MicroRNA-214 Suppresses Oncogenesis and Exerts Impact on Prognosis by Targeting PDRG1 in Bladder Cancer

<div><p>MicroRNA-214 (miR-214) has been reported to be dysregulated in human bladder cancer tissues. We aimed to investigate the clinical correlation, biological significance and molecular network of miR-214 in bladder cancer. Our results showed miR-214 was down-regulated in bladder cancer tissues and significantly associated with tumor stage, lymph node status, grade, multifocality, history of non-muscle-invasive bladder cancer (NMIBC). Moreover, miR-214 could serve as an independent factor of recurrence-free survival (RFS) and overall survival (OS) for patients with muscle-invasive bladder cancer (MIBC). Restoration of miR-214 expression in bladder cancer cell lines inhibited cell proliferation, migration, invasion and markedly promoted apoptosis. Dual-luciferase reporter assay recognized PDRG1 as direct downstream target gene of miR-214. PDRG1 was significantly increased in tumors low of miR-214 and knockdown of PDRG1 mimicked the effects of miR-214 overexpression. Our findings manifest that miR-214 could exert tumor-suppressive effects in bladder cancer by directly down-regulating oncogene PDRG1 and suggest an appealing novel indicator for prognostic and therapeutic intervention of bladder cancer.</p></div

miR-214 restoration inhibited cell migration and invasion <i>in vitro</i>.

<p>(<b>A</b>) Wound healing assay, (<b>B</b>) Transwell migration assay and (<b>C</b>) Transwell invasion assay in mock/miR-NC/miR-214 transfected bladder cancer cells. Error bars correspond to mean ± SEM. ***<i>P</i> < 0.001, t-test, n = 6.</p

PDRG1 knockdown inhibited cell migration and invasion <i>in vitro</i>.

<p>(<b>A</b>)Wound healing assay, (<b>B</b>) Transwell migration assay and (<b>C</b>) Transwell invasion assay in bladder cancer cells transfected with mock/si-NC/si-PDRG1. Error bars correspond to mean ± SEM. ***<i>P</i> < 0.001, t-test, n = 6.</p

miR-214 reintroduction inhibited cell proliferation and promoted apoptosis <i>in vitro</i>.

<p>(<b>A</b>) The miR-214 expression was significantly downregulated in bladder cancer cell lines than that in normal bladder cell line (SV-HUC-1). (<b>B</b>) Relative expression of miR-214 after miRNA mimic transfections as determined by qRT- PCR. (<b>C</b>) Cell viability assay in mock/miR-NC/miR-214 transfected bladder cancer cells (statistical analysis between miR-NC and miR-214: ***<i>P</i> < 0.001, t-test, n = 4). (<b>D</b>) Flow cytometry analysis showed that enforced expression of miR-214 significantly promoted apoptosis. Error bars correspond to mean ± standard error of the mean (SEM). ***<i>P</i> < 0.001, t-test, n = 6.</p

PDRG1 knockdown inhibited cell proliferation and promoted apoptosis <i>in vitro</i>.

<p>(<b>A</b>) Relative expression of PDRG1 mRNA after siRNA transfections as determined by qRT- PCR. (<b>B</b>) Protein levels of PDRG1 were assessed by western blot in si-NC/si- PDRG1 bladder cancer cells with β-actin as a loading control. (<b>C</b>) Cellular proliferation assay in bladder cancer cells transfected with mock/si-NC/si-PDRG1 (statistical analysis between si-NC and si-PDRG1: ***<i>P</i> < 0.001, t-test, n = 4). (<b>D</b>) Flow cytometry analysis indicated that PDRG1 knockdown significantly promoted apoptosis. Error bars correspond to mean ± SEM. ***<i>P</i> < 0.001, t-test, n = 6.</p

Clinicopathologic parameters and expression of miR-214 and PDRG1 in bladder cancer.

