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Updated: Oct 24, 2025

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells
Published on: May 21, 2019
miR-141-3p suppresses development of clear cell renal cell carcinoma by regulating NEK6
Yifei Liu1, Wenqiang Fu1, Feng Yin1
1Department of Urology, Tangshan Central Hospital, Tangshan.
Abstract:
Currently, there have been few studies on the function and molecular mechanism of miR-141-3p in the development of clear cell renal cell carcinoma (CCRCC). This study aimed to explore the relationship between miR-141-3p and NIMA (never in mitosis, gene A)-related kinase-6 (NEK6) and investigate the role of the interaction in CCRCC cell proliferation, migration, invasion and apoptosis.Starbase database was used to predict the target gene of miR-141-3p in CCRCC and dual-luciferase reporter assay was performed to verify the targeting relationship between miR-141-3p and the target gene. Real-time quantitative PCR was conducted to detect the expression of miR-141-3p and NEK6 mRNA in cells. Western blot was carried out to detect the protein level of NEK6 in cells. Cell Counting Kit-8 assay, transwell assay and wound healing assay were conducted to detect CCRCC cell proliferation, invasion and migration abilities. Flow cytometry was performed to detect CCRCC cell apoptosis. miR-141-3p was markedly lowly expressed, and NEK6 was a target of miR-141-3p and was remarkably highly expressed in CCRCC cells. Over-expressing miR-141-3p could inhibit CCRCC cell proliferation, migration, invasion and promote apoptosis. The inhibitory effect of miR-141-3p over-expression on cell proliferation, migration and invasion was significantly weakened by over-expressing NEK6. miR-141-3p could regulate CCRCC cell proliferation, migration, invasion and apoptosis by targeting NEK6. This study lays the basis for the exploration of the molecular mechanism underlying CCRCC pathogenesis and research on targeted therapies for CCRCC.
Insights
MicroRNA-141-3p is downregulated in clear cell renal cell carcinoma (CCRCC) and inhibits tumor progression by targeting NEK6. Restoring miR-141-3p levels offers potential for CCRCC targeted therapy.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Clear cell renal cell carcinoma (CCRCC) pathogenesis involves complex molecular mechanisms.
- The role of microRNA-141-3p (miR-141-3p) in CCRCC development remains underexplored.
- NIMA-related kinase-6 (NEK6) is implicated in various cancers.
Purpose of the Study:
- To investigate the relationship between miR-141-3p and NEK6 in CCRCC.
- To elucidate the functional role of the miR-141-3p/NEK6 interaction in CCRCC cell behavior.
- To explore the therapeutic potential of targeting this pathway in CCRCC.
Main Methods:
- Bioinformatic prediction (Starbase) and dual-luciferase reporter assay to confirm miR-141-3p targeting of NEK6.
- Quantitative PCR and Western blot to assess miR-141-3p and NEK6 expression levels.
- In vitro assays (Cell Counting Kit-8, Transwell, wound healing, flow cytometry) to evaluate cell proliferation, migration, invasion, and apoptosis.
Main Results:
- miR-141-3p was significantly downregulated, while NEK6 was upregulated in CCRCC cells.
- Overexpression of miR-141-3p suppressed CCRCC cell proliferation, migration, and invasion, while promoting apoptosis.
- Upregulation of NEK6 partially reversed the inhibitory effects of miR-141-3p, confirming NEK6 as a direct target mediating these functions.
- miR-141-3p regulates CCRCC progression by targeting NEK6.
Conclusions:
- miR-141-3p acts as a tumor suppressor in CCRCC by inhibiting proliferation, migration, and invasion, and inducing apoptosis.
- The miR-141-3p/NEK6 axis is a critical molecular mechanism in CCRCC development.
- Targeting the miR-141-3p/NEK6 pathway represents a promising therapeutic strategy for CCRCC.
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