Mechanism of LINC01410 promoting proliferation and migration in esophageal squamous cell carcinoma

doi:10.19401/j.cnki.1007-3639.2028.08.005

Abstract

Abstract:

Background and purpose: Long non-coding RNA (lncRNA) LINC01410, with a length of 647 bp, participates in a variety of tumor biological processes. However, the role and mechanism of LINC01410 involved in esophageal squamous cell carcinoma (ESCC) remain unclear. This study aimed to explore the potential mechanism of LINC01410 promoting ESCC proliferation and invasion, to provide a potential prognostic indicator and therapeutic target for individuals with ESCC. Methods: Gene Expression Profiling Interactive Analysis 2 (GEPIA2) databases were used to analyze the expression of LINC01410 and overall survival in esophageal squamous cell carcinoma data set in the Cancer Genome Atlas (TCGA). Gene Set Enrichment Analysis (GSEA) was performed to identify the underlying signaling pathways involved in the biological effects of LINC01410 in ESCC. A total of 62 pairs of ESCC tissues and paracancerous tissues from ESCC patients who underwent radical surgery in the Department of Thoracic Surgery at the First Affiliated Hospital of Xinxiang Medical College and the First People's Hospital of Pingdingshan City from January 2020 to December 2021 were collected. This project has been approved by the Hospital Ethics Committee (First Affiliated Hospital of Xinxiang Medical College, No. 2018036; First People's Hospital of Pingdingshan City, No. 2019-018). The expression of LINC01410 in ESCC tissues was detected by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR). We transfected EC109 cells with LV-NC or LV-over/LINC01410 and EC9706 cells with shRNA-NC or shRNA-LINC01410. Stable transfected cells (EC109/NC, EC109/OE, EC9706/NC and EC9706/KD) were selected in primary cell culture medium containing puromycin. The expression of LINC01410 was detected by RTFQ-PCR. The impact of LINC01410 on ESCC cell proliferation was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assays. The effect of LINC01410 on ESCC cell invasion was detected by transwell migration assay. T cell factor/lymphoid enhancer factor 1 (TCF/LEF1) luciferase reporter assay was performed to validate the effect of LINC01410 on the activity of canonical Wnt/β-catenin signaling pathway. The expressions of Wnt/β-catenin and epithelial-mesenchymal transition (EMT) signal pathway related proteins in ESCC cells were detected by Western blot. Results: By analyzing the LINC01410 expression from ESCC samples in TCGA by GEPIA2, we found LINC01410 was consistently increased in ESCC tumors compared with normal tissues (P<0.05), and high LINC01410 expression was associated with poorer overall survival (OS). RTFQ-PCR assay showed that expressions of LINC01410 were higher in esophageal cancer tissues and esophageal cancer cells (EC109 and EC9706) than in precancerous tissues and HEEC cells (P<0.05). The expression level of LINC01410 was significantly correlated with invasion range, lymph node metastasis and TNM stage in ESCC patients (P<0.01). LINC01410 expression was also upregulated in EC109/OE, however the expression of LINC01410 in EC9706/KD was decreased (P<0.01). MTT assay showed overexpression of LINC01410 increased the viability of EC109 cells, while knockdown of LINC01410 decreased the viability of EC9706 cells (P<0.01). Colony formation assay indicated that overexpression of LINC01410 enhanced the clonogenic ability of ESCC cells, while knockdown of LINC01410 reduced colony formation (P<0.01). Transwell migration assay showed that LINC01410 overexpression drastically increased the number of migratory cells, while silencing of LINC01410 suppressed the migration in EC9706 cells (P<0.01). GSEA revealed that Wnt/β-catenin and EMT pathways were significantly enriched in ESCC samples with a high level of LINC01410. TCF/LEF1 luciferase reporter assay showed higher levels of Wnt-dependent activities were observed in EC109/OE cells, whereas silenced LINC01410 in EC9706 cells led to contrary results (P<0.01). Western blot analysis showed that overexpression of LINC01410 in EC109 cell significantly increased the expression levels of N-cadherin, β-catenin, cyclin D1, c-Myc and decreased E-cadherin expression, while knockdown LINC01410 resulted in opposite results. Conclusion: LINC01410 promotes proliferation and metastasis of ESCC, which might be caused by activation of Wnt/β-catenin and EMT signaling pathways.

Key words: Esophageal squamous cell carcinoma, Long non-coding RNA, LINC01410, Proliferation, Invasion, Wnt/β-catenin signaling pathway

CLC Number:

R735.1

CAO Fei, YU Wenhao, TANG Xiaonan, MA Zidong, CHANG Tingmin, GONG Yabin, LIAO Mingjuan, KANG Xiaohong. Mechanism of LINC01410 promoting proliferation and migration in esophageal squamous cell carcinoma[J]. China Oncology, 2024, 34(8): 753-762.

Figures/Tables 8

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Clinical parameters	n	LINC01410 expression		χ²	P value
Clinical parameters	n	High (n=31)	Low (n=31)	χ²	P value
Gender				2.312	0.381
Male	33	17	16
Female	29	14	15
Age/year				0.781	0.233
≤60	22	10	12
>60	40	21	19
Tumor size				6.214	0.021
T₁+T₂	52	22	30
T₃	10	9	1
Metastatic lymph nodes				9.324	<0.000 1
No	43	15	28
Yes	19	16	3
TNM stage				12.117	0.002
Ⅰ-Ⅱ	46	18	28
Ⅲ	16	13	3