Radiotranscriptomics identified new mRNAs and miRNA markers for distinguishing prostate cancer from benign prostatic hyperplasia

Abstract The present study investigated ultrasound (US) phenotypes reflecting prostate cancer (PCa)‐related genetic mutations. Herein, integration of radiotranscriptomic data, US and contrast‐enhanced ultrasound (CEUS) radiomic images, and RNA sequencing was performed with the aim of significantly improving the accuracy of PCa prognosis. We performed radiotranscriptomic analysis of clinical, imaging, and two genomic (mRNA and microRNA expression) datasets from 48 and 22 men with PCa and benign prostatic hyperplasia (BPH), respectively. Twenty‐three US texture features and four microvascular perfusion features were associated with various patterns of 52 differentially expressed genes related to PCa (p < 0.05); 17 overexpressed genes were associated with two key texture features. Twelve overexpressed genes were identified using microvascular perfusion features. Furthermore, mRNA and miRNA biomarkers could be used to distinguish between PCa and BPH. Compared with RNA sequencing, B‐mode and CEUS features reflected genomic alterations associated with hormone receptor status, angiogenesis, and prognosis in patients with PCa. These findings indicate the potential of US to assess biomarker levels in patients with PCa

RhoB Acts as a Tumor Suppressor That Inhibits Malignancy of Clear Cell Renal Cell Carcinoma.

This study aims to investigate the biological role of RhoB in clear cell renal cell carcinoma (ccRCC). The expression of RhoB was examined in specimens of patients and cell lines by Western blot and Immunohistochemistry. The correlation between RhoB expression and clinicopathologic variables was also analyzed. The effects of RhoB on cell proliferation, cell cycle, cell apoptosis, and invasion/migration were detected by over-expression and knockdown of RhoB level in ccRCC cells via plasmids and RNAi. The results showed that RhoB was low-expressed in ccRCC surgical specimens and cell lines compared with adjacent normal renal tissues and normal human renal proximal tubular epithelial cell lines (HKC), and its protein expression level was significantly associated with the tumor pathologic parameter embracing tumor size(P = 0.0157), pT stage(P = 0.0035), TNM stage(P = 0.0024) and Fuhrman tumor grade(P = 0.0008). Further, over-expression of RhoB remarkably inhibited the cancer cell proliferation, colony formation and promoted cancer cell apoptosis, and aslo reduced the invasion and migration ability of ccRCC cells. Interestingly, up-regulation of RhoB could induce cell cycle arrest in G2/M phase and led to cell cycle regulators(CyclineB1,CDK1) and pro-apoptotic protein(casp3,casp9) aberrant expression. Moreover, knockdown of RhoB in HKC cells promoted cell proliferation and migration. Taken together, our study indicates that RhoB expression is decreased in ccRCC carcinogenesis and progression. Up-regulation of RhoB significantly inhibits ccRCC cell malignant phenotype. These findings show that RhoB may play a tumor suppressive role in ccRCC cells, raising its potential value in futural therapeutic target for the patients of ccRCC

MicroRNA-19a and microRNA-19b promote the malignancy of clear cell renal cell carcinoma through targeting the tumor suppressor RhoB

<div><p>Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma, which shows high aggressiveness and lacks biomarkers. RhoB acts as a tumor suppressor that inhibits the progression of ccRCC. In the present study, we examined the effects of oncogenic microRNAs, miR-19a and miR-19b, on RhoB expression in ccRCC cells. The results showed that both miR-19a and miR-19b could directly target the 3′untranslated region (3’UTR) of RhoB, resulting in the reduced expression of RhoB. With RT-PCR analysis, we detected the increased expression of miR-19a and miR-19b in ccRCC tissues compared to adjacent non-tumor renal tissues. These data also demonstrated an exclusive negative correlation between miR-19a/19b and RhoB expression in ccRCC specimens and cell lines. In addition, the knockdown of RhoB or overexpression of miR-19a and miR-19b in ccRCC cells could promote cell proliferation, migration and invasion. These data demonstrate the direct roles of miR-19a and miR-19b on the repression of RhoB and its consequences on tumorigenesis, cancer cell proliferation and invasiveness. These results suggest the potential clinical impact of miR-19a and miR-19b as molecular targets for ccRCC.</p></div

MiR-19a and miR-19b inhibitors reduce cell migration and invasiveness, and inhibit cell proliferation.

