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Journal of Southern Medical University logoLink to Journal of Southern Medical University
. 2023 Jan 20;43(1):68–75. [Article in Chinese] doi: 10.12122/j.issn.1673-4254.2023.01.09

miR-125b-5p通过负调控RAB3D基因表达抑制骨肉瘤细胞增殖与迁移

miR-125b-5p inhibits proliferation and migration of osteosarcoma cells by negatively regulating RAB3D expression

Qiuyu TAN 1, Xu WU 1, Fang JI 2, Shaoyun WU 3, Liang YAN 1,*
PMCID: PMC9978723  PMID: 36856212

Abstract

Objective

To investigate the inhibitory effect of miR-125b-5p on proliferation and migration of osteosarcoma and the role of RAB3D in mediating this effect.

Methods

The expression level of miR-125b-5p was detected by qRT-PCR in a normal bone cell line (hFOB1.19) and in two osteosarcoma OS cell lines (MG63 and HOS). A miR-125b-5p mimic or inhibitor was transfected in the osteosarcoma cell lines via liposome and the changes in cell proliferation and migration were detected with EDU and Transwell experiments. Bioinformatic analysis was conducted for predicting the target gene of miR-125b-5p, and the expression level of RAB3D in hFOB1.19, MG63, and HOS cells was detected by Western blotting. In the two osteosarcoma cell lines transfected with miR-125b-5p mimic or inhibitor, the expression levels of RAB3D mRNA and protein in osteosarcoma cells were examined with qRT-PCR and Western blotting. The effects of RAB3D overexpression, RAB3D knockdown, or overexpression of both miR-125b-5p and RAB3D on the proliferation and migration of cells were assessed using EDU and Transwell experiments.

Results

The two osteosarcoma cell lines had significantly lower expression levels of miR-125b-5p (P < 0.05). Bioinformatic analysis predicted that RAB3D was a possible target gene regulated by miR-125b-5p. In osteosarcoma cells, overexpression of miR-125b-5p significantly lowered the expression of RAB3D protein (P < 0.05); inhibiting miR-125b-5p expression significantly decreased RAB3D expression only at the protein level (P < 0.05) without obviously affecting its mRNA level. Modulation of miR-125b-5p and RAB3D levels produced opposite effects on proliferation and migration of osteosarcoma cells, and in cells with overexpression of both miR-125b-5p and RAB3D, the effect of RAB3D on cell proliferation and migration was blocked by miR-125b-5p overexpression (P < 0.05).

Conclusion

Overexpression of miR-125b-5p inhibits the proliferation and migration of osteosarcoma cells by regulating the expression of RAB3D at the post-transcriptional level.

Keywords: miR-125b-5p, RAB3D, osteosarcoma cells, cell proliferation, cell migration

骨肉瘤是一种主要发生于青少年的骨组织恶性肿瘤，其结缔组织能直接诱导产生肿瘤骨或骨样组织，常发生于管状骨，且预后不良[1, 2]。虽然现在骨肉瘤的临床治疗方法不断完善，但由于骨肉瘤的高度异质性，经常出现转移性病例，而转移性病例的生存率仅仅只有20%[3, 4]。因此，对骨肉瘤发展的具体机制进行进一步研究尤为重要。

微RNA（miRNA）是一类长度为18~24核苷酸的单链非编码RNA分子，通过序列互补特定识别并结合到靶基因mRNA的3′末端非翻译区域(UTR），抑制靶基因mRNA翻译，从而起到调控基因表达的作用[5]。现有研究证实miRNAs在细胞增殖、分化、凋亡和迁移等细胞进程方面发挥了重要作用，在多种癌症中也发现miRNAs起到抑癌或促癌的作用[6, 7]。在已有的研究中[8]，miR-125b-5p在多种肿瘤的发生与发展中发挥着重要的作用。有研究表明[9]，miR-125b-5p可以下调TPD52基因表达，从而影响乳腺癌细胞的增殖、迁移和侵袭。另有研究表明，miR-125b-5p在膀胱癌中呈现异常表达[10]。以往研究表明[11]，miR-125b通过抑制STAT3基因表达抑制了骨肉瘤细胞的增殖及迁移。但由于miR-125b的前体经过加工剪切后会形成miR-125b-5p及miR-125b-3p二种产物，因此有必要探讨哪一种miRNA在骨肉瘤中发挥具体作用。

