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. 2017 May 11;36(19):2667-2679.
doi: 10.1038/onc.2016.419. Epub 2016 Nov 28.

MicroRNA-383 Located in Frequently Deleted Chromosomal Locus 8p22 Regulates CD44 in Prostate Cancer

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Free PMC article

MicroRNA-383 Located in Frequently Deleted Chromosomal Locus 8p22 Regulates CD44 in Prostate Cancer

N Bucay et al. Oncogene. .
Free PMC article

Abstract

A major genomic alteration in prostate cancer (PCa) is frequent loss of chromosome (chr) 8p with a common region of loss of heterozygosity (LOH) at chr8p22 locus. Genomic studies implicate this locus in the initiation of clinically significant PCa and with progression to metastatic disease. However, the genes within this region have not been fully characterized to date. Here we demonstrate for the first time that a microRNA component of this region-miR-383-is frequently downregulated in prostate cancer, has a critical role in determining tumor-initiating potential and is involved in prostate cancer metastasis via direct regulation of CD44, a ubiquitous marker of PCa tumor-initiating cells (TICs)/stem cells. Expression analyses of miR-383 in PCa clinical tissues established that low miR-383 expression is associated with poor prognosis. Functional data suggest that miR-383 regulates PCa tumor-initiating/stem-like cells via CD44 regulation. Ectopic expression of miR-383 inhibited tumor-initiating capacity of CD44+ PCa cells. Also, 'anti-metastatic' effects of ectopic miR-383 expression were observed in a PCa experimental metastasis model. In view of our results, we propose that frequent loss of miR-383 at chr8p22 region leads to tumor initiation and prostate cancer metastasis. Thus, we have identified a novel finding that associates a long observed genomic alteration to PCa stemness and metastasis. Our data suggest that restoration of miR-383 expression may be an effective therapeutic modality against PCa. Importantly, we identified miR-383 as a novel PCa tissue diagnostic biomarker with a potential that outperforms that of serum PSA.

Conflict of interest statement

Conflict of Interest: The authors have no conflicts of interest to disclose.

