甲状腺乳头状癌细胞中长链非编码RNA FoxP4-AS1的表达及其生物学功能

Expression of long non-coding RNA FoxP4-AS1 in papillary thyroid carcinoma cells and its biological function

背景与目的:笔者前期研究发现,长链非编码RNA Fox P4-AS1 (lnc RNA Fox P4-AS1)在甲状腺乳头状癌(PTC)组织中异常表达,其低表达是PTC区域淋巴结转移的独立危险因素,但其功能及作用机制尚不清楚。因此,本研究探讨lnc RNA Fox P4-AS1在PTC细胞中的表达,初步分析其对PTC细胞生物学行为的影响。方法:用q RT-PCR检测PTC细胞系(TPC-1、K1细胞)和正常甲状腺滤泡上皮细胞(Nthy-ori3-1细胞)中lnc RNA Fox P4-AS1的表达;将TPC-1、K1细胞分别转染Fox P4-AS1过表达慢病毒载体和空载病毒载体(阴性对照)后,并分别用CCK-8实验、克隆形成实验、Ed U、Transwell实验、流式细胞术分析细胞生物学功能的变化;通过GEPIA数据库和Lnc Tar网站预测Fox P4-AS1的靶基因,并用Western blot进行验证。用以上转染后的细胞构建小鼠皮下移植瘤模型,观察移植瘤的生长情况,免疫组化检测瘤组织Ki-67的表达。通过KEGG数据库初步分析Fox P4-AS1可能影响的信号通路。结果:q RT-PCR结果显示,与正常甲状腺滤泡上皮细胞比较,lnc RNA Fox P4-AS1在两种PTC细胞中的表达水平均明显降低(均P<0.05)。细胞功能实验显示,转染Fox P4-AS1过表达慢病毒载体的TPC-1、K1细胞增殖活力明显减弱、侵袭和迁移能力均较阴性对照组细胞明显降低,且细胞周期阻滞在G0/G1期(均P<0.01)。GEPIA数据库和Lnc Tar网站预测Fox P4-AS1与CDK4存在潜在的相互作用靶点,Western blot结果显示,转染Fox P4-AS1过表达慢病毒载体两种细胞的CDK4/cyclin D1表达水平较各自阴性对照组细胞明显降低(均P<0.05)。皮下移植瘤实验结果显示,转染Fox P4-AS1过表达慢病毒载体的PTC细胞较转染空载病毒载体的PTC细胞在小鼠体内生长慢,形成的瘤体小,且瘤组织中Ki-67表达减少。KEGG通路富集分析显示,Fox P4-AS1低表达主要富集于PI3K-Akt-m TOR通路、细胞周期、细胞凋亡、免疫调节相互作用,Fox P4-AS1高表达主要富集于DNA甲基化、氧化应激通路等。结论:lnc RNA Fox P4-AS1在PTC细胞中表达下调,且与PTC细胞的恶性生物学行为密切相关,Fox P4-AS1可能通过抑制CDK4/cyclin D1表达抑制PTC细胞增殖,作为保护性因素发挥作用。

Background and Aims:The authors have previously found that the long non-coding RNA FoxP4-AS1(lnc RNA FoxP4-AS1) is abnormally expressed in papillary thyroid carcinoma(PTC) tissue,and its low expression is an independent risk factor for regional lymph node metastasis in PTC.However,its function and action mechanism remain unclear.Therefore,this study was conducted to investigate the expression of lnc RNA FoxP4-AS1 in PTC cells,and make a preliminary analysis of its effect on the biological behavior of PTC cells.Methods:The expressions of lnc RNA FoxP4-AS1 in PTC cell lines(TPC-1,K1 cells) and normal thyroid follicular epithelial cells(Nthy-ori3-1 cells) were determined by q RT-PCR.In TPC-1 and K1 cells after transfection with FoxP4-AS1 overexpressing lentiviral vector or empty virus vector(negative control),the changes in biological functions were analyzed by CCK-8 assay,colony formation assay,Ed U incorporation assay,Transwell assay and flow cytometry,respectively.The potential target genes of FoxP4-AS1 were predicted by GEPIA database and Lnc Tar website,and then verified by Western blot analysis.Using above transfected cells,subcutaneous tumor models in mice were created,then,the growth of the xenograft tumors was observed and Ki-67 expressions in the tumors were determined by immunohistochemical staining.A preliminary analysis of the signaling pathways potentially associated with FoxP4-AS1 was performed by using the KEGG database.Results:q RT-PCR results showed that the expression levels of lnc RNA FoxP4-AS1 in the two types of PTC cells were significantly lower than that in the normal thyroid follicular epithelial cells(both P<0.05).Cell function experiments showed that the proliferation activity as well as invasion and migration abilities were significantly decreased with G0/G1 phase arrest in TPC-1 and K1 cells after transfection with FoxP4-AS1 overexpressing lentivirus vector compared with the negative control cells(all P<0.01).The GEPIA database and Lnc Tar website predicted that there were potential interaction targets between FoxP4-AS1 and CDK4.Western blot results showed that the expression levels of CDK4/cyclin D1 in the two types of cells transfected with FoxP4-AS1 overexpressing lentivirus vector were significantly lower than those in their corresponding negative control cells(all P<0.05).In the subcutaneous tumor transplantation models,the PTC cells transfected with FoxP4-AS1 overexpressing lentivirus vector showed a slow growth in the mice,small volume of the formed tumor and low Ki-67 expression in the tumor compared with PTC cells transfected with empty virus vector.KEGG pathway enrichment analysis showed that the FoxP4-AS1 low expression was mainly enriched in PI3 K-AKT-MTOR pathway,cell cycle,apoptosis,and immune regulation interactions,and the FoxP4-AS1 high expression was mainly enriched in DNA methylation and oxidative stress pathways.Conclusion:The expression of lnc RNA FoxP4-AS1 is down-regulated in PTC cells and is closely related to the malignant biological behavior of PTC cells.FoxP4-AS1 may exert its actions as a protective factor by suppressing the expression of CDK4/cyclin D1 and thereby inhibiting the proliferation of PTC cells.