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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-17-5p post-transcriptionally regulates p21 expression in irradiated betel quid chewing-related oral squamous
1Department of Radiation-oncology, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan.
Background And Purpose:
Betel nut chewing is associated with oral cavity cancer in Taiwan. OC3 is an oral carcinoma cell line that was established from cells collected from a long-term betel nut chewer who does not smoke. After we found that microRNA-17-5p (miR-17-5p) is induced in OC3 cells, we used this cell line to examine the biological role(s) of this microRNA in response to exposure to ionizing radiation.
Materials And Methods:
A combined SYBR green-based real-time PCR and oligonucleotide ligation assay was used to examine the expression of the miR-17 polycistron in irradiated OC3 cells. The roles of miR-17-5p and p21 were evaluated with specific antisense oligonucleotides (ODN) that were designed and used to inhibit their expression. Expression of the p21 protein was evaluated by Western blotting. The clonogenic assay and annexin V staining were used to evaluate cell survival and apoptosis, respectively. Cells in which miR-17-5p was stably knocked down were used to create ectopic xenografts to evaluate in vivo the role of miR-17-5p.
Results:
A radiation dose of 5 Gy significantly increased miR-17-5p expression in irradiated OC3 cells. Inhibition of miR-17-5p expression enhanced the radiosensitivity of the OC3 cells. We found that miR-17-5p downregulates radiation-induced p21 expression in OC3 cells and, by using a tumor xenograft model, it was found that p21 plays a critical role in increasing the radiosensitivity of OC3 cells in vitro and in vivo.
Conclusion:
miR-17-5p is induced in irradiated OC3 cells and it downregulates p21 protein expression, contributing to the radioresistance of OC3 cells.
Insights
microRNA-17-5p (miR-17-5p) is induced by radiation in oral cancer cells. Inhibiting miR-17-5p increases radiosensitivity by restoring p21 expression, crucial for cancer cell death.
Area of Science:
- Oncology
- Molecular Biology
- Radiotherapy Research
Background:
- Betel nut chewing is a risk factor for oral cavity cancer.
- OC3 is an oral carcinoma cell line derived from a betel nut chewer.
- microRNA-17-5p (miR-17-5p) expression is induced in OC3 cells.
Purpose of the Study:
- To investigate the biological role of miR-17-5p in OC3 cells exposed to ionizing radiation.
- To determine the relationship between miR-17-5p, p21 expression, and radiosensitivity.
Main Methods:
- Real-time PCR and oligonucleotide ligation assay to measure miR-17 polycistron expression.
- Antisense oligonucleotides used to inhibit miR-17-5p and p21.
- Western blotting for p21 protein analysis.
- Clonogenic assays and annexin V staining for cell survival and apoptosis.
- In vivo studies using ectopic xenografts.
Main Results:
- A 5 Gy radiation dose significantly increased miR-17-5p expression in OC3 cells.
- Inhibition of miR-17-5p enhanced OC3 cell radiosensitivity.
- miR-17-5p was found to downregulate radiation-induced p21 expression.
- p21 plays a critical role in increasing radiosensitivity both in vitro and in vivo.
Conclusions:
- miR-17-5p is induced by radiation in OC3 cells.
- miR-17-5p downregulates p21 protein expression, contributing to radioresistance.
- Targeting miR-17-5p may enhance radiotherapy efficacy for oral cancer.
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