lncRNA FOXP4-AS1通过miR-507调控甲状腺乳头状癌细胞生物学行为的作用机制研究
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1.贵州医科大学临床医学院,贵州 贵阳550000;2.贵州省人民医院 血管甲状腺外科,贵州 贵阳550499;3.贵州医科大学附属医院 甲状腺外科,贵州 贵阳 550000;4.贵州省第二人民医院 甲状腺外科,贵州 贵阳 550004;5.贵黔国际总医院 乳腺甲状腺科,贵州 贵阳 550000

作者简介:

朱雪音,贵州医科大学临床医学院硕士研究生,主要从事甲状腺癌基础方面的研究。

基金项目:

国家自然科学基金资助项目(81860478)。


Mechanism of lncRNA FOXP4-AS1 regulating the biological behavior of papillary thyroid carcinoma cells via miR-507
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1.Clinical Medical College of Guizhou Medical University, Guiyang 550000, China;2.Department of Vascular and Thyroid Surgery, Guizhou Provincial People's Hospital, Guiyang 550499, China;3.Department of Thyroid Surgery, the Affiliated Hospital of Guizhou Medical University, Guiyang 550000, China;4.Department of Thyroid Surgery, the Second People's Hospital of Guizhou Province, Guiyang 550004, China;5.Department of Breast and Thyroid Surgery, Guiqian International General Hospital, Guiyang 550000, China

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    摘要:

    背景与目的 长链非编码RNA(lncRNA)可通过结合mircoRNA(miRNA)来间接调控下游mRNA的转录及降解,从而调控肿瘤发生与发展。lncRNA FOXP4-AS1是近年来发现的一种新的肿瘤相关生物标志物,在不同的肿瘤中发挥着不同的调控作用,笔者前期研究发现,FOXP4-AS1在甲状腺乳头状癌(PTC)中呈低表达,发挥抑癌作用。此外,笔者通过数据库预测miR-507可与FOXP4-AS1互补结合。因此,本研究探讨FOXP4-AS1通过调控miR-507及其下游靶mRNA抑制PTC细胞的生长的作用与机制。方法 通过TCGA数据库分析miR-507在甲状腺癌(TC)中的表达水平及其在TC中的临床意义。qRT-PCR检测PTC细胞系(TPC-1、K1)和正常甲状腺滤泡上皮细胞(Nthy-ori3-1)中miR-507的表达水平,以及过表达及敲低FOXP4-AS1后检测miR-507表达水平的变化。用双荧光素酶报告基因实验验证FOXP4-AS1与miR-507靶向关系。分别在FOXP4-AS1过表达及敲低稳转株上转染miR-507的模拟物、抑制物,并分别用CCK-8实验、克隆形成实验、Transwell实验、划痕愈合实验以及流式细胞术检测细胞功能的变化。用生物信息学方法分析miR-507下游靶点并用qRT-PCR验证。结果 TCGA数据库分析结果显示,miR-507在TC中高表达,且其表达水平TC患者与临床病理分期、T分期、腺外浸润等临床病理特征有关(均P<0.05)。qRT-PCR结果显示,与Nthy-ori3-1细胞比较,miR-507在两种PTC细胞中呈高表达,且过表达和敲低FOXP4-AS1后,两种PTC细胞中miR-507的表达水平随之反向改变(均P<0.05)。双荧光素酶报告基因实验结果显示,FOXP4-AS1与miR-507靶向结合,并抑制miR-507的表达。细胞功能实验及功能回复实验显示,FOXP4-AS1过表达后,PTC细胞的增殖活力、迁移能力和抗凋亡能力明显减弱,同时加入miR-507的模拟物后,PTC细胞以上功能回复(均P<0.05);敲低FOXP4-AS1后,PTC细胞的增殖活力、迁移能力和抗凋亡能力明显升高,同时加入miR-507的抑制物后,PTC细胞以上功能回复(均P<0.05)。数据库预测与GO、KEGG富集分析结果显示,miR-507下游可能涉及CAMK4,qRT-PCR验证结果显示,CAMK4的表达水平随FOXP4-AS1表达水平的上调、下调呈同向改变,且其表达水平随miR-507模拟物和抑制物的加入而反向改变(均P<0.05)。结论 FOXP4-AS1可以靶向结合miR-507,并可能通过海绵作用抑制miR-507表达水平调控PTC细胞的增殖、迁移及细胞凋亡。CAMK4可能是FOXP4-AS1/miR-507通路发挥抑癌作用的下游靶点之一。

