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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
Inducing cell proliferative prevention in human acute promyelocytic leukemia by miR-182 inhibition through modulation
Mahdi Fasihi-Ramandi1, Abbas Moridnia1, Ali Najafi1
1Molecular Biology Research Center, Baqiyatallahc University of Medical Sciences, Tehran, Iran.
Abstract:
MicroRNAs (miRNAs) are one class of endogenous non-coding RNAs that involved in post-transcriptional regulation of the gene. MiRNAs through interaction with messenger RNA (mRNA) involved in several biological processes such as cell cycle, differentiation, growth, metabolism, aging and apoptosis. MiRNAs may act as an oncogene or a tumor suppressor via up or down regulation in cancerous cells. MiR-182 located in a miR-183/-96/-182 cluster, this is the highly conserved cluster to have an important role in cancer development and tumorigenesis. Abnormal expression of miR-182 in a variety of human cancers has reported. Oncogenic features of miR-182 confirmed through negative regulation of various tumor suppressor genes. In this study, miR-182 inhibition in acute promyelocytic leukemia (APL) cell line (HL60) was performed by locked nucleic acid (LNA) anti-miR. MTT assay in three-time points 24, 48 and 72h after LNA-anti-miR-182 transfection was performed. Our study demonstrated inhibition of miR-182 can expansively decrease cell proliferation of APL cells. The Western blotting analysis presents that CASP9 expression associated with inhibition of miR-182. CASP9 protein has an important role in the mitochondrial cell death pathway as the initiator of apoptosis. These results can offer a way for inhibition of APL cells proliferates and produce translational medicine based on microgenomics and antisense therapy.
Insights
Inhibition of microRNA-182 (miR-182) using antisense therapy significantly reduces acute promyelocytic leukemia cell proliferation. This approach also increases the expression of CASP9, a key protein in apoptosis, offering a potential new treatment strategy.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- MicroRNAs (miRNAs) are crucial regulators of gene expression, influencing processes like cell cycle and apoptosis.
- Aberrant miRNA expression, particularly miR-182, is implicated in various human cancers, acting as an oncogene.
- The miR-183/-96/-182 cluster plays a significant role in cancer development and tumorigenesis.
Purpose of the Study:
- To investigate the effect of inhibiting miR-182 in acute promyelocytic leukemia (APL) cells.
- To explore the potential of antisense therapy targeting miR-182 for APL treatment.
- To elucidate the molecular mechanisms underlying miR-182's role in APL proliferation and apoptosis.
Main Methods:
- Utilizing locked nucleic acid (LNA) anti-miR-182 to inhibit miR-182 in the HL60 APL cell line.
- Performing MTT assays at 24, 48, and 72 hours post-transfection to assess cell proliferation.
- Conducting Western blotting to analyze the expression of CASP9 protein.
Main Results:
- Inhibition of miR-182 significantly decreased the proliferation of APL cells (HL60).
- Western blotting revealed an association between miR-182 inhibition and increased CASP9 protein expression.
- CASP9, an initiator of apoptosis in the mitochondrial pathway, showed elevated levels upon miR-182 suppression.
Conclusions:
- Targeting miR-182 with antisense therapy is a promising strategy for inhibiting APL cell proliferation.
- The observed increase in CASP9 expression suggests a mechanism involving the induction of apoptosis.
- These findings support the development of translational medicine approaches based on microgenomics and antisense therapy for APL.
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