<p>(A) Representative images showing that the transfection of miR-19a and miR-19b inhibitors decreases the migration of 786-O cells by wound healing assay (Left); quantification of relative migration at 12 and 24 h (Right). (B) Representative images and quantification of relative migration, showing that the transfection of miR-19a and miR-19b inhibitors reduces the invasiveness of 786-O cells by transwell assay. The migratory activities of 786-O cells transfected with control oligo were was included as a negative inhibitor control. (C) The proliferation potential of 786-O cells transfected with miR-19a and miR-19b inhibitors or control oligo was determined by the MTS Assay. The data are representative of three independent experiments. *Significant differences from control oligo-transfected cells (P<0.05).</p

Primer sequences used in the present study.

<p>Primer sequences used in the present study.</p

MiR-19a and miR-19b inhibitors induce cell apoptosis.

<p>(A) Flow cytometry results of Annexin-V5 analysis in 786-O cells transfected with miR-19a and miR-19b inhibitors. An inhibitor control was included as a negative control. Graphs showing changes of the apoptotic percentage of the cells (left); quantification of relative apoptosis (right). The data represent the means ± S.D. from three independent experiments performed in triplicate. *Significant differences from control oligo-transfected cells (P<0.05) (B) Western blot results showing the effect of miR-19a and miR-19b inhibitors on the expression of cleaved caspase-9 in 786-O cells. (C) Potential mechanism showing the effect of the negative regulation of RhoB by miR-19a/b on the proliferation, migration, invasion and apoptosis of ccRCC.</p

miR-19a/19b and RhoB expression levels are negatively correlated in patient tissues samples and cell lines.

<p>(A) QRT-PCR analysis for miR19a/b and RhoB expression in paired patient specimens (n = 70). (B) QRT-PCR analysis for miR19a/b in patient specimens (n = 8). (C) Western blot analysis for RhoB expression in the same specimens as panel B. N1-8 are normal tissues and A1-8 are paired tumor tissues. (D and E) QRT-PCR and Western blot analysis for miR19a/b and RhoB expression in normal renal cells and ccRCC cell lines. β-actin was used as loading control for western blot. The data represent the average of three independent experiments. *Indicates statistical significance (P<0.05).</p

Overexpression of miR-19a and miR-19b or RhoB knockdown show similar phenotypes in promoting the growth, migration and mobility of ccRCC cells.

<p>(A) MTS results showing the promotion potential of miR-19a and miR-19b mimics overexpression or RhoB knockdown on the proliferation of A498 cells. (B) Representative images and quantification show the clonogenic plating efficacy of A498 cells overexpressing miR-19a and miR-19b mimics. (C and D) Representative images (right) and quantification (left) of wound healing assay (C) and transwell assay (D) showing the potential effects of the overexpression of miR-19a and miR-19b mimics on the migration and invasiveness of the cells. Control oligo was included as a negative control. (E and F) Representative images (right) and quantification (left) of wound healing assay (E) and transwell assay (F) showing the potential effects of RhoB knockdown on the migration and invasiveness of the cells. Control siRNA was included as a negative control. The data are representative of three independent experiments performed in triplicate. *Significant differences from control oligo-transfected cells (P<0.05).</p

miR-19a and miR-19b regulate RhoB expression.

<p>(A and B) QRT-PCR analysis for miR19a/b (top) and Western -blot analysis of RhoB (bottom) expression in A498 and 786-O cells after transfection with miR-19a and miR-19b mimics or inhibitors for 48 h. Control mimics or inhibitors were included as a negative control. β-actin was included for equal protein loading. (C) The predicted binding motif sequence of miR-19a and miR-19b in the 3’-UTR region of RhoB. (D) Reporter assay showing the effect of miR19a/b mimics on the luciferase activity of reporter plasmids containing RhoB-3’UTR-wt, RhoB-3’UTR-mut or empty plasmid psi-check2 in A498 cells transfected with miR19a/b mimics. Control oligo was included as a negative control. (E) Reporter assay showing the effect of miR-19a and miR-19b inhibitors on luciferase activity of reporter plasmids containing RhoB-3’UTR-wt, RhoB-3’UTR-mut or empty plasmid psi-check2 in 786-O cells. The data represent the average of three independent experiments. *Indicates statistical significance (P<0.05).</p