    Abstract:

    Background and Aims Long non-coding RNAs (lncRNAs) can indirectly regulate the transcription and degradation of downstream mRNAs by binding to microRNAs (miRNAs), thereby regulating the occurrence and development of tumors. LncRNA FOXP4-AS1 is a recently discovered tumor-related biomarker, playing different regulatory roles in different tumors. Our previous study found that FOXP4-AS1 is downregulated in papillary thyroid carcinoma (PTC) and is a tumor suppressor. In addition, bioinformatics analysis predicted that miR-507 could complementarily bind to FOXP4-AS1. Therefore, this study was conducted to explore the role and mechanism of FOXP4-AS1 in inhibiting the growth of PTC cells by regulating miR-507 and its downstream target mRNA.Methods The expression levels of miR-507 in thyroid cancer (TC) and its clinical significance were analyzed using the TCGA database. The expression levels of miR-507 in PTC cell lines (TPC-1, K1) and normal thyroid follicular epithelial cells (Nthy-ori3-1) were detected by qRT-PCR and the changes in miR-507 expression levels after overexpression and knockdown of FOXP4-AS1 were measured. The dual-luciferase reporter gene assay was used to verify the targeting relationship between FOXP4-AS1 and miR-507. miR-507 mimic and inhibitor were transfected into stable cell lines overexpressing or knockdown of FOXP4-AS1, and changes in cell function were detected by CCK-8 assay, colony formation assay, Transwell assay, scratch healing assay, and flow cytometry. Bioinformatics analysis was used to predict the downstream targets of miR-507, and qRT-PCR was used for validation.Results Analysis of the TCGA database showed that miR-507 was highly expressed in TC, and its expression level was associated with clinical pathological features such as clinical stage, T stage, and extrathyroidal infiltration (all P<0.05). qRT-PCR results showed that compared with Nthy-ori3-1 cells, miR-507 was highly expressed in both PTC cell lines, and the expression levels of miR-507 in both PTC cells changed inversely after overexpression and knockdown of FOXP4-AS1 (all P<0.05). The results of the dual-luciferase reporter gene assay showed that FOXP4-AS1 targeted and inhibited the expression of miR-507. Cell function experiments and functional recovery experiments showed that after overexpression of FOXP4-AS1, the proliferation, migration, and anti-apoptotic ability of PTC cells were significantly weakened, and these functions were restored after the addition of miR-507 mimic (all P<0.05); knockdown of FOXP4-AS1 in PTC cells resulted in a significant increase in proliferation, migration, and anti-apoptotic ability, and these functions were restored after the addition of the miR-507 inhibitor (all P<0.05). Bioinformatics prediction and GO, KEGG enrichment analysis results showed that miR-507 downstream may involve CAMK4. qRT-PCR validation results showed that the expression level of CAMK4 changed in the same direction as the expression level of FOXP4-AS1, and its expression level changed inversely with the addition of miR-507 mimic and inhibitor (all P<0.05).Conclusion FOXP4-AS1 can target miR-507, and may regulate the proliferation, migration, and apoptosis of PTC cells by inhibiting the expression level of miR-507 through a sponge mechanism. CAMK4 may be one of the downstream targets of the FOXP4-AS1/miR-507 pathway in exerting its anticancer effects.