RAB3D是分泌组织中表达的一类小分子GTP结合蛋白，该蛋白参与膜运输，并在细胞增殖、凋亡及迁移方面发挥了重要作用[12]。研究表明，RAB3D在多种癌症的发展中发挥了作用，如：RAB3D促进了食管鳞状细胞癌细胞的增殖和迁移[13]；另有研究表明，RAB3D通过影响CDK4和CDK6信号传导来刺激肿瘤细胞周期进程，从而影响肿瘤细胞增殖[14]。而在骨肉瘤中有关miR-125b-5p调控RAB3D的研究还未见报道，亟需进一步研究。

本文通过生物信息学预测，miR-125b-5p可能结合的靶基因为RAB3D，并研究了RAB3D对骨肉瘤细胞增殖及迁移的影响，在体外探讨了miR-125b-5p通过调控RAB3D抑制骨肉瘤增殖及迁移的具体机制。

1. 材料和方法

1.1. 材料

1.1.1. 细胞株

人骨肉瘤细胞MG63、HOS、永生化人软骨细胞hFOB1.19（中科院上海细胞库）。

1.1.2. 试剂

Lipofectamine 3000（Thermo）；DMEM培养基（Gibco）；胰酶（Gibco）；胎牛血清（Sigma）；RIPA裂解液（碧云天）；BCA蛋白浓度检测试剂（Thermo Fisher）；DMSO（Sigma）；GAPDH I抗、RAB3D I抗（Proteintech）；山羊抗兔II抗（Santa cruz）；山羊抗鼠II抗（Santa cruz）；G418（碧云天）。

1.2. 方法

1.2.1. 细胞培养

人骨肉瘤细胞系MG63、HOS细胞及人软骨细胞hFOB1.19培养于37 ℃的恒温培养箱中，用含有10%的血清的DMEM培养，待细胞融合度长至80~90%时，进行下一步实验。

1.2.2. 细胞转染

将细胞在六孔板中培养，待细胞融合度达到60%~70%时，按照Lipofectamine 3000的说明书将等量的miR-125b-5p过表达（miR-125b-5p mimic，广州锐博生物科技有限公司）及抑制剂（miR-125b-5p inhibitor，广州锐博生物科技有限公司）、RAB3D过表达质粒（OE-RAB3D，上海吉玛制药有限公司）及siRNA（si-RAB3D，上海吉玛制药有限公司）转染到骨肉瘤细胞中。细胞分组为：Control mimic；miR-125b-5p mimic；Control inhibitor；miR-125b-5p inhibitor；Si-control；Si-RAB3D；OE-control；OE-RAB3D；Control mimic + OE-control；Control mimic+OE-RAB3D；miR-125b-5p mimic+OE-control；miR-125b-5p mimic+OE-RAB3D。

1.2.3. 生物信息学分析筛选miR-125b-5p可能结合的靶基因

为找出miR-125b-5p可能调控的靶基因，通过3种不同的生物信息学软件[15]（TargetScan、miRanda及PicTar）筛选可能结合miR-125b-5p的靶基因。在候选靶基因中，3种软件一致预测RAB3D是miR-125b-5p结合的靶基因。

1.2.4. 细胞迁移实验

在24孔板中每孔加入600 μL的含有20%血清的DMEM，将MG63细胞用不含血清的DMEM重悬，含有5×103 MG63细胞的Transwell小室插入24孔板中。24 h后将Transwell小室取出，PBS清洗一遍后放入4%多聚甲醛中，室温固定20 min，用PBS清洗3遍，放入0.5%的结晶紫溶液中染色15 min，1×PBS清洗3遍后，使用电子显微镜（Olympus BX51）进行拍照。

1.2.5. 细胞增殖实验

细胞增殖实验使用EdU试剂盒法，根据说明书按照以下步骤操作：加入稀释的EdU溶液（每1 mL完全培养基中加人EdU溶液1 μL）1 mL于37 ℃孵育2 h；随后，PBS清洗3遍，加入150 μL的4%多聚甲醛固定30 min；吸掉培液，再加入浓度为2 mg /mL的甘氨酸溶液150 μL，室温孵育5 min；PBS清洗3遍，随后加入250 μL的渗透液（0.5% Triton×100）孵育10 min；PBS清洗1遍；加入250 μL /孔的染色液（Appollo染色液）室温避光30 min，渗透液清洗3次，10 min/次；随后用1×DAPI进行细胞核染色30 min，再用PBS清洗2次；最后采用Olympus BX51荧光显微镜察并拍照。

1.2.6. 总RNA提取和qRT-PCR

细胞总RNA提取使用RNA快速提取试剂盒进行，并使用逆转试剂盒将总RNA逆转录为cDNA。通过qRT-PCR法测定miR-125b-5p表达量（U6作为内参基因）及RAB3D mRNA表达量（GAPDH作为内参基因）。RAB3D和GAPDH引物序列如表 1所示。数据计算使用2-△△法。

1.

