RNAi silencing targeting RNF8 enhances radiosensitivity of a non-small cell lung cancer cell line A549.

Abstract

PURPOSE:

The E3 ubiquitin ligase RNF8 regulates the accumulation or removal of a number of proteins at DNA lesions, thereby playing a critically important role in DNA damage response. The present study investigated the possibility of using RNF8 as a new target in the radiation treatment of human non-small cell lung cancer.

METHODS AND MATERIALS:

We used RNA interference technology to silence the expression of RNF8 in A549 cells, and then detected the radiation response by colony forming assays. DNA repair was monitored by γ-H2AX foci formation after RNF8 depletion. Expression of Ku70 and Rad51 were assessed by immunofluorescent staining and Western blotting. Cell cycle and apoptosis were measured by flow cytometry assays.

RESULTS:

After lentivirus-mediated siRNA transfection, expression of RNF8 in A549 cells downregulated which led to an increased radiosensitivity and impaired DNA repair. RNF8 knockdown did not affect Ku70 expression, however, Rad51, a key player in homologous recombination (HR) repair, was abrogated at sites of DNA damage. Furthermore, we observed an extended G2/M arrest and an increased induction of apoptosis after ionizing radiation in the absence of RNF8.

CONCLUSIONS:

RNF8 silencing effectively downregulates Rad51 therefore maybe impairing HR repair, and prolongs the G2/M accumulation as well as cell apoptosis upon radiation, which all suggest an enhanced radiosensitivity on A549 cells.

• Authors: Hongxia Zhou, Xiaoqian Mu, Jing Chen, Hongli Liu, Wei Shi, Enming Xing, Kunyu Yang, Gang Wu
• Journal: International journal of radiation biology (Int J Radiat Biol) Vol. 89 Issue 9 Pg. 708-15 (Sep 2013) ISSN: 1362-3095 [Electronic] England
• PMID: 23578064 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• DNA-Binding Proteins
• H2AX protein, human
• Histones
• RNF8 protein, human
• Ubiquitin-Protein Ligases
• Rad51 Recombinase

Topics

• Apoptosis (radiation effects)
• Carcinoma, Non-Small-Cell Lung (radiotherapy)
• Cell Cycle (radiation effects)
• Cell Line, Tumor
• DNA Breaks, Double-Stranded
• DNA-Binding Proteins (antagonists & inhibitors, genetics)
• Down-Regulation
• Histones (analysis)
• Humans
• Lentivirus (genetics)
• Lung Neoplasms (radiotherapy)
• RNA Interference
• Rad51 Recombinase (genetics)
• Radiation Tolerance
• Ubiquitin-Protein Ligases