    表 1 FOXP4-AS1引物及内参引物序列Table 1 The primer sequences of FOXP4-AS1 and internal reference
    图1 FOXP-AS1与miR-507的结合靶点预测Fig.1 Prediction of the binding sites between FOXP4-AS1 and miR-507
    图2 TCGA数据库分析miR-507的表达 A:miR-507在TC中的表达;B:miR-507在泛癌中的表达Fig.2 Analysis of miR-507 expression in TCGA database A: Expression of miR-507 in TC; B: Expression of miR-507 in pan-cancer
    图3 miR-507的表达在TC中的临床意义 A:miR-507低表达与高表达患者的PFI曲线;B:miR-507表达与TC关系的ROC曲线;C-I:miR-507表达与临床病理分期、T分期、N分期、M分期、腺外浸润、性别、年龄的关系Fig.3 Clinical significance of miR-507 expression in TC A: PFI curves of miR-507 low expression compared to high expression patients; B: ROC curve of miR-507 expression related to TC; C-I: Relationship of miR-507 expression with clinicopathologic stage, T stage, N stage, M stage, extrathyroidal infiltration, gender, and age
    图4 miR-507在PTC细胞及正常甲状腺上皮细胞中的表达水平比较Fig.4 Comparison of miR-507 expression levels in PTC cells and normal thyroid epithelial cells
    图5 过表达效率分析 A:TPC-1和K1细胞转染过表达FOXP4-AS1慢病毒后的明场及荧光图;B:TPC-1和K1细胞过表达FOXP4-AS1效率Fig.5 Overexpression efficiency analysis A: Bright-field and fluorescent images of TPC-1 and K1 cells transfected with overexpressed FOXP4-AS1 lentivirus; B: Efficiency of FOXP4-AS1 overexpression in TPC-1 and K1 cells
    图6 敲低效率分析 A:TPC-1和K1细胞转染敲低FOXP4-AS1慢病毒后的明场及荧光图;B:TPC-1和K1细胞敲低FOXP4-AS1效率Fig.6 Knockdown efficiency analysis A: Bright-field and fluorescent images of TPC-1 and K1 cells transfected with FOXP4-AS1 knockdown lentivirus; B: Efficiency of FOXP4-AS1 knockdown in TPC-1 and K1 cells
    图7 TPC-1和K1细胞过表达及敲低FOXP4-AS1后miR-507表达水平的变化Fig.7 The changes in miR-507 expression levels after overexpression and knockdown of FOXP4-AS1 in TPC-1 and K1 cells
    图8 FOXP4-AS1与miR-507的双荧光素酶报告基因实验结果Fig.8 Results of the dual-luciferase reporter gene assay for the interaction between FOXP4-AS1 and miR-507
    图9 CCK-8实验检测PTC细胞的增殖能力Fig.9 CCK-8 assay to detect the proliferation ability of PTC cells
    图10 克隆形成实验检测PTC细胞的增殖能力Fig.10 Clone formation to detect the proliferation ability of PTC cells
    图11 Transwell实验检测PTC细胞的迁移能力Fig.11 Transwell assay to detect the migration ability of PTC cells
    图12 划痕愈合实验检测PTC细胞的迁移能力Fig.12 Scratch healing assay to detect the migration ability of PTC cells
    图13 流式细胞术检测PTC细胞的凋亡Fig.13 Apoptosis of PTC cells detected by flow cytometry
    图14 miR-507靶基因预测 A:利用Targetscan、miRDB、miRDIP和miRWalk数据库预测miR-507靶基因的Venn图;B:GO、KEGG富集分析预测miR-507可能参与的功能Fig.14 Prediction of miR-507 target genes A: Venn diagram predicting miR-507 target genes using TargetScan, miRDB, miRDIP, and miRWalk databases; B: GO and KEGG enrichment analysis predicting the potential functions of miR-507
    图15 qRT-PCR验证FOXP4-AS1通过miR-507影响CAMK4的转录水平Fig.15 Validation of the effect of FOXP4-AS1 on CAMK4 transcription through miR-507 using qRT-PCR
    图1 FOXP-AS1与miR-507的结合靶点预测Fig.1 Prediction of the binding sites between FOXP4-AS1 and miR-507
