cc-RCC is the most common type in sporadic renal cancer and ~60% of cc-RCC patients have mutated or inactivated VHL
genes. Usually, pVHL inhibits the HIF-1 and beta-catenin signaling pathway. Thus, loss of pVHL results in the aberrant accumulation of cytoplasmic and nuclear beta-catenin and HIF, accelerating tumorigenesis (6
CTNNB1 (beta-catenin) has been known as a key player in the Wnt/beta-catenin signaling pathway in several cancers including renal cancer (16
). MEK1 (MAP2K1) acts as a mitogen-activated protein (MAP kinase kinase) and plays an important role as an essential component of the MAP kinase signal transduction pathway. MEK1 is also involved in several cellular processes, including proliferation and transcription regulation in several cancers including renal cancer. In the present study, we found that aberrant MEK1 expression in human renal cancer tissues was correlated with higher pathological stage (pTNM) and shorter overall survival and recurrence interval after radical nephrectomy. Kaplan–Meier plots also showed an inverse correlation between MEK1 expression and renal cancer patients’ outcomes. Several MEK1 inhibitors have been identified and have been evaluated in phases I, II and III clinical trials in several cancers (17
). The effect of MEK1 inhibitors alone or in combination with others, such as mammalian target of rapamycin inhibitors or epidermal growth factor receptor inhibitors has been reported in the treatment of several cancers, including RCC (18
). However, problems remain regarding the side effect and efficacy of these treatments. Thus, it is important to find new and safe options or approaches to achieve renal cancer remission. Therefore, we focused on miRNAs as a potential new treatment strategy. We initially searched for tumor-suppressive miRNAs inhibiting the two major cancer pathways, including beta-catenin and HIF-1 downstream genes (MEK1
) with miRDB. This led to the identification of miR-1826 that only targets beta-catenin and MEK1.
Regarding the relationship between miRNA and CTNNB1, Xia et al.
) has previously reported that miR-200a functions as a tumor suppressor by directly regulating CTNNB1 expression in nasopharyngeal carcinoma. Saydam et al.
) found that miR-200a plays an important role as a tumor suppressor and directly targets CTNNB1 mRNA. They also showed that miR-200a blocks Wnt/beta-catenin signaling in meningioma cells (21
). However, there have been no reports related to the role of miRNA and CTNNB1 in renal cancer.
Similarly, miRNAs studies involving MEK1 have found that miR-34a inhibits cell proliferation by repressing MEK1 during megakaryocytic differentiation (22
). Also miR-424 regulates cell proliferation via the silencing of MEK1 and cyclin E1 in senile hemangioma (23
). As far as we know, there have been no reports concerning the relationship of miRNA, MEK1 and renal cancer.
Only one miRNA-1826 was found to target both CTNNB1 and MEK1. This was also shown by 3′ UTR luciferase assays, indicating that relative luciferase levels were significantly lower in miR-1826-transfected renal cancer cells compared with miR-NC-transfected controls. Though mRNA expression of CTNNB1 and MEK1 was not changed in miR-1826-transfected cells (data not shown), the protein expression of CTNNB1 and MEK1 was significantly downregulated compared with miR-NC-transfected cells. Our results are consistent with the fact that miRNAs can bind to the 3′ UTR of target mRNA and repress translation from mRNA to protein (11
). Thus, our results suggest that CTNNB1 and MEK1 are direct targets of miR-1826. We also performed an immunohistochemical study of CTNNB1 and MEK1 to examine the relationship between miR-1826 and CTNNB1 or MEK1 expression levels in human renal cancer tissues. We found an inverse correlation between miR1826 and CTNNB1 or MEK1 protein expression.
During renal cancer progression, beta-catenin (CTNNB1) is translocated into the nucleus, binds to T cell-factor/lymphoid-enhancer-factor transcriptional factors, activates target genes and thereby promotes tumorigenesis (8
). Thus, we performed western analysis of survivin, a member of the T cell-factor/lymphoid-enhancer-factor downstream effectors, to assess whether miR-1826 inhibits downstream Wnt/beta-catenin signaling by beta-catenin downregulation. We found that expression of survivin was also downregulated by miR-1826 in transfected cells. Parker et al.
) reported that high survivin expression is an independent predictor of cc-RCC progression and death from RCC. Moreover, several other laboratories have reported that the expression of survivin is associated with renal cancer aggressiveness and an important prognostic marker for renal cancer (26
). Although other factors, such as insulin-like growth factor 1, interferon and nuclear factor-kappaB also regulate survivin protein expression (28
), miR-1826 may be an additional important inhibitor of survivin in cc-RCC.
As a next step, we found that the expression of miR-1826 was significantly downregulated in renal cancer tissues compared with matched normal kidney tissues (n = 46). These results are consistent with those of miR-1826 expression in normal kidney and renal cancer cell lines, suggesting that miR-1826 may have tumor-suppressive functions in renal cancer. We did find a significantly shorter overall survival and earlier recurrence after radical nephrectomy in patients with ‘lower expression of miR-1826’. This result suggests that low miR-1826 expression in renal cancer tissues may contribute to poor patient prognosis. However, our sample number is relatively small, a larger study will be needed to look at the correlation between miR-1826 expression and clinical parameters. We also performed several functional assays using miR-1826 or miR-NC-transfected VHL-inactivated renal cancer cells (A-498 and 786-O). As expected, we found that overexpression of miR-1826 significantly inhibited renal cancer cell proliferation and also significantly inhibited renal cancer cell migration and invasion abilities. We also found that miR-1826 induced significant G1 cell cycle arrest and apoptosis in VHL-inactivated renal cancer cells. To validate whether miR-1826 plays a tumor-suppressive role by inhibiting CTNNB1 and MEK1 expression, we performed functional analyses of CTNNB1 and MEK1 in A-498 cells using a siRNA technique. The knockdown of CTNNB1 and MEK1 was confirmed at the mRNA (data not shown) and protein levels. We, then performed functional analyses (MTS and invasion analyses) and found an effect similar to that of miR-1826 overexpression. Namely, CTNNB1 or MEK1 knockdown resulted in inhibition of renal cancer cell proliferation and invasion ability. Taken together, this evidence suggests that miR-1826 exerts its tumor-suppressive effects through beta-catenin and MEK1 downregulation in renal cancer cells.
In conclusion, this is the first report documenting that miR-1826 expression is significantly decreased in human renal cancer tissues where it functions as a tumor suppressor by inhibiting CTNNB1 and MEK1 expression. Our results suggest that miR-1826 may play a therapeutically important role in renal cancer patients, especially in those with VHL-inactivated renal cancer.