Ionizing Radiation-induced Novel MicroRNA Regulates Cellular Radiation Response

Aims:The aims of this work were to identify novel human microRNAs (miRNAs) whose expression level could be increased by ionizing irradiation such as X-rays and to reveal novel radiation responding pathways by confirming interactions between novel miRNAs and its target genes.Materials and Methods:Human cervix carcinoma HeLa cell line was used in this study. After exposure to X-rays, expression level of small RNAs was detected by using Solexa deep sequencing. Novel human miRNA candidates were predicted by bioinformatics analysis and their expression was comfirmed by RT-PCR.Online softwares were used to predict the targets of these novel miRNA candidates and miR-5094was chosen for further investigation. Then, we constructed dual-Luciferase miRNA target expression vectors to prove the interaction between miR-5094and the3’-UTR of its potential target gene STAT5b. X-rays and carbon beams were used to treat cells and miR-5094cellular concentration was artificially changed by lipo-transfection of miR-5094mimics or inhibitor. Subsequently, we carried out Western blot and real time PCR to detect the expression of miR-5094and its predicted target STAT5b. At last, we tested the survival, viability, metastasis and cell cycle proportion to confirm the participation of miR-5094-STAT5b pathway in cellula radiation response.Results:1. By Solexa deep sequencing, we detected421known miRNAs in irradiated cells and sham control, among which190miRNAs were up-regulated after irradiation and231miRNAs down-regulated. There were22known miRNAs with altered expression level, which was consistent with earlier reports.2.337novel miRNA candidates were predicted,13out of28candidates were identified by RT-PCR, among which10of them were identified as novel miRNAs cloned for the first time.3. miR-5094negatively regulated the translation of STAT5b mRNA by direct interaction with its3’-UTR, which was proved by using dual-Luciferase report vectors.4. A negative correlation between miR-5094and STAT5b was detected in cells transfected with miR-5094mimics or its inhibitor and subsequently exposed to X-rays or carbon ion beam.5. miR-5094repressed cell survival and cell proliferation by inhibiting the expression of STAT5b, whereas inhibitor worked oppositly.6. The miR-5094-STAT5b pathway regulated the expression of downstream gene Bcl-2.Conclusion:Ionizing irradiation changes the expression level of miRNAs including enriching the abundance of miRNAs rarely expressing in normal conditions, which makes it a powerful tool for identifying novel miRNAs. In this study, we identified10novel human miRNAs with X-ray irradiation, among which miR-5094targets STAT5b. The miR-5094-STAT5b pathway is involving in cellular radiation response by regulating cell survival and viability.