探讨ALKBH5对子痫前期作用机制提供实验依据，为寻找子痫前期新的预测标志物及潜在药物靶点提供依据。

Biographies

何建萍，副主任医师，E-mail: 2311378101@qq.com

李晓娟，主治医师，E-mail: liyuwei0300@163.com

Funding Statement

国家自然科学地区基金（81760273，81360103）；昆明市科技计划项目（2019-1-S-25318000001501）；云南省科技计划面上项目（2017FB107）；云南省“万人计划”青年拔尖人才专项；云南省卫生科技计划项目（2018NS0112）；昆明医科大学研究生创新基金项目（2019S119）

Contributor Information

何 建萍 (Jianping HE), Email: 2311378101@qq.com.

李 晓娟 (Xiaojuan LI), Email: liyuwei0300@163.com.

钱 源 (Yuan QIAN), Email: yuanqian2x@hotmail.com.

References

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