miR-1247-5p/Smad2轴对胃癌细胞迁移、侵袭和上皮间质转化的影响

doi:10.11958/20240634

摘要/Abstract

摘要：

目的探讨miR-1247-5p在胃癌中的表达情况及其对胃癌细胞迁移、侵袭的影响机制。方法分析癌症基因组图谱（TCGA）数据库癌旁组织与胃癌组织中差异表达的miRNA；受试者工作特征（ROC）曲线分析miR-1247-5p对恶性肿瘤预后的预测价值；实时荧光定量PCR（RT-qPCR）检测本院收集的10例胃癌和癌旁组织中miR-1247-5p的表达水平；蛋白质-蛋白质相互作用（PPI）分析miR-1247-5p下游靶基因；双萤光素酶实验观察miR-1247-5p与Smad2的结合情况；Transwell实验检测过表达miR-1247-5p对胃癌细胞迁移和侵袭能力的影响；RT-qPCR、Western blot实验检测miR-1247-5p对Smad2和上皮间质转化（EMT）标记分子表达水平的影响。结果 TCGA数据库及本院收集的胃癌组织中miR-1247-5p表达水平均低于癌旁正常组织；ROC曲线分析表明miR-1247-5p表达水平是绝大部分肿瘤类型患者预后的独立预测指标。过表达miR-1247-5p显著抑制胃癌细胞的迁移和侵袭能力。生物信息学分析提示miR-1247-5p下游靶基因参与EMT等与肿瘤转移密切相关的通路。PPI分析表明Smad2是EMT信号通路中的Hub基因。双萤光素酶实验表明miR-1247-5p和Smad2存在结合位点。RT-qPCR、Western blot实验证实miR-1247-5p可抑制Smad2表达和EMT过程。结论 miR-1247-5p在胃癌组织中低表达，过表达miR-1247-5p可抑制EMT信号通路，降低胃癌细胞迁移和侵袭能力。

关键词: 胃肿瘤, 上皮-间质转化, 细胞运动, 肿瘤浸润, Smad2蛋白质, miR-1247-5p

Abstract:

Objective To explore the expression of miR-1247-5p in gastric cancer and its effect on migration and invasion of gastric cancer cells. Methods The differentially expressed miRNAs in adjacent non-cancerous tissue versus gastric cancer tissue of TCGA database were analyzed. The prognostic value of miR-1247-5p in malignant tumor was analyzed by receiver operating characteristic (ROC) curve. RT-qPCR assay was used to detect expression levels of miR-1247-5p of gastric cancer tissue and adjacent non-cancerous tissue collected in 10 cases from our hospital. Protein-Protein Interaction (PPI) analysis was used to identify downstream target genes of miR-1247-5p. Dual-luciferase reporter assay was used to observe the binding of miR-1247 to Smad2. Transwell assay was used to investigate the effect of miR-1247-5p overexpression on migration and invasion abilities of gastric cancer cells. RT-qPCR and Western blot experiments were used to examine the impact of miR-1247-5p on expression levels of Smad2 and epithelial-mesenchymal transition (EMT) marker molecules. Results The expression levels of miR-1247-5p were lower in gastric cancer tissue collected from both TCGA database and our hospital than those in adjacent normal tissue. ROC curve analysis indicated that the expression level of miR-1247-5p was an independent prognostic indicator for most tumor types. Overexpression of miR-1247-5p significantly inhibited the migration and invasion abilities of gastric cancer cells. Bioinformatics analysis suggested that the downstream target genes of miR-1247-5p were involved in EMT and other pathways closely related to tumor metastasis. PPI analysis revealed that Smad2 was a Hub gene in the EMT signaling pathway. Dual-luciferase reporter assay demonstrated the existence of binding sites between miR-1247-5p and Smad2. RT-qPCR and Western blot experiments confirmed that miR-1247-5p can inhibit the expression of Smad2 and the process of EMT. Conclusion miR-1247-5p is lowly expressed in gastric cancer tissue, and overexpression of miR-1247-5p can inhibit EMT signaling pathway and reduce the invasive metastatic potential of gastric cancer cells.

