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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
Overexpressed miR-494 down-regulates PTEN gene expression in cells transformed by
Linhua Liu1, Yiguo Jiang, Hongyu Zhang
1Institute for Chemical Carcinogenesis, State Key Laboratory of Respiratory Diseases, Guangzhou Medical University, Guangzhou 510182, PR China.
Aims:
We investigated the functionality of miR-494 in anti-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (anti-BPDE)-transformed human bronchial epithelial cell 16HBE to reveal its potential target coding-gene.
Main Methods:
The expression of mature miR-494 in cells was detected by miRNA-specific quantitative real-time polymerase chain reaction (QRT-PCR). QRT-PCR and Western blot were used to identify the expression of phosphatase and tensin homolog (PTEN) mRNA and protein. Following activation or inhibition of mature miRNA expression with precursors or antisense inhibitors, PTEN expression, luciferase activities, cell apoptosis, cell growth in soft agar and cell motility were analyzed.
Key Findings:
The expression of miR-494 increased while PTEN protein appeared to be lower in malignant transformed 16HBE cells. Enforced miR-494 level decreased PTEN protein expression compared to a negative precursor control group. Inhibition of miR-494 expression increased PTEN protein expression compared to negative inhibitor control group. Decreased expression of miR-494 increased caspase-3/7 activities in transformed 16HBE cells, and increased expression of miR-494 decreased this activity. Inhibition of miR-494 also decreased the malignancy of transformed cells.
Significance:
MiR-494 regulates the expression of PTEN post-transcriptionally and functions as a micro-oncogene in carcinogenesis induced by anti-BPDE. MiR-494 may be a useful target for gene therapy.
Insights
MicroRNA-494 (miR-494) acts as a micro-oncogene in benzo(a)pyrene-induced lung cancer, regulating PTEN expression. Targeting miR-494 offers a potential gene therapy strategy for lung carcinogenesis.
Area of Science:
- Molecular biology
- Cancer research
- Gene regulation
Background:
- Benzo(a)pyrene is a carcinogen that can induce malignant transformation in human bronchial epithelial cells.
- MicroRNAs play crucial roles in gene regulation and are implicated in various diseases, including cancer.
Purpose of the Study:
- To investigate the role of miR-494 in anti-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (anti-BPDE)-transformed 16HBE cells.
- To identify the target gene regulated by miR-494 and elucidate its function in carcinogenesis.
Main Methods:
- Quantitative real-time PCR (QRT-PCR) to measure miR-494 expression.
- Western blot and QRT-PCR to assess PTEN mRNA and protein levels.
- Functional assays including cell apoptosis, soft agar growth, and cell motility analyses following miR-494 modulation.
Main Results:
- miR-494 expression was upregulated, and PTEN protein was downregulated in anti-BPDE-transformed 16HBE cells.
- miR-494 directly targets PTEN, regulating its post-transcriptional expression.
- Modulation of miR-494 affected cell apoptosis, proliferation, and motility, indicating its role in malignant transformation.
Conclusions:
- MiR-494 functions as a micro-oncogene in anti-BPDE-induced lung carcinogenesis by downregulating PTEN.
- MiR-494 represents a potential therapeutic target for gene therapy in lung cancer.
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