Figures

Fig. 1
Fig. 1. MicroRNA-383 located in frequently deleted chr8p22 region is under expressed in prostate cancer
A. Schematic representation of chr8p22 region highlighting the location of miR-383 within the host gene SGCZ. B. CNAs at miR-383 locus in prostate adenocarcinomas in the TCGA cohort. Table above summarizes the observed CNAs. C. Relative miR-383 expression levels in the TCGA training cohort (n=187). z-scores were calculated for tumor tissues and plotted along x-axis. D. Relative miR-383 expression levels in training-validation SFVAMC cohort (n=112). miR-383 expression was analyzed in laser capture microdissected (LCM) PCa tissues (n=112) and matched adjacent normal regions by real-time PCR. Data were normalized to RNU48 control. E. Average miR-383 expression in LCM microdissected PCa tissues and adjacent normal regions of SFVAMC cohort as assessed by RT-PCR in Fig. 1D. Data are represented as mean ± S.D. F. CNAs at miR-383 locus in prostate adenocarcinomas in a subset of the SFVAMC cohort (n=24). G. Correlation between CNAs and miR-383 expression in SFVAMC cohort (n=24). H. Relative miR-383 expression levels in primary prostate epithelial cells (PPEC) and prostate cancer cell lines as assessed by RT-PCR. Data were normalized to RNU48 control. (* P< .05).
Fig. 2
Fig. 2. Low miR-383 expression is associated with poor survival outcome in prostate cancer
A. Kaplan-Meier survival curve for PCa patients (SFVAMC and TCGA cohorts), stratified based on relative miR-383 levels. P-value based on a log rank test. B. Correlation of miR-383 expression with clinicopathological parameters in PCa patients. P-values are based on Chi square test.
Fig. 3
Fig. 3. miR-383 is a potential novel diagnostic biomarker for prostate cancer
A. ROC curve analysis showing the ability of miR-383 to discriminate between tumor and normal samples. B. Diagnostic utility measures of miR-383 as a prostate cancer biomarker. C. ROC curve analysis for PSA to discriminate between tumor and normal samples. D. Logistic regression analyses to compare the ability of miR-383 and PSA to identify a tumor sample. Probabilities of tumor conditional on miR-383 values are plotted along y-axis while that of PSA along x-axis. E. ROC curve analysis for combined potential of miR-383 and PSA to distinguish between malignant and normal tissues. (*P< .05).
Fig. 4
Fig. 4. miR-383 overexpresssion suppresses in vitro attributes of tumorigenicity in prostate cancer cell lines
miR-383 mimic/control miR/mock was transfected in Du145/ PC3/ LNCaP cell lines followed by functional assays (performed 72 hrs post-transfection) (*P< .05). A. Cell cycle assay in Du145/ PC3/ LNCaP cells after miR-CON (left panel) or miR-383 (right panel) transfections. B. Apoptosis assay in Du145/ PC3/ LNCaP cells after miR-CON (left panel) or miR-383 (right panel) transfections as assessed by ANNEXIN V-FITC /7-AAD staining. C. MTS cellular viability assays, D. Colony formation assays, and E. Transwell invasion and migration assays in Du145/ PC3/ LNCaP cells transfected with mock/ miR-CON/ miR-383.
Fig. 5
Fig. 5. miR-383 directly regulates prostate cancer stem cell marker CD44
A. Immunoblots of endogenous CD44 in DU145/PC3 cells transfected with mock/miR-CON/miR-383. Right panels show endogenous CD44 in PC-3M-luc-C6 cells stably transfected with control miR/miR-383. GAPDH was used a loading control. B. Relative CD44 mRNA expression in DU145/PC3/LNCaP cells transfected with mock/miR-CON/miR-383. Data were normalized to GAPDH control. C. Schematic representation of the CD44 3′-UTR showing the putative miR-383 binding site. Mutant CD44 3′ UTR is represented below. D. Luciferase reporter assays with the indicated wt and mutated 3′ UTR constructs or control luciferase construct co-tranfected with miR-CON/ miR-383 in DU145 (left panels) and PC3 cells (right panels). Firefly luciferase values were normalized to Renilla luciferase activity and plotted as relative luciferase activity (* P< .05 as compared to miR-CON). E. Relative miR-383 levels in purified CD44- and CD44+ subpopulations of PCa xenografts (DU145, PC3, LAPC9). Data were normalized to RNU48 control. F. DU145/PC3 cells were transfected with two sets of siRNAs specific to CD44 (si1 and si2) or a nonspecific (NS) control siRNA/mock transfected for 72 h followed by functional assays. Left panels: Relative CD44 mRNA expression after siRNA transfections as assessed by RT-PCR. p-values are relative to NS siRNA. GAPDH was used as a control. Right panels: Immunoblot analyses for CD44 protein expression after NS/CD44 siRNA transfections. GAPDH was used as a loading control. G. Cellular viability assay, H. Clonogenicity assay, I. Transwell invasion assay and migration assay after NS/CD44 siRNA-1 and siRNA-2 transfections. J. Apoptosis assay in DU145 and PC3 cells upon NS siRNA (left panel) or CD44 siRNA-1 (middle panel) or CD44 siRNA-2 (right panel) transfections. K. Invasion and migration assays in Du145 (upper panels) and PC3 (lower panels) after indicated transfections. (*P< .05).
Fig. 6
Fig. 6. miR-383 inhibits tumor initiating capacity of CD44+ prostate cancer cells in vitro
CD44+ subpopulations were purified from DU145 xenograft tumors followed by miR-383 overexpression and in vitro functional assays. A. Assessment of miR-383 overexpression in CD44+ cells by real time PCR. Data were normalized to RNU48 control. B. Phase contrast images of CD44+ cells transfected with miR-CON/miR-383. C. Clonogenicity assay in miR-CON/miR-383 transfected CD44+ cells. D. Sphere formation assay in miR-CON/miR-383 transfected CD44+ cells. E. Western Blot analysis of miR-CON/miR-383 transfected CD44+ xenograft cells. Band intensities were determined by Image J and relative ratios (CD44/GAPDH) were calculated and are shown below the blot.
Fig. 7
Fig. 7. miR-383 overexpression inhibits primary tumor growth and metastasis of prostate cancer in vivo
Bioluminescent PC-3M-luc-C6 cells were stably transfected with control miR/miR-383 and were either subcutaneously injected (Fig. 7B-C) or were used to generate mouse PCa experimental metastasis model (Fig. 7D). (* P= .05). A. Relative miR-383 expression levels in PC-3M-luc-C6 cells stably transfected with control miR/miR-383 as assessed by RT-PCR. Data were normalized to RNU48 control. B. Tumor volumes of xenograft tumors from miR-CON (blue) and miR-383 (red) groups, at the indicated time points. Data represent the mean of each group ±SD. Right panels: Representative images of mice from miR-CON/ miR-383 group on day 45. Arrows indicate tumors. C. Upper panels: CD44 protein levels in control (C1-C2) and miR-383 prostate cancer xenografts (T1-T4) as assessed by immunoblot analysis. GAPDH was used as a loading control. Lower panels: Relative miR-383 expression as assessed by real-time PCR in xenograft tumors represented in upper panels. Data were normalized to RNU48 control. D. miR-CON/miR-383 expressing PC-3M-luc-C6 were injected in the left ventricles of nu/nu mice (day 0) followed by periodic monitoring of metastases by in vivo BLI. Average bioluminescence of mice from miR-CON/ miR-383 groups on day 45 are plotted. Data represent the mean of each group ±SD Right panels: Representative bioluminescence images from miR-CON/miR-383 groups at indicated time-points. The scale bar on the right represents the relative intensity of bioluminescence. E. Correlation between miR-383 expression and lymph node metastasis in TCGA cohort of prostate adenocarcinomas.

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