The impact of miR-1247-5p/Smad2 axis on gastric cancer cell migration, invasion and epithelial-to-mesenchymal transition

doi:10.11958/20240634

Abstract

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

CLC Number:

R735.2

Figures/Tables 8

References 16

[1]	DUWE L, MUNOZ GARRIDO P, LEWINSKA M, et al. MicroRNA-27a-3p targets FoxO signalling to induce tumour-like phenotypes in bile duct cells[J]. J Hepatol, 2023, 78(2):364-375. doi:10.1016/j.jhep.2022.10.012.
[2]	MO Y, LEUNG L L, MAK C S L, et al. Tumor-secreted exosomal miR-141 activates tumor-stroma interactions and controls premetastatic niche formation in ovarian cancer metastasis[J]. Molecular Cancer, 2023, 22(1):4-10. doi:10.1186/s12943-022-01703-9.
[3]	ZENG B, LI Y, FENG Y, et al. Downregulated miR-1247-5p associates with poor prognosis and facilitates tumor cell growth via DVL1/Wnt/β-catenin signaling in breast cancer[J]. Biochem Biophys Res Commun, 2018, 505(1):302-308. doi:10.1016/j.bbrc.2018.09.103.
[4]	CHU Y, FAN W, GUO W, et al. miR-1247-5p functions as a tumor suppressor in human hepatocellular carcinoma by targeting Wnt3[J]. Oncol Rep, 2017, 38(1):343-351. doi:10.3892/or.2017.5702.
[5]	LI R, QU H, WANG S, et al. CancerMIRNome:an interactive analysis and visualization database for miRNome profiles of human cancer[J]. Nucleic Acids Res, 2022, 50(D1):D1139-D1146. doi:10.1093/nar/gkab784.
[6]	LI J H, LIU S, ZHOU H, et al. starBase v2.0:decoding miRNA-ceRNA,miRNA-ncRNA and protein-RNA interaction networks from large-scale CLIP-Seq data[J]. Nucleic Acids Res, 2014, 42(Database issue):D92-D97. doi:10.1093/nar/gkt1248.
[7]	LIU C J, HU F F, XIE G Y, et al. GSCA:an integrated platform for gene set cancer analysis at genomic,pharmacogenomic and immunogenomic levels[J]. Brief Bioinform, 2023, 24(1):112-124. doi:10.1093/bib/bbac558.
[8]	OKUNO K, KANDIMALLA R, MENDIOLA M, et al. A microRNA signature for risk-stratification and response prediction to FOLFOX-based adjuvant therapy in stage Ⅱ and Ⅲ colorectal cancer[J]. Molecular Cancer, 2023, 22(1):13-21. doi:10.1186/s12943-022-01699-2.
[9]	DONG X, CHANG M, SONG X, et al. Plasma miR-1247-5p,miR-301b-3p and miR-105-5p as potential biomarkers for early diagnosis of non-small cell lung cancer[J]. Thorac Cancer, 2021, 12(4):539-548. doi:10.1111/1759-7714.13800.
[10]	PREETHI K A, SELVAKUMAR S C, ROSS K, et al. Liquid biopsy:Exosomal microRNAs as novel diagnostic and prognostic biomarkers in cancer[J]. Mol Cancer, 2022, 21(1):54-60. doi:10.1186/s12943-022-01525-9.
[11]	GRAZIANI V, RODRIGUEZ HERNANDEZ I, MAIQUES O, et al. The amoeboid state as part of the epithelial-to-mesenchymal transition programme[J]. Trends Cell Biol, 2022, 32(3):228-242. doi:10.1016/j.tcb.2021.10.004.
[12]	JEHANNO C, VULIN M, RICHINA V, et al. Phenotypic plasticity during metastatic colonization[J]. Trends Cell Biol, 2022, 32(10):854-867. doi:10.1016/j.tcb.2022.03.007.
[13]	JUNG H Y, FATTET L, TSAI J H, et al. Apical-basal polarity inhibits epithelial-mesenchymal transition and tumour metastasis by PAR-complex-mediated SNAI1 degradation[J]. Nat Cell Biol, 2019, 21(3):359-371. doi:10.1038/s41556-023-01223-8.
[14]	LONDERO M, GALLO A, CATTANEO C, et al. NF-YAl drives EMT in Claudinlow tumours[J]. Cell Death Dis, 2023, 14(1):65-76. doi:10.1038/s41419-023-05591-9.
[15]	QIU Y, ZHANG Y, TIAN L, et al. Identification of a comprehensive alternative splicing function during epithelial-mesenchymal transition[J]. iScience, 2023, 26(4):10651-10661. doi:10.1016/j.isci.2023.106517.
[16]	HUANG T, BAO H, MENG Y H, et al. Tumour budding is a novel marker in breast cancer:the clinical application and future prospects[J]. Ann Med, 2022, 54(1):1303-1312. doi:10.1080/07853890.2022.2070272.

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

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

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^＊＊

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^＊＊

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^＊＊