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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
An In Vivo Method to Identify microRNA Targets Not Predicted by Computation Algorithms: p21 Targeting by miR-92a in
Xiaoping Su1, Huaming Wang2, Wei Ge3
1National Key Laboratory of Medical Immunology and Institute of Immunology, Second Military Medical University, Shanghai, P. R. China.
Abstract:
microRNA (miRNA) dysregulation is involved in the development and progression of various human cancers, including hepatocellular carcinoma (HCC). However, how to identify the miRNAs targeting specific mRNA in cells is a significant challenge because of the interaction complexity and the limited knowledge of rules governing these processes. Some miRNAs are not predictable by current computer algorithms available. Here, using p21 mRNA as target, we established a new method, called miRNA in vivo precipitation (miRIP), to identify which kind of miRNAs can actually bind to the specific mRNA in cells. Several unpredictable miRNAs that bound p21 mRNA in HepG2 and PC-3 cells were identified by the miRIP method. Among these miRNAs identified by miRIP, miR-92a was found and confirmed to interact robustly with p21 mRNA, both in HepG2 and PC-3 cells. miR-92a was found to be remarkably increased in HCC tissues, and higher expression of miR-92a significantly correlated with lower expression of p21, which is related to poor survival of HCC patients. Moreover, inhibition of miR-92a could significantly suppress HCC growth in vitro and in vivo by upregulating p21. Together, miR-92a, which is identified by miRIP, is functionally shown to be associated with HCC growth as an oncogenic miRNA by inhibiting expression of targeting gene p21. In addition, several unpredictable miRNAs that target STAT3 mRNA were also identified by the miRIP method in HepG2 cells. Our results demonstrated that the miRIP approach can effectively identify the unpredictable but intracellular existing miRNAs that target specific mRNA in vivo.
Insights
A new method, miRNA in vivo precipitation (miRIP), identifies previously unpredictable microRNAs (miRNAs) targeting specific mRNAs. miRIP identified miR-92a as an oncogenic miRNA promoting hepatocellular carcinoma (HCC) by inhibiting p21 expression.
Area of Science:
- Molecular Biology
- Oncology
- Biochemistry
Background:
- MicroRNA (miRNA) dysregulation is implicated in human cancers, including hepatocellular carcinoma (HCC).
- Identifying specific miRNA-mRNA interactions within cells is challenging due to complexity and limitations of predictive algorithms.
- Existing computational methods often fail to predict all functional miRNA-mRNA interactions.
Purpose of the Study:
- To establish a novel method, miRNA in vivo precipitation (miRIP), for identifying intracellular miRNAs that bind specific mRNAs.
- To investigate the role of miR-92a in hepatocellular carcinoma (HCC) progression using the miRIP method.
- To explore the therapeutic potential of targeting miR-92a in HCC.
Main Methods:
- Development and application of the miRNA in vivo precipitation (miRIP) technique to capture endogenous miRNA-mRNA complexes.
- Validation of miR-92a interaction with p21 mRNA in HepG2 and PC-3 cell lines using miRIP.
- Analysis of miR-92a and p21 expression in HCC tissues and correlation with patient survival.
- In vitro and in vivo experiments to assess the effect of miR-92a inhibition on HCC growth.
Main Results:
- The miRIP method successfully identified unpredictable miRNAs binding to p21 mRNA in human cancer cell lines.
- miR-92a was confirmed to interact with p21 mRNA and its expression was significantly elevated in HCC tissues.
- High miR-92a expression correlated with low p21 expression and poor survival in HCC patients.
- Inhibition of miR-92a suppressed HCC growth by upregulating p21, demonstrating its oncogenic role.
Conclusions:
- The miRIP approach is an effective tool for identifying functionally relevant, yet computationally unpredictable, intracellular miRNA-mRNA interactions.
- miR-92a acts as an oncogenic miRNA in HCC by targeting and inhibiting p21 expression, contributing to tumor growth.
- Targeting miR-92a represents a potential therapeutic strategy for hepatocellular carcinoma.
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