Key words: stomach neoplasms, epithelial-mesenchymal transition, cell movement, neoplasm invasiveness, Smad2 protein, miR-1247-5p

中图分类号:

R735.2

图/表 8

表1 引物序列

Tab.1 Primer sequences

基因	引物序列（5'→3'）	产物大小/bp
miR-1247-5p	上游：ACCCGTCCCGTTCGTC	90
miR-1247-5p	下游：TGCAGGGTCCGAGGTATTC	90
U6	上游：CTCGCTTCGGCAGCACA	694
U6	下游：AACGCTTCACGAATTTGC	694
Smad2	上游：CTCACTGTAGATGGCTTTACAG	156
Smad2	下游：GATCTTCAGATTACAGCCTGG	156
Vimentin	上游：AGTCCACTGAGTACCGGAGAC	781
Vimentin	下游：CATTTCACGCATCTGGCGTTC	781
E-cadherin	上游：AGGCCAAGCAGCAGTACATT	186
E-cadherin	下游：ATTCACATCCAGCACATCCA	186
N-cadherin	上游：GACAATGCCCCTCAAGTGTT	345
N-cadherin	下游：CCATTAAGCCGAGTGATGGT	345
GAPDH	上游：GGTCTCCTCTGACTTCAACA	558
GAPDH	下游：GTGAGGGTCTCTCTCTTCCT	558

图1 miR-1247-5p在肿瘤组织中的表达情况及其与患者预后的关系 A：TCGA数据库中胃癌与正常组织中差异表达的miRNA；B：TCGA数据库胃癌与正常组织中miR-1247-5p的表达差异分析；C：miR-1247-5p表达水平预测不同肿瘤患者不良预后的ROC曲线；D：泛癌分析miR-1247-5p在各种肿瘤和正常组织中的表达情况（＊P<0.05，＊＊P<0.01，）；E：本院胃癌及癌旁组织中miR-1247-5p表达差异分析。

Fig.1 Expression of miR-1247-5p in tumor tissue and its relationship with patient prognosis

图2 miR-1247-5p下游靶基因生物信息学分析 A：miR-1247-5p下游靶基因的KEGG富集分析；B：miR-1247-5p下游靶基因的GSCA分析（＊P<0.05；#FDR≤0.05）。

Fig.2 Bioinformatics analysis of miR-1247-5p downstream target genes

图3 Smad2是miR-1247-5p的下游靶基因之一 A：PPI网络分析；B：miR-1247-5p与Smad2的结合位点；C：双萤光素酶报告基因观察miR-1247-5p对Smad2的调控作用，＊＊P<0.01。

Fig.3 Smad2 is one of the downstream target genes of miR-1247-5p

图4 Transwell实验观察2组胃癌细胞的迁移和侵袭情况

Fig.4 The invasion and metastasis of gastric cancer cells of two groups observed by Transwell assay

表2 2组胃癌细胞迁移和侵袭能力的比较（n=4，个/视野，$\bar{x}±s$）

Tab.2 Comparison of invasive metastatic ability between the two groups of gastric cancer cells

组别	细胞迁移	细胞侵袭
mimics-NC组	47.50±1.13	35.25±2.46
mimics-miR-1247-5p组	27.75±1.89	24.25±1.11
t	8.964^＊＊	4.076^＊＊

图5 各组胃癌细胞的Smad2和EMT标记分子的蛋白印迹图

Fig. 5 Protein immunoblotting profiles of Smad2 and EMT marker molecules in various gastric cancer cell lines

表3 2组胃癌细胞中Smad2和EMT标记分子mRNA和蛋白表达水平比较（n=4，$\bar{x}±s$）

Tab.3 Comparison of mRNA and protein expression levels of Smad2 and EMT-labeled molecules between two groups of gastric cancer cells

组别	mRNA
组别	Smad2	Vimentin	E-cadherin	N-cadherin
mimics-NC组	1.00±0.05	1.00±0.14	1.00±0.13	1.01±0.12
mimics-miR-1247-5p组	0.34±0.02	0.51±0.04	2.01±0.07	0.58±0.19
t	23.335^＊＊	5.718^＊＊	11.885^＊＊	3.339^＊
组别	蛋白
组别	Smad2	Vimentin	E-cadherin	N-cadherin
mimics-NC组	1.00±0.10	1.34±0.03	0.53±0.03	1.05±0.01
mimics-miR-1247-5p组	0.61±0.06	0.93±0.03	1.73±0.17	0.82±0.03
t	5.905^＊＊	14.871^＊＊	12.732^＊＊	11.819^＊＊

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