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. 2008 Nov 1;68(15):1599-606.
doi: 10.1002/pros.20827.

Radiation modulation of microRNA in prostate cancer cell lines

Sajni Josson  1 , Shian-Ying Sung, Kaiqin Lao, Leland W K Chung, Peter A S Johnstone
Affiliations

Radiation modulation of microRNA in prostate cancer cell lines

Sajni Josson et al. Prostate. .

Abstract

Background: MicroRNAs (miRNA) are gene regulators and play an important role in response to cellular stress.

Methods: Using multiplexed quantitative real-time PCR we performed global miRNA screening of prostate cancer cells in response to radiation treatment.

Results: Several miRNA were significantly altered in response to radiation treatment. Significant changes were observed in miR-521 and miR-34c. To determine the role of miR-521 in radiation response we transiently overexpressed miR-521 using miR-521 mimic. The miR-521 mimic significantly sensitized prostate cancer cells to radiation treatment. Conversely, ectopic inhibition of miR-521 resulted in radiation resistance of prostate cancer cells. To determine the mechanism by which miR-521 modulates radiation sensitivity we measured the expression levels of one of its predicted target protein, Cockayne syndrome protein A (CSA). CSA is a DNA repair protein, and its levels correlated inversely with the levels of miR-521. Radiation treatment downregulated the levels of miR-521 and upregulated CSA protein. Similarly, ectopic inhibition of miR-521 resulted in increased CSA protein levels. Therefore by altering the levels of CSA protein, miR-521 sensitized prostate cancer cells to radiation treatment.

Conclusion: miR-521 modulates the expression levels of DNA repair protein, CSA and plays an important role, in the radio-sensitivity of prostate cancer cell lines. Thus miR-521 can be a potential target for enhancing the effect of radiation treatment on prostate cancer cells.

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Figures

Figure 1
Figure 1
a. Unsupervised hierarchical clustering heat map of miRNA expression profile at 4 h in response to ionizing radiation. The groups are: LNCaP, LNCaP + 6 Gy, C4-2 cells and C4-2 + 6 Gy. Quantitative real time PCR analysis was used to measure miRNA levels. The cluster heat map was produced using expression levels (Ct value) of 182 miRNAs. Note that a higher Ct value means a lower expression level. If the values between 0-20, are in blue; if 20-35 in yellow and 35-40 is red. All experiments were performed in duplicates. b. Clongenic survival of C4-2 and LNCaP prostate cancer cells in response to radiation treatment.
Figure 2
Figure 2
Overexpression of miR-521 sensitizes LNCaP cell to radiation treatment. a. Quantitative real time PCR analysis of miR-521 levels in LNCaP cells treated with a miR-521 mimic and radiation. b. Cell viability of LNCaP cells overexpressing miR-521 in response to radiation treatment using trypan blue exclusion assay. c. Cell viability response of LNCaP cells overexpressing miR-521 mimic and their response to radiation using MTS assay, five days after treatment. Percent viability was determined. All experiments were performed in triplicate, repeated at least two to three times, and representative findings are shown. Student’s t-test was used to determine the statistical significance.
Figure 3
Figure 3
a. Cell viability (Trypan Blue exclusion) assay was used to determine the role of miR-521 inhibitor in radiation resistance in LNCaP prostate cancer cells. b. Cell viability (MTS) assay was used to determine the role of miR-521 inhibitor in radiation resistance in LNCaP prostate cancer cells. c. Clonogenic assay was performed to determine the role of miR-521 in radiation resistance in LNCaP prostate cancer cells.
Figure 4
Figure 4
a. Quantified western analysis of miR-521 predicted target, CSA in response to miR-521 inhibitor and radiation treatment in LNCaP prostate cancer cells. b. Quantified western analysis of MnSOD in response to miR-521 inhibitor and radiation treatment in LNCaP prostate cancer cells. Representative western blots are attached below the graphs.
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