Mechanism Study Of MicroRNA-449a Enhancing Radiosensitivity In Prostate Cancer Cells

Prostate cancer is the most commonly diagnosed cancer in worldwide men and is the third most common cause of cancer related deaths.Currently,radiation therapy is one of the most common definitive treatment options for localized prostate cancer.However,radiation therapy in prostate cancer is by no means innocuous and is associated with urinary and bowel side effects.Significant number of patients undergoing radiation therapy will develop locally resistent or recurrent tumors.Therefore,how to reduce tumor radioresistance and improve tumor radiosensitivity is a hot topic in the radiotherapeutic field of prostate cancer.MicroRNAs(miRNAs)are classes of non-coding RNA molecules with ~22 nucleotides in length,that regulate the stability and translation of mRNA by perfect or imperfect base pairing at the 3'-untranslated region(3'-UTR)of their target mRNA.In the human genome,more than 2,578 mi RNAs have been identified and are predicted to target at least 60% of all protein coding genes.No surprising,miRNAs,as critical gene regulators,can influence signaling pathways that alter multiple cellular processes,including the DNA damage response(DDR)after ionizing radiation(IR).Recently,increasing evidence showed miRNAs are involved in the regulation of tumor radiosensitivity,which maybe new potential clinical target for radiation sensitization.To explore the relationship between miRNA and tumor radio sensitivity and illustrate the molecular mechanism how miRNA regulates tumor cells resistance to IR,we firstly analyzed the miRNA expression profile in LNCaP cells that treated with different linear energy transfer(LET)12C6+ ion beams and X-rays irradiation by human microarray.Selected miRNAs were further validated by qRT-PCR in LNCaP cells.The results showed that miRNA expression differently altered in LNCaP cells following different LET 12C6+ ion beams and X-rays irradiation.High LET 12C6+ ion beams radiation significantly altered more miRNAs as compared to low LET 12C6+ ion beams radiation.Upregulation of mi R-17-92 clusters and miR-15/16 cluster were observed in LNCaP cells treated with X-ray irradiation,while downregualtion of these miRNAs were observed in LNCaP cells treated with 70keV/?m 12C6+ ion beams.The alteration of let-7 family and miR-34 family miRNAs expression are reverse to miR-17-92 and mi R-15/16 clusters.Notably,upregulation of mi R-449 a wereobserved in all cells treated with X-ray and different LET 12C6+ ion beams irradiation.Using TargetScan online software to predict the target of miR-449 a,we found that c-Myc is a positive target of mi R-449 a.It has been reported that miR-449 a is downregulated and c-Myc is amplificated in various malignancies,including prostate cancer.According qRT-PCR results of prostate cancer tissue samples and matched normal prostate tissue samples,we found that miR-449 a was significantly down-regulated and c-Myc was upregulated in prostate cancer tissue compared with normal prostate tissues.c-Myc expression levels were negatively correlated with miR-449 a expression in prostate cancer tissue or cells.In response to IR,we found miR-449 a was upregulated and c-Myc was downregulated in LNCaP cells.By establishing c-Myc as a direct target of miR-449 a,we revealed that miR-449 a directly regulated the expression of c-Myc by binding the 3'-UTR of its mRNA.Overexpression of miR-449 a or knockdown of c-Myc sensitized prostate cancer cells to X-ray radiation.MiR-449 a enhanced radiosensitivity of LNCaP cells by targeting c-Myc.However,the effect of miR-449a/c-Myc enhancing radiosensitivity is not far-ranging.In wild-type Rb LNCaP and PC-3 cells,overexpression of miR-449 a enhances radiation-induced G2/M arrest and apoptosis and promotes the sensitivity to X-rays radiation,while mutant Rb DU-145 cells are resistant to the X-rays radiation despite in the presence of miR-449 a.The cell cycle distribution and apoptosis of DU-145 cells is not significantly altered by mi R-449 a in the response to ionizing radiation.We found the LNCaP,PC-3 and DU-145 cells have different state of p53 and Rb protein through analyzing the molecular biological characteristic of prostate cancer cells and the WB results.LNCaP cells express wild-type p53,DU-145 contains p53233 leu and p53274 phe mutation,while no p53 protein expression in PC-3 cells.In addition,LNCaP and PC-3 cells express wild type Rb protein,whereas DU-145 cell line has a 105 base pair deletion in exon 21 of the Rb gene.Thus,we speculated that overexpression of miR-449 a or knockdown of c-Myc may inactivate RB/E2 F signaling and then increased the radiosensitivity of LNCaP and PC-3 cells to IR.We further identified that mi R-449a/c-Myc enhances radiation-induced G2/M phase arrest and apoptosis through modulating CDC25A/Rb/E2F1 and sensitizes LNCaP and PC-3 cells to X-rays radiation.Through analysis of biological properties and detecting the expression of proteins involved in cell cycle and apoptosis by western blot in miR-449 a overexpression cells,we found miR-449a/c-Myc enhanced IR-induced G2/M arrest by inhibiting CDC25A/Rb/E2F1 signaling pathway to control CyclinB1/Cdc2 activity.On the other hand,Bcl-2 is an apoptotic target suppressed by both c-Myc and E2F1,therefore,mi R-449a/c-Myc enhanced IR-induced apoptosis by directly or indirectly down-regulating Bcl-2 to activate proteins involved in mitochondrial apoptosis pathway and induce apoptosis.These findings highlighted an unrecognized mechanism of mi R-449a-mediated c-Myc regulation in response to IR,which provides a support for the combination of ionizing radiation with miRNA regulation as a therapeutic strategy for patients with prostate cancer.