The purpose of this study was to identify prostate cancer (PC) oncogenic microRNAs (miRs) based on miR microarray and to investigate whether these oncogenic miRs may be useful as PC biomarkers.
...Initially, we carried out miR microarray and real-time PCR using RWPE-1, PC-3, DU-145 and LNCaP cells. To investigate the function of miR-183, we used a miR-183 knockdown inhibitor in cell growth and wound-healing assays. We used several algorithms and confirmed that they are directly regulated by miR-183.
We identified three potential oncogenic miRs (miR-146a, miR-183 and miR-767-5P). The expression of miR-183 in PC cells (PC-3, DU-145 and LNCaP) was upregulated compared with RWPE-1 cells. MiR-183 expression was also significantly higher in PC tissues compared with that in matched normal prostate tissues. Additionally, miR-183 expression was correlated with higher prostate-specific antigen, higher pT and shorter overall survival. MiR-183 knockdown decreased cell growth and motility in PC cells and significantly decreased prostate tumour growth in in vivo nude mice experiments. We identified Dkk-3 and SMAD4 as potential target genes of miR-183.
Our data suggest that oncogenic miR-183 may be useful as a new PC biomarker and that inhibition of miR-183 expression may be therapeutically beneficial as a PC treatment.
Recently several microRNAs (miRNAs) have been found to be regulated by genistein in cancer cells. In this study, we focused on the gene regulatory effect of genistein on microRNA and its target genes ...in prostate cancer (PC).
Initially, we investigated the effect of genistein on prostate cancer cells and identified that the expression of miRNA-1260b was decreased by genistein. We performed functional analyses and investigated the relationship between miRNA-1260b expression and prostate cancer patient outcomes. Two target genes (sFRP1 and Smad4) of miR-1260b were identified based on computer algorithm and 3'UTR luciferase assay was carried out to determine direct miRNA regulation of the genes.
Genistein promoted apoptosis while inhibiting prostate cancer cell proliferation, invasion and TCF reporter activity in PC cells. MiR-1260b was highly expressed in prostate cancer tissues and significantly downregulated by genistein in PC cells. After knocking down miR-1260b, cell proliferation, invasion, migration and TCF reporter activity were decreased in PC cells. Western analysis and 3'UTR luciferase assay showed that the two target genes (sFRP1 and Smad4) were directly regulated by miR-1260b. The expression of sFRP1 and Smad4 was significantly decreased in prostate cancer tissues. Genistein also increased expression of these two genes via DNA demethylation and histone modifications.
Our data suggest that genistein exerts its anti-tumour effect via downregulation of miR-1260b that targeted sRRP1 and Smad4 genes in prostate cancer cells. The expression of sFRP1 and Smad4 was also modulated by genistein via DNA methylation or histone modifications in PC cell lines.
Wnt-signalling has an important role in renal cancer and it is modulated by genistein in other cancers. Recently, microRNAs (miRNAs) have emerged as new regulators of gene expression. Thus, we ...focused on miRNAs to examine the regulatory mechanism of genistein on the Wnt-signalling pathway in renal cell carcinoma (RCC).
Initially, we investigated the effect of genistein on Wnt-signalling (TOPflash reporter assay (TCF reporter assays)) in renal cancer cells, and using microarray identified candidate miRNAs whose expression was decreased by genistein. We performed functional analyses and investigated the relationship between miRNA expression and renal cancer patient outcomes. We also did 3'UTR luciferase assays to look at direct miRNA regulation of Wnt-signalling-related genes.
Genistein promoted apoptosis while inhibiting RCC cell proliferation and invasion. Genistein also decreased TCF reporter activity in RCC cells. We found that miR-1260b was highly expressed and significantly downregulated by genistein in RCC cells. The expression of miR-1260b was significantly higher in renal cancer tissues compared with normal, and significantly related to overall shorter survival. In addition, miR-1260b promoted renal cancer cell proliferation and invasion in RCC cells. The 3'UTR luciferase activity of target genes (sFRP1, Dkk2, Smad4) was significantly decreased and their protein expression significantly upregulated in miR-1260b inhibitor-transfected renal cancer cells.
Our data suggest that genistein inhibited Wnt-signalling by regulating miR-1260b expression in renal cancer cells.
The purpose of this study was to identify new tumour suppressor microRNAs (miRs) in clear cell renal cell carcinoma (ccRCC), carry out functional analysis of their suppressive role and identify their ...specific target genes.
To explore suppressor miRs in RCC, miR microarray and real-time PCR were performed using HK-2 and A-498 cells. Cell viability, invasion and wound healing assays were carried out for functional analysis after miR transfection. To determine target genes of miR, we used messenger RNA (mRNA) microarray and target scan algorithms to identify target oncogenes. A 3'UTR luciferase assay was also performed. Protein expression of target genes in ccRCC tissues was confirmed by immunohistochemistry and was compared with miR-584 expression in ccRCC tissues.