<p>Clinicopathologic parameters and expression of miR-214 and PDRG1 in bladder cancer.</p

Aberrant CCR4 Expression Is Involved in Tumor Invasion of Lymph Node-Negative Human Gastric Cancer

<div><p>Cellular chemotaxis is the best-known function of chemokine receptors which are closely linked with tumor metastasis. In fact, positive expression of chemokine receptors could also be identified even in some patients without metastatic tumors, while the clinical relevance of this data has not been fully established. Our studies were designed to clarify the CCR4 expression profiles and to explore its potential role in histologically node-negative (pN0) gastric cancer (GC). Immunohistochemistry (IHC) or immunohistofluorescence (IHF) analysis was performed on specimens obtained from 108 patients with pN0 GC. We found that CCR4 was aberrantly over-expressed inpN0 GC tissues, with different expression patterns on tumor cells and being associated with T-stage (<i>P</i> = 0.002). The matrigel <i>in vitro</i> invasion assay revealed that over-expression of CCR4 in GC cell lines significantly enhanced the invasive capacity of these cells. Results from real-time RT-PCR and gelatinzymography showed a significant increase in matrix metalloproteinase (MMP)-9 production induced by the forced expression of CCR4 in GC cell lines. Our data suggest that the aberrant CCR4 expression is involved in tumor invasion of pN0 GC and, conceivably, antagonists of CCR4 might be useful candidates for controlling early events in tumor progression.</p></div

miR-214 negatively regulated PDRG1 expression by directly binding to its 3′-UTR complimentary sequences.

<p>(<b>A</b>) Sketch of the presumptive miR-214 binding sequences in PDRG1 3′-UTR and structure of wild-type or mutant PDRG1 pmiR-REPORT vectors. The mutant binding site was italicized and underlined. (<b>B</b>) Dual-Luciferase activity assay was performed in bladder cancer cells cotransfected with miR-NC/ miR-214 and pmiR-REPORT vectors containing WT- PDRG1 3′-UTR/ MUT-PDRG1 3′-UTR sequences. Data were displayed as normalized fold change in relative luciferase activity (Rluc/Luc). Error bars correspond to mean ± SEM. ***<i>P</i> < 0.001, t-test, n = 6. (<b>C</b>) The PDRG1 mRNA was down-regulated in bladder cancer cells transfected with miR-214 by qRT-PCR. Error bars correspond to mean ± SEM. ***<i>P</i> < 0.001, t-test, n = 6. (<b>D</b>) Western blot results of endogenous PDRG1 protein expression in bladder cancer cells transfected with miR-NC/ miR-214 with β-actin as a loading control. (<b>E</b>) Spearman’s correlation analysis indicated an inverse correlation between miR-214 expression and PDRG1 mRNA levels in bladder cancer tissues.</p

miR-214 expression was frequently attenuated in bladder cancer tissues and associated with poor progonosis.

<p><b>(A)</b> Expression level of miR-214 was determined by means of qRT-PCR and normalized against U6 RNA, an endogenous control, in 106 pairs of human bladder cancer tissues (BC) and matched adjacent normal tissues (NT). ***<i>P</i> < 0.001, Wilcoxon test, n = 106. (<b>B</b>) The attenuated expression of miR-214 was found in 74% (78 of 106) of bladder cancer tissues compared with adjacent normal specimens. (<b>C</b>) Relative expression of miR-214 was presented as log₂ fold change (BC/NT) and the log₂ fold change was defined as follows: <1, underexpression; >1, overexpression; the remaining was defined as unchanged. (<b>D</b>) Kaplan-Meier RFS and OS curves based on the miR-214 expression levels of patients with bladder cancer. The cut-off point was the median miR-214 expression level in the bladder cancer tissue samples. (<b>E</b>) Kaplan-Meier RFS and OS curves based on the miR-214 expression levels of patients with MIBC. The cut-off point was the median miR-214 expression level in the MIBC tissue samples.</p

CCR4 expression levels in human gastric cancer cells and non-cancerous gastric tissues.

<p>CCR4 was detected at different levels, including negative expression (-) (A), weak expression (+) (B), moderate expression (++) (C) and strong expression (+++) (D). The expression was negative in non-neoplastic cells (the right area in the image) adjacent to CCR4 positive gastric cancer cells (E), and weak expression of membrane CCR4 was found in gastric preneoplastic lesions (F). Magnification, ×400 (A-D, F), ×200 (E).</p