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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
MicroRNA-155-5p Diminishes in Vitro Ovarian Cancer Cell Viability by Targeting HIF1α Expression
Ysrafil Ysrafil1, Indwiani Astuti1, Sumadi Lukman Anwar2
1Departement of Pharmacology and Therapy, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Farmako Yogyakarta 55281, Indonesia.
Abstract:
Ovarian cancer is the most lethal of gynecological malignancies. Recently, the development of microRNA (miRNA) -based therapeutics that could impact broad cellular programs, leading to inhibition of cancer cell viability, is gaining attention in the therapeutic landscape. The therapy is based on the presence of aberrant expressions of miRNA in cancer cells. Decreasing of tumor suppressor miRNA expression causes upregulation of oncoprotein, which worsens the prognosis of the ovarian cancer. miR-155-5p mimics were carried by chitosan nanoparticles using new nanotechnology methods. Cellular uptake of miRNA was assessed by fluorescence microscope while MTT and qPCR assay were used to determine miRNA profile and the effect of CS-NP/miRNA on SKOV3 cells. Results of profiling validated using quantitative realtime-polymerase chain reaction (PCR) found one of the most altered tumor suppressor miRNAs, miR-155-5p was downregulated 892.15-fold. According to bioinformatic analysis we identified the miRNA could recognize and regulate HIF1α expression. Transfection of mimics for miR-155-5p showed significantly increased miR-155-5p endogen SKOV3 expression level compared to the control group. We found differences after transfection mimics for miR-155-5p 31.5 and 63 nanoMolar. Increasing of miR-155-5p endogen lead to diminished SKOV3 viability (by 30%; <0.05 at concentration 80 nanoMolar). These mimics may cause an increase in upregulated miR-155-5p endogen that can reduce HIF1α expression. Here we found 2-fold and 2.8-fold reduction of HIF1α expression level after transfection compared to the control group. According to these findings, the mimics miR-155-5p can inhibit ovarian cancer cell proliferation by regulating HIF1α expression.
Insights
MicroRNA (miRNA) mimics, delivered via chitosan nanoparticles, show promise in treating ovarian cancer by restoring tumor suppressor miR-155-5p levels and reducing cancer cell viability through HIF1α regulation.
Area of Science:
- Oncology
- Biotechnology
- Molecular Biology
Background:
- Ovarian cancer is a leading cause of gynecological cancer deaths.
- Aberrant microRNA (miRNA) expression is implicated in cancer development and progression.
- Restoring tumor suppressor miRNA function is a potential therapeutic strategy.
Purpose of the Study:
- To develop and evaluate chitosan nanoparticle-encapsulated miR-155-5p mimics for ovarian cancer therapy.
- To investigate the effect of miR-155-5p restoration on ovarian cancer cell viability and HIF1α expression.
Main Methods:
- Chitosan nanoparticles (CS-NP) were used to deliver miR-155-5p mimics.
- Cellular uptake was confirmed using fluorescence microscopy.
- Quantitative real-time PCR (qPCR) and MTT assays were employed to assess miRNA expression, cell viability, and HIF1α levels.
Main Results:
- miR-155-5p was found to be significantly downregulated in ovarian cancer cells.
- Transfection with miR-155-5p mimics increased endogenous miR-155-5p levels in SKOV3 cells.
- miR-155-5p mimic treatment diminished SKOV3 cell viability by 30% and reduced HIF1α expression by 2-2.8 fold.
Conclusions:
- miR-155-5p mimics delivered by chitosan nanoparticles can effectively inhibit ovarian cancer cell proliferation.
- The therapeutic effect is mediated through the regulation of HIF1α expression.
- This nanotechnology-based approach holds potential for novel ovarian cancer treatments.
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