RAB3D及GAPDH引物序列

Primer sequences for RAB3D and GAPDH

Upstream sequence Downstream sequence
GAPDH 5'-CGAGCCACATCGCTCAGACA-3' 5'-GTGGTGAAGACGCCAGTGGA-3'
RAB3D 5'-TTCCTGTTCCGATACGCGG-3' 5'-TACATGAGCAGGAAGCCCAT-3'
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1.2.7. 蛋白提取和Western blot

使用RIPA裂解细胞，冰上裂解40 min，4 ℃、12 000 r/min离心20 min；取上清，使用BCA法对蛋白质进行定量，随后加入1/5体积的5×SDS上样缓冲液，100 ℃金属浴，5 min。10% SDS-PAGE电泳后，开始转膜（300 mA，90 min），转膜结束后，将膜放入5%脱脂奶粉中，封闭1 h。最后加入RAB3D I抗（1∶1000稀释），4 ℃孵育过夜。第2天，将膜取出加入1×TBST清洗6遍（5 min/遍）。随后，加入II抗（1∶5000稀释），常温孵育1 h，将膜取出，1×TBST清洗6遍（5 min/遍）。将膜置于曝光板，确保正面朝上，加入发光底物，化学发光成像仪曝光。

1.2.8. 数据统计

数据统计与分析运用SPSS v17.0软件，数据以均数±标准差表示，组间比较用单因素方差分析，P < 0.05表示差异具有统计学意义。

2. 结果

2.1. miR-125b-5p抑制骨肉瘤细胞的增殖和迁移

qRT-PCR结果表明（图 1A），与正常的人软骨细胞hFOB1.19相比，在MG63与HOS细胞中miR-125b-5p的表达量分别只有人软骨细胞hFOB1.19的67.7%（P < 0.05）及3.27%（P < 0.05）。细胞增殖实验结果（图 1B~E）显示，在过表达miR-125b-5p时，与对照组相比，MG63细胞和HOS细胞的增殖能力分别被抑制了36.1%（P < 0.05）及29.4%（P < 0.05）；而在抑制miR-125b-5p时，与对照组相比，MG63细胞和HOS的增殖能力分别升高了1.32倍（P < 0.05）及1.26倍（P < 0.05）。细胞迁移实验的结果（图 1F~I）显示，在过表达miR-125b-5p时，与对照组相比，MG63细胞和HOS细胞的迁移能力分别被抑制了50.3%（P < 0.05）及50.8%（P < 0.05）；，而在抑制miR-125b-5p时，与对照组相比，MG63细胞和HOS细胞的迁移能力分别升高了2.15倍（P < 0.05）及1.41倍（P < 0.05）。

1.

1

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miR-125b-5p对骨肉瘤MG63细胞和HOS细胞增殖及迁移的影响

Effect of miR-125b-5p expression level on proliferation and migration of osteosarcoma cells. A: qRT-PCR of miR-125b-5p expression in hFOB1.19, MG63 and HOS cells. B, C: EdU assay of MG63 cells transfected with miR-125b-5p mimic or inhibitor (Original magnification: × 100). D, E: EdU assay of HOS cells transfected with miR-125b-5p mimic or inhibitor (×100). F, G: Migration assay of MG63 cells transfected with miR-125b-5p mimic or inhibitor (× 100). H, I: Migration assay of HOS cells transfected with miR-125b-5p mimic or inhibitor.*P < 0.05, **P < 0.01, ***P < 0.001.

2.2. 生物信息学预测并验证miR-125b-5p调控RAB3D基因表达

通过3种生物信息学软件，预测了miR-125b-5p可能结合的靶基因。3种软件一致得出RAB3D是miR-125b-5p可能结合的靶基因（图 2A），且RAB3D与miR-125b-5p的结合能为-20.6 kcal/mol，符合miRNAs与靶基因结合的结合能范围之内。qRT-PCR结果表明（图 2B），转染miR-125b-5p mimic到骨肉瘤细胞后，与对照组相比，细胞内的m125b-5p明显升高（P < 0.05）；而转染miR-125b-5p inhibitor到细胞后，细胞内的miR-125b-5p明显降低（P < 0.05）。Western blot实验显示（图 2D、E），转染miR-125b-5p mimic到骨肉瘤细胞后，RAB3D的蛋白表达水平与对照组相比显著降低（P < 0.05）；转染miR-125b-5p inhibitor到骨肉瘤细胞后，RAB3D的蛋白表达水平与对照组相比显著升高（P < 0.05）；而RAB3D的mRNA水平，与对照组相比，变化无统计学意义（图 2C）。

2.

