P71.04 MiR-1243 Increases Radiosensitivity in EGFR-TKI Resistant Non-Small Cell Lung Cancer Cells

Abstract

Acquired resistance is an important issue which limits the application of EGFR-TKI in advanced non-small cell lung cancer (NSCLC). Our data showed nearly half of EGFR-mutant patients develop local relapse or limited metastases at disease progression. Previous studies have confirmed the value of radiotherapy in advanced lung cancer patients. EGFR-TKI resistant cells with T790M exhibited enhanced sensitivity to radiation. Studies have shown that microRNAs are involved in DNA damage repair mechanisms and thus affect the sensitivity of radiotherapy. Therefore, it is important to explore the detailed mechanism on miRNAs regulating radiosensitivity in EGFR-TKI resistant NSCLC. We performed miRNA microarray to screen miRNAs differentially expressed in the paired gefitinib-sensitivity PC-9 cells and gefitinib-resistant PC-9-GR cells (T790M mutation). Furthermore, we use bioinformatics to screen out target proteins and verify the expression differences of these target proteins. The proliferation of lung cancer cells was determined by CCK-8 assay. Proteins and the phosphorylation of proteins were evaluated by western blot assay. Colony formation assay showed PC-9-GR cells were more sensitive to radiation than PC-9 cells. MiRNA microarray showed 156 differentially expressed miRNAs between PC-9 and PC-9-GR cells. miR-1243 was in the top10 differentially expressed miRNAs and it was up-regulated in PC-9-GR cells. Stable knock-down miR-1243 PC-9-GR cells were further constructed using, termed as PC-9-GR-miR1243KD. CCK-8 assay showed similar proliferation kinetics between PC-9-GR and PC-9-GR-miR1243KD. It was found that PC-9-GR-miR1243KD cells showed a significant reduction in clone formation compared with PC-9-GR and PC-9-GR-NC cells after radiation, indicating higher radiosensitivity. Bioinformatics showed miR-1243 may function as a tumor oncogene to regulate apoptosis of gefitinib resistant cell with T790M mutation. SPOCK1 was one of its targeting gene. Western blots showed SPOCK1 was high expression in PC-9-GR-miR1243KD cells. This study found novel molecular mechanism might correlate with increase of radiosensitivity in EGFR-TKI resistant NSCLC cells. It could be a new therapeutic strategy for patients in advanced NSCLC to aid expansion of the effectiveness of TKI treatment through radiotherapy.