Related Experiment Video
Updated: Aug 3, 2025
In vitro Cell Migration and Invasion Assays
Published on: June 1, 2014
Long non-coding RNA colon cancer-associated transcript 1-Vimentin axis promoting the migration and invasion of HeLa
Zhangfu Li1, Jiangbei Yuan1, Qingen Da1,2
1Department of Hepato-Pancreato-Biliary Surgery, Peking University Shenzhen Hospital, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen, Guangdong 518036, China.
Background:
Long non-coding RNA colon cancer-associated transcript 1 (CCAT1) is involved in transforming multiple cancers into malignant cancer types. Previous studies underlining the mechanisms of the functions of CCAT1 primarily focused on its decoy for miRNAs (micro RNAs). However, the regulatory mechanism of CCAT1-protein interaction associated with tumor metastasis is still largely unknown. The present study aimed to identify proteome-wide CCAT1 partners and explored the CCAT1-protein interaction mediated tumor metastasis.
Methods:
CCAT1-proteins complexes were purified and identified using RNA antisense purification coupled with the mass spectrometry (RAP-MS) method. The database for annotation, visualization, and integrated discovery and database for eukaryotic RNA binding proteins (EuRBPDB) websites were used to bioinformatic analyzing CCAT1 binding proteins. RNA pull-down and RNA immunoprecipitation were used to validate CCAT1-Vimentin interaction. Transwell assay was used to evaluate the migration and invasion abilities of HeLa cells.
Results:
RAP-MS method worked well by culturing cells with nucleoside analog 4-thiouridine, and cross-linking was performed using 365 nm wavelength ultraviolet. There were 631 proteins identified, out of which about 60% were RNA binding proteins recorded by the EuRBPDB database. Vimentin was one of the CCAT1 binding proteins and participated in the tumor metastasis pathway. Knocked down vimetin ( VIM ) and rescued the downregulation by overexpressing CCAT1 demonstrated that CCAT1 could enhance tumor migration and invasion abilities by stabilizing Vimentin protein.
Conclusion:
CCAT1 may bind with and stabilize Vimentin protein, thus enhancing cancer cell migration and invasion abilities.
Insights
Long non-coding RNA colon cancer-associated transcript 1 (CCAT1) stabilizes Vimentin protein, enhancing cancer cell migration and invasion. This study identifies CCAT1-protein interactions critical for tumor metastasis.
Area of Science:
- Molecular Biology
- Cancer Research
- RNA Biology
Background:
- Long non-coding RNA colon cancer-associated transcript 1 (CCAT1) promotes malignancy in various cancers.
- Previous research focused on CCAT1's role as a microRNA decoy.
- The interaction mechanisms between CCAT1 and proteins in tumor metastasis remain largely unexplored.
Purpose of the Study:
- To identify CCAT1-binding proteins across the proteome.
- To investigate CCAT1-protein interactions driving tumor metastasis.
Main Methods:
- RNA antisense purification coupled with mass spectrometry (RAP-MS) to identify CCAT1-protein complexes.
- Bioinformatic analysis using EuRBPDB to assess RNA-binding proteins.
- RNA pull-down, immunoprecipitation, and Transwell assays to validate CCAT1-Vimentin interaction and cell migration/invasion.
Main Results:
- The RAP-MS method successfully identified 631 proteins, with ~60% identified as RNA-binding proteins.
- Vimentin was identified as a CCAT1-binding protein involved in tumor metastasis.
- CCAT1 enhances cancer cell migration and invasion by stabilizing Vimentin.
Conclusions:
- CCAT1 interacts with and stabilizes Vimentin protein.
- This interaction promotes cancer cell migration and invasion, contributing to tumor metastasis.
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