Expression of miR-584 in RCC (A-498 and 769-P) cells was downregulated compared with HK-2 cells. Transfection of miR-584 dramatically decreased cell motility. The ROCK-1 mRNA was inhibited by miR-584 and predicted to be target gene. The miR-584 decreased 3'UTR luciferase activity of ROCK-1 and ROCK-1 protein expression. Low expression of miR-584 in ccRCC tissues was correlated with high expression of ROCK-1 protein. The knockdown of ROCK-1 by siRNA inhibited cell motility.
miR-584 is a new tumour suppressor miR in ccRCC and inhibits cell motility through downregulation of ROCK-1.
The canonical Wnt signalling pathway is activated in most sporadic colorectal cancers (CRCs). We previously reported that FZD7 functions as a receptor for the canonical Wnt signalling pathway in ...colon cancer cells.
In this study, we examined the function of FZD7 in survival, invasion and metastatic capabilities of colon cancer cells. FZD7_siRNA transfection decreased cell viability of HT-29 and HCT-116 colon cancer cells. Expression of c-Jun, phosphorylation of JNK and c-Jun, and activation of RhoA were suppressed after FZD7_siRNA transfection into HCT-116 cells. In vitro invasion activity and Wnt target gene expression were also reduced in HCT-116 cells transfected with FZD7_siRNA. Liver metastasis of stable FZD7_siRNA HCT-116 cell transfectants in scid mice was decreased to 40-50% compared to controls. The mRNA levels of FZD7 in 135 primary CRC tissues were examined by real-time PCR. FZD7 mRNA levels were significantly higher in stage II, III or IV tumours than in non-tumour tissues (P<0.005), and overall survival was shorter in those patients with higher FZD7 expression (P<0.001).
These data suggest that FZD7 may be involved in enhancement of survival, invasion and metastatic capabilities of colon cancer cells through non-canonical Wnt signalling pathways as well as the canonical pathway.
Ras association domain family 1A (RASSF1A) is a tumor suppressor that regulates the cell cycle, apoptosis, and microtubule stability. The association between the methylation levels of RASSF1A and the ...prognosis of clear-cell renal cell carcinoma (CCRCC) remains unclear. Therefore, we investigated this relationship to determine the prognostic value of RASSF1A methylation levels for CCRCC.
The study comprised 179 Japanese patients who underwent radical or partial nephrectomy for CCRCC. The methylation level of 5′ CpG islands in the RASSF1A was evaluated using combined bisulfite restriction analysis and bisulfite sequencing.
High levels of methylation in the RASSF1A promoter were significantly more frequent in grade 3 compared with grade 1 or 2 tumors (P = 0.028) and in patients with stage III or IV compared with patients with stage I or II (P = 0.043). Patients with high methylation levels had a significantly less favorable prognosis compared with those with low methylation levels (P = 0.040). Higher methylation levels were independently associated with a poor prognosis following multivariate analysis (P = 0.0053).
These results indicate that quantitative promoter methylation levels of the RASSF1A gene may be a useful marker to predict the prognosis of CCRCC.
BACKGROUND/AIM Matrilysin is one of the matrix metalloproteinases that has a critical role in tumour invasion, and is often expressed in gastrointestinal cancers. The aim of this study was to examine ...the role of matrilysin in metastasis of human colorectal cancers. PATIENTS (SUBJECTS)/METHODS The relation between matrilysin expression and Dukes’s type was investigated immunohistochemically in 83 surgically resected colorectal cancers, including five with liver metastasis. Moreover, the effects of matrilysin on the in vivo invasive and metastatic potential of colon cancer cells transfected with matrilysin cDNA were examined after subcutaneous injection into SCID mice. RESULTS In 46% of primary and all of metastatic liver tumours, over 10% of cancer cells were stained positively for matrilysin. The expression of matrilysin correlated significantly with the presence of nodal or distant metastases (p<0.05). In addition, matrilysin transfectants formed invasive tumours and multiple liver metastases in SCID mice, without producing any significant difference in the subcutaneous tumour growth from mock transfectants. Casein zymography showed that the invading and metastasised tumours showed conspicuous matrilysin activity, which correlated with the number of metastatic lesions (p<0.001). CONCLUSIONS Matrilysin showed a correlation with metastasis in a cohort of 83 colorectal cancer patients and marked metastatic potentiation in human colorectal cancer xenografts, indicating that it may play a critical role in the metastatic pathway of colorectal cancers.
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