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.

Keywords::

• RNF8
• radiosensitivity
• Rad51
• ubiquitylation
• DNA damage response

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This work was supported by grants from National Natural Sciences Foundation of China (No. 30870739) and Independent Innovation Research Foundation of Huazhong University of Science and Technology (No. 2011JC074).