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An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells
Published on: May 21, 2019
The ceRNA PVT1 inhibits proliferation of ccRCC cells by sponging miR-328-3p to elevate FAM193B expression
Guohai Xie1,2,3, Xinyi Zheng4, Zhong Zheng5
1Department of Urology, Ningbo First Hospital, Ningbo 315000, Zhejiang, China.
Abstract:
Clear cell renal cell carcinoma (ccRCC) is a common and fatal malignancy. Long noncoding RNAs (lncRNAs) have emerged as crucial biomarkers and regulators in many cancers, warranting the detailed investigation of their biological functions and molecular mechanisms. In this study, we explored the role and mechanism of plasmacytoma variant translocation 1 (PVT1), a competitive endogenous RNA (ceRNA) in ccRCC tissues in vitro and in vivo. We found that PVT1 is upregulated in ccRCC cells and promoted cell proliferation. Bioinformatic analysis, dual-luciferase reporter assays, argonaute 2-RNA immunoprecipitation (AGO2-RIP), quantitative PCR arrays, western blot assay, and rescue experiments were conducted to explore the underlying mechanisms of PVT1. Our analyses revealed that miR-328-3p was a direct target of PVT1 and that FAM193B was a direct target of miR-328-3p. FAM193B is upregulated in ccRCC tissues and promotes cell proliferation by activating the MAPK/ERK and PI3K/AKT pathways. Our results indicated that PVT1 promotes ccRCC cells proliferation by sponging miR-328-3p to upregulate FAM193B and activate the MAPK/ERK and PI3K/AKT pathways. Collectively, these results suggest that PVT1- miR-328-3p-FAM193B loop could serve as a potential biomarker and therapeutic target for ccRCC.
Insights
Plasmacytoma variant translocation 1 (PVT1) promotes clear cell renal cell carcinoma (ccRCC) cell proliferation by regulating the miR-328-3p/FAM193B axis. This PVT1-miR-328-3p-FAM193B pathway represents a potential therapeutic target for ccRCC.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Clear cell renal cell carcinoma (ccRCC) is a prevalent and lethal cancer.
- Long noncoding RNAs (lncRNAs) are increasingly recognized as key regulators and biomarkers in various cancers, including ccRCC.
Purpose of the Study:
- To investigate the role and molecular mechanism of plasmacytoma variant translocation 1 (PVT1) in ccRCC.
- To elucidate the PVT1-mediated competitive endogenous RNA (ceRNA) network in ccRCC progression.
Main Methods:
- Bioinformatic analysis, dual-luciferase reporter assays, Argonaute 2-RNA immunoprecipitation (AGO2-RIP).
- Quantitative PCR arrays, Western blot assays, and in vitro/in vivo rescue experiments were employed.
- Analysis included ccRCC tissues and cell lines.
Main Results:
- PVT1 was found to be upregulated in ccRCC tissues and cells, promoting tumor cell proliferation.
- PVT1 directly targets miR-328-3p, which in turn targets FAM193B.
- FAM193B upregulation in ccRCC activates the MAPK/ERK and PI3K/AKT pathways, contributing to proliferation.
Conclusions:
- PVT1 acts as an oncogenic lncRNA in ccRCC by sponging miR-328-3p, leading to FAM193B upregulation.
- The PVT1-miR-328-3p-FAM193B axis activates oncogenic signaling pathways (MAPK/ERK, PI3K/AKT).
- This regulatory loop presents a potential diagnostic biomarker and therapeutic target for ccRCC.
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