2

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骨肉瘤中miR-125b-5p调控RAB3D基因表达

miR-125b-5p regulates RAB3D gene expression. A: Duplex structures of miR-125b-5p and the 3′UTR of RAB3D. B: qRT-PCR of miR-125b-5p expression in cells transfected with control mimic/inhibitor or miR-125b-5p mimic/inhibitor. C: qRT-PCR of RAB3D mRNA in cells transfected with control mimic/inhibitor or miR-125b-5p mimic/inhibitor. D, E: Western blotting of cells transfected with control mimic/inhibitor or miR-125b-5p mimic/inhibitor. *P < 0.05, **P < 0.01, ***P < 0.001.

2.3. RAB3D促进骨肉瘤细胞的增殖和迁移

Western blot实验结果（图 3A）显示在骨肉瘤细胞HOS、MG63细胞中，RAB3D的蛋白表达量较正常人软骨细胞分别升高了3.37倍（P < 0.05）及2.07倍（P < 0.05）。Western blot实验结果显示，在MG63细胞（图 3B~E）与HOS细胞内过表达RAB3D后，其蛋白表达量与对照组相比明显升高（P < 0.05），在细胞内干扰RAB3D的表达后，其蛋白表达量明显降低（P < 0.05）。细胞增殖实验结果（图 3F~I）显示，RAB3D蛋白水平过表达后，骨肉瘤细胞MG63与HOS的增殖能力与对照组相比，分别升高了1.67倍（P < 0.05）及1.21倍（P < 0.05）；而RAB3D蛋白表达水平被抑制后，骨肉瘤细胞MG63与HOS的增殖能力与对照组相比，分别抑制了36%（P < 0.05）及29.3%（P < 0.05）。细胞迁移实验的结果显示（图 3J~M），RAB3D蛋白水平过表达后，骨肉瘤细胞MG63与HOS的迁移能力与对照组相比，分别升高了1.63倍（P < 0.05）及1.35倍（P < 0.05）；而RAB3D蛋白表达水平被抑制后，骨肉瘤细胞MG63与HOS的迁移能力与对照组相比，分别抑制了65%及57.4%（P < 0.05）。

3.

3

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RAB3D促进了骨肉瘤MG63细胞和HOS细胞的增殖及迁移

Effect of RAB3D expression level on proliferation and migration of osteosarcoma cells. A: Western blotting for detecting RAB3D expression in hFOB1.19, HOS and MG63 cells. B, C: Western blotting of RAB3D expression in MG63 cells transfected with RAB3D siRNA or overexpression vector. D, E. Western blotting of RAB3D expression in HOS cells transfected with RAB3D siRNA or overexpression vector. F, G: EdU assay of MG63 cells transfected with RAB3D siRNA or overexpression vector (×100). H, I: EdU assay of HOS cells transfected with RAB3D siRNA or overexpression vector (×100). J, K: Migration assays of MG63 cells transfected with RAB3D siRNA or overexpression vector (×100). L, M: Migration assay of HOS cells transfected with RAB3D siRNA or overexpression vector (×100). *P < 0.05, **P < 0.01, ***P < 0.001.

2.4. miR-125b-5p调控RAB3D基因表达抑制了骨肉瘤细胞的增殖和迁移

因此本研究在两种骨肉瘤细胞MG63和HOS中设置4个不同的组别：Control mimic+OE-Control，OE-RAB3D + Control mimic，miR-125b-5p + OE-Control，miR-125b-5p mimic+OE-RAB3D。细胞增殖实验结果（图 4A~D）显示，提高RAB3D的表达明显促进了骨肉瘤细胞的增殖能力（P < 0.05），而这种被提高的增殖能力在同时过表达miR-125b-5p后被阻断。细胞迁移实验结果（图 4E~H）显示，提高RAB3D的表达明显促进了骨肉瘤细胞的迁移能力（P < 0.05），而这种被提高的迁移能力在同时过表达miR-125b-5p后被阻断。

4.

4

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miR-125b-5p调控RAB3D基因表达抑制骨肉瘤细胞的增殖及迁移

Overexpression of miR-125b-5p inhibits proliferation and migration of osteosarcoma cells by downregulating RAB3D expression. A, B: EdU assay of MG63 transfected with control mimic+OE-control, control mimic+OE-RAB3D, miR-125b-5p mimic + OE-control, or miR-125b-5p mimic + OE-RAB3D (× 100). C, D: EdU assay of HOS cells with different transfections (×100). E, F: Migration assay of MG63 cells with different transfections (× 100). G, H: Migration assay of HOS cells with different transfections (×100). *P < 0.05, **P < 0.01, ***P < 0.001.