    图2 TCGA数据库分析miR-507的表达 A:miR-507在TC中的表达;B:miR-507在泛癌中的表达Fig.2 Analysis of miR-507 expression in TCGA database A: Expression of miR-507 in TC; B: Expression of miR-507 in pan-cancer
    图3 miR-507的表达在TC中的临床意义 A:miR-507低表达与高表达患者的PFI曲线;B:miR-507表达与TC关系的ROC曲线;C-I:miR-507表达与临床病理分期、T分期、N分期、M分期、腺外浸润、性别、年龄的关系Fig.3 Clinical significance of miR-507 expression in TC A: PFI curves of miR-507 low expression compared to high expression patients; B: ROC curve of miR-507 expression related to TC; C-I: Relationship of miR-507 expression with clinicopathologic stage, T stage, N stage, M stage, extrathyroidal infiltration, gender, and age
    图4 miR-507在PTC细胞及正常甲状腺上皮细胞中的表达水平比较Fig.4 Comparison of miR-507 expression levels in PTC cells and normal thyroid epithelial cells
    图5 过表达效率分析 A:TPC-1和K1细胞转染过表达FOXP4-AS1慢病毒后的明场及荧光图;B:TPC-1和K1细胞过表达FOXP4-AS1效率Fig.5 Overexpression efficiency analysis A: Bright-field and fluorescent images of TPC-1 and K1 cells transfected with overexpressed FOXP4-AS1 lentivirus; B: Efficiency of FOXP4-AS1 overexpression in TPC-1 and K1 cells
    图6 敲低效率分析 A:TPC-1和K1细胞转染敲低FOXP4-AS1慢病毒后的明场及荧光图;B:TPC-1和K1细胞敲低FOXP4-AS1效率Fig.6 Knockdown efficiency analysis A: Bright-field and fluorescent images of TPC-1 and K1 cells transfected with FOXP4-AS1 knockdown lentivirus; B: Efficiency of FOXP4-AS1 knockdown in TPC-1 and K1 cells
    图7 TPC-1和K1细胞过表达及敲低FOXP4-AS1后miR-507表达水平的变化Fig.7 The changes in miR-507 expression levels after overexpression and knockdown of FOXP4-AS1 in TPC-1 and K1 cells
    图8 FOXP4-AS1与miR-507的双荧光素酶报告基因实验结果Fig.8 Results of the dual-luciferase reporter gene assay for the interaction between FOXP4-AS1 and miR-507
    图9 CCK-8实验检测PTC细胞的增殖能力Fig.9 CCK-8 assay to detect the proliferation ability of PTC cells
    图10 克隆形成实验检测PTC细胞的增殖能力Fig.10 Clone formation to detect the proliferation ability of PTC cells
    图11 Transwell实验检测PTC细胞的迁移能力Fig.11 Transwell assay to detect the migration ability of PTC cells
    图12 划痕愈合实验检测PTC细胞的迁移能力Fig.12 Scratch healing assay to detect the migration ability of PTC cells
    图13 流式细胞术检测PTC细胞的凋亡Fig.13 Apoptosis of PTC cells detected by flow cytometry
    图14 miR-507靶基因预测 A:利用Targetscan、miRDB、miRDIP和miRWalk数据库预测miR-507靶基因的Venn图;B:GO、KEGG富集分析预测miR-507可能参与的功能Fig.14 Prediction of miR-507 target genes A: Venn diagram predicting miR-507 target genes using TargetScan, miRDB, miRDIP, and miRWalk databases; B: GO and KEGG enrichment analysis predicting the potential functions of miR-507
    图15 qRT-PCR验证FOXP4-AS1通过miR-507影响CAMK4的转录水平Fig.15 Validation of the effect of FOXP4-AS1 on CAMK4 transcription through miR-507 using qRT-PCR
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朱雪音,马宁,陈松,高庆军,赵代伟. lncRNA FOXP4-AS1通过miR-507调控甲状腺乳头状癌细胞生物学行为的作用机制研究[J].中国普通外科杂志,2024,33(5):796-814.
DOI:10.7659/j. issn.1005-6947.2024.05.013

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  • 收稿日期:2023-09-12
  • 最后修改日期:2024-04-17
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  • 在线发布日期: 2024-06-06