MiR-34a attenuates paclitaxel-resistance of hormone-refractory prostate cancer PC3 cells through direct and indirect mechanisms.

Patients with hormone-refractory prostate cancer are treated with taxane drugs, but eventually become drug resistant. We aimed to elucidate the molecular mechanisms underlying paclitaxel resistance of hormone-refractory prostate cancer with a special focus on the roles of miR-34a and SIRT1. Paclitaxel-resistant cells (PC3PR) were generated from hormone-refractory PC3 cells. The expression levels of mRNA and miRNA were determined by reverse transcriptase PCR and those of protein were by Western blot analysis. Transfection of miRNA precursor or siRNA was performed using the liposome-mediated method. MiR-34a over-expression and SIRT1 knockdown attenuated paclitaxel resistance of PC3PR cells. MiR-34a expression was reduced in PC3PR cells compared with PC3 cells, while the expression levels of HuR and Bcl2 as well as SIRT1 were elevated in PC3PR cells. Luciferase reporter assays revealed that both SIRT1 3'-UTR and promoter activities were higher in PC3PR cells than in PC3 cells. Introduction of miR-34a precursor into PC3PR cells resulted in decreases in HuR, Bcl2, and SIRT1 expression and inhibition of the SIRT1 3'-UTR activity. HuR knockdown reduced SIRT1 and Bcl2 expression. These results suggest that miR-34a not only directly but also indirectly via regulating HuR expression acts on the 3'-UTR of SIRT1 and Bcl2 mRNAs, thereby controlling their expression. Thus, in PC3PR cells, reduced expression of miR-34a confers paclitaxel resistance via up-regulating SIRT1 and Bcl2 expression. MiR-34a and its downstream targets SIRT1 and Bcl2 play important roles in the development of paclitaxel resistance, all of which can be useful biomarkers and promising therapeutic targets for the drug resistance in hormone-refractory prostate cancer.

Kojima K ，Fujita Y ，Nozawa Y ，Deguchi T ，Ito M ... -  《-》

Effects of miR-34a on cell growth and chemoresistance in prostate cancer PC3 cells.

Fujita Y ，Kojima K ，Hamada N ，Ohhashi R ，Akao Y ，Nozawa Y ，Deguchi T ，Ito M ... -  《-》

MicroRNA-34a: a novel tumor suppressor in p53-mutant glioma cell line U251.

Previous studies showed that microRNA-34 (miR-34a) family was found to be a direct target of p53, functioning downstream of the p53 pathway as tumor suppressors. MiR-34a was identified to represent the status of p53 and participate in initiation and progress of cancers. We undertook this study to investigate the role of miR-34a in glioma cells. Expression levels of miR-34a in glioma cell lines and normal brains were detected using qRT-PCR. Human U251 glioma cells were transfected with miR-34a mimics, and the effects of miR-34a restoration were assessed by MTT assays, cell cycle analysis, caspase-3 activation, and in vitro migration and invasion assays. A computational search revealed a conserved target site of miR-34a within the 3'-untranslated region of SIRT1. Luciferase reporter assay was performed to examine the effects of miR-34a on expression of potential target gene SIRT1, and mRNA and protein expression of SIRT1 after miR-34a transfection were detected by qRT-PCR and Western blot analysis. MiR-34a expression was markedly reduced in p53-mutant cells U251 compared with A172 and SHG-44 cells expressing wild-type p53 and normal brains. Overexpression of miR-34a in U251 cells resulted in inhibition of cell growth and arrest in G0-G1 phase and induced apoptosis. Also, restoration of miR-34a significantly reduced in vitro migration and invasion capabilities. Reporter assays indicated that SIRT1 was a direct target of miR-34a. In U251 cells, overexpression of miR-34a decreased SIRT1 protein levels but not mRNA expressions, which demonstrated miR-34a-induced SIRT1 inhibition occurred at the posttranscriptional level. Our results demonstrate that miR-34a acts as a tumor suppressor in p53-mutant glioma cells U251, partially through regulating SIRT1.

Luan S ，Sun L ，Huang F 《-》

ETS1 promotes chemoresistance and invasion of paclitaxel-resistant, hormone-refractory PC3 prostate cancer cells by up-regulating MDR1 and MMP9 expression.

ETS1, which belongs to the ETS transcription factor family, plays important roles in diverse aspects of cancer such as drug resistance and metastasis. In the present study, we examined the functional roles of ETS1 in paclitaxel resistance and invasion using human prostate cancer PC3 cells and paclitaxel-resistant PC3PR cells established from PC3 cells. Our results showed that ETS1mRNA and protein expression was markedly up-regulated in paclitaxel-resistant PC3PR cells compared with paclitaxel-sensitive PC3 cells. The mRNA levels of MDR1 as well as MMP1, MMP3, MMP9 and uPA were positively correlated with that of ETS1. In PC3PR cells, silencing of ETS1 expression by siRNAs inhibited the activity of the MDR1 promoter containing ETS binding sites, reduced the mRNA and protein levels of MDR1 and suppressed paclitaxel resistance. Furthermore, ETS1 knockdown decreased secretion of MMP9 as well as its intracellular mRNA level, and dramatically inhibited invasion of PC3PR cells. Our results suggest that ETS1 promotes paclitaxel resistance and invasion in part by up-regulating MDR1 and MMP9 expression. Taken together, a novel therapeutic strategy targeting the ETS1 gene could be designed to overcome chemoresistance and metastasis of taxane-resistant, hormone-refractory prostate cancer.

Kato T ，Fujita Y ，Nakane K ，Kojima T ，Nozawa Y ，Deguchi T ，Ito M ... -  《-》

Dysregulation of microRNA-34a expression causes drug-resistance to 5-FU in human colon cancer DLD-1 cells.

MiR-34a was identified as one of the down-regulated micro-RNAs (miRs) in human colorectal cancer 5-fluorouracil (5-FU)-resistant DLD-1 cells compared with those in the parental DLD-1 cells. Exposure to 5-FU at 30 μM activated phosphoinositide 3-kinase (PI3K)/Akt signaling markedly from 12h up to 48 h in the 5-FU-resistant cells compared with that in the parental cells and resulted in an overt difference in growth at those times. Furthermore, the expression of miR-34a in the 5-FU-resistant cells was sustained at a low-level, whereas it was up-regulated in the parental cells after the 5-FU treatment. Sirt1, which is one of the target genes for miR-34a and related to drug-resistance, was strikingly up-regulated in the 5-FU-resistant cells. The ectopic expression of miR-34a in the 5-FU-resistant cells inhibited growth, as in the parental cells, and attenuated the resistance to 5-FU through the down-regulation of Sirt1 and E2F3. Moreover, the silencing of Sirt1 significantly canceled the resistance to 5-FU in the 5-FU-resistant cells. These findings suggest that miR-34a targeting the Sirt1 and E2F3 genes could negatively regulate, at least in part, the resistance to 5-FU in human colorectal cancer DLD-1 cells.

Akao Y ，Noguchi S ，Iio A ，Kojima K ，Takagi T ，Naoe T ... -  《-》