3. 讨论

有研究表明，miR-125b-5p在多种癌症中发挥了调控作用[16]。如miR-125b-5p通过调控SOX12基因的表达抑制了乳腺癌细胞的转移[17]。miR-125b-5p通过调控TXNRD1基因的表达抑制了肝癌细胞的增殖、迁移及侵袭[18]。上述研究表明，miR-125b-5p在多种肿瘤中抑制了肿瘤的发展。虽有研究表明，Circular RNA 0086996通过吸附miR-125b-5p在骨肉瘤中调控了肿瘤的生长及迁移[19]，但miR-125b-5p在骨肉瘤中发挥功能的具体作用机制还不明确。本研究以此作为切入点，选择miR-125b-5p作为研究对象。我们比较了正常人软骨细胞与骨肉瘤细胞中的miR-125b-5p的含量，发现在骨肉瘤细胞系中低表达，随后我们通过干预miR-125b-5p的表达证实了miR-125b-5p抑制骨肉瘤细胞的增殖及迁移，这与已有的研究结果相一致[19]。

随后，本研究通过3种生物信息学软件，预测了miR-125b-5p可能结合的靶基因为RAB3D。RAB3D作为RAS癌基因家族成员之一，在多种癌症进展中起到了重要的作用[20-22]。目前，已有报道在多种癌症中存在miRNAs调控RAB3D基因表达的机制，如miR-30b-5p通过靶向RAB3D抑制肾癌细胞增殖和迁移能力[23, 24]。但关于骨肉瘤中miR-125b-5p调控RAB3D的研究还未见报道。本研究通过在二种骨肉瘤细胞中干预RAB3D基因的表达，通过体外功能实验，发现RAB3D促进了骨肉瘤细胞的增殖与迁移，这与miR-125b-5p对骨肉瘤细胞增殖及迁移的作用相反。

随后，本研究在骨肉瘤两个细胞系中通过western blot实验证实了骨肉瘤中miR-125b-5p可以在转录后水平调控RAB3D基因的表达，同时通过细胞回复实验也证实了miR-125b-5p通过调控RAB3D的表达进而抑制骨肉瘤细胞的增殖和迁移。研究表明，多个miRNA可以调控一个基因的表达进而抑制或者促进肿瘤进程[25]。结合文献及我们的研究成果，本研究发现miR-125b-5p通过调控RAB3D的表达抑制了骨肉瘤细胞的增殖与迁移，为miR-125b-5p抑制骨肉瘤细胞增殖与迁移提供了一个新的作用机制。

RAB3D是原癌基因RAS基因家族的成员，参与调控信号传导、细胞增殖等生物过程[26, 27]。在先前的报告中，RAB3D在各种类型的癌症中表达失调[20, 27]。骨肉瘤的不断增殖与远处转移是骨肉瘤患者预后不良、生存期短的主要原因。已有研究表明RAB3D可以通过调节PI3K/Akt信号通路参与到肿瘤细胞的增殖和转移[28, 29]。而PI3K/Akt信号通路在调节细胞功能方面起着关键作用，包括生存、分化、血管生成、侵袭和转移[30]，且越来越多的证据表明PI3K/Akt信号通路在骨肉瘤中起重要作用[31]。结合现有文献与我们的研究成果，推测miR-125b-5p可以通过调控RAB3D的表达抑制了骨肉瘤细胞的增殖与迁移，是由于PI3K/Akt信号通路的改变。因此在今后的研究中我们将进一步深入探讨。

综上所述，本文新发现并证实miR-125b-5p可以通过靶向调控RAB3D基因的表达从而抑制骨肉瘤细胞的功能，该发现阐明了miR-125b-5p抑制骨肉瘤细胞功能的具体分子机制。该研究结果也补充了既往关于骨肉瘤miRNA调控RAB3D表达的研究，为未来miR-125b-5p作为骨肉瘤治疗的潜在核酸药物提供新的理论基础。

Biography

谈秋瑜，硕士研究生，E-mail: tanqiuyu20209010@163.com

Funding Statement

国家自然科学基金（81872171）；安徽省科研编制计划项目科学研究项目—重大项目（2022AH040179）；安徽省高校自然科学重点项目（KJ2021A0852, KJ2021A0856）；皖南医学院青年卓越人才项目（Wyqnyx202002）

Supported by National Natural Science Foundation of China (81872171)

Contributor Information

谈 秋瑜 (Qiuyu TAN), Email: tanqiuyu20209010@163.com.

颜 亮 (Liang YAN), Email: lyan@wnmc.edu.cn.

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