In the current study, we have found for the first time that miR-424, a direct target of HIF-1α, decreases CDDP sensitivity of bladder cancer cells through down-regulation of pro-apoptotic
UNC5B and
SIRT4 (Fig.
6f). Our present results unveiled the molecular mechanisms how bladder cancer cells acquire and/or maintain CDDP resistance under normoxia, and also might provide a clue to develop a promising strategy to treat CDDP-resistant bladder cancer patients.
miR-424 has been initially identified as a differentiation-specific miRNA, which played an important role in the regulation of the monocyte/macrophage differentiation program [
27]. In addition to differentiation, a growing body of evidence suggests that miR-424 has a dual function (oncogene or tumor-suppressor) during carcinogenesis, dependent on its target genes [
28‐
33]. According to our present results, miR-424 was significantly increased in bladder cancer tissues, especially in high-grade tumors, and its higher expression level was associated with the poor clinical outcome, indicative of its oncogenic role in bladder cancer. Forced expressions of miR-424 and its mimics conferred CDDP resistance on bladder cancer 5637 and T24 cells in vitro and in vivo, while miR-424 inhibitor enhanced the cytotoxic activity of CDDP in these bladder cancer cells, suggesting that miR-424 contributes to the acquired CDDP resistance of bladder cancer. Notably, CDDP treatment induced miR-424 expression in a HIF-1α-dependent manner in bladder cancer 5637 cells. Collectively, our findings imply that targeting miR-424 might be an effective therapeutic strategy against refractory bladder cancers.
Another new finding of the present study was that pro-apoptotic
UNC5B and
SIRT4 are novel downstream target genes of miR-424. Based on our present results, forced expression of miR-424 mimics reduced
UNC5B and
SIRT4 expressions in bladder cancer 5637 and T24 cells, whereas miR-424 inhibitor increased
UNC5B and
SIRT4 expressions in these cells. Consistent with these results, we have identified a potential miR-424-binding site within the 3′-UTR of
UNC5B and
SIRT4. Luciferase reporter assay revealed that miR-424 remarkably prohibits the reporter activity driven by the wild-type but not the mutated 3′-UTR of these genes, suggesting that
UNC5B and
SIRT4 are the direct downstream targets of miR-424. Inverse correlations between miR-424 and
UNC5B or
SIRT4 expression levels in bladder cancer tissues further supported the negative regulation of
UNC5B and
SIRT4 by miR-424 in vivo. Additionally, siRNA-mediated silencing of either
UNC5B or
SIRT4 resulted in a significant decrease in the sensitivity to CDDP of bladder cancer 5637 and T24 cells, whereas miR-424-mediated decrease in CDDP sensitivity was partially restored by forced expression of UNC5B or SIRT4. Thus, these results indicate that
UNC5B and
SIRT4 play a vital role in the regulation of CDDP-mediated apoptotic response in bladder cancer cells. In support of this notion, it has been shown that UNC5B has an ability to potentiate tumor suppressor p53-dependent apoptotic cell death in response to DNA damage [
17]. UNC5B, a dependence receptor, was capable to trigger apoptotic response in the absence of its ligand netrin-1 by the activation of DAPK (death associated protein kinase) through PP2A (protein phosphatase 2A)-mediated its dephosphorylation, which subsequently induced caspase-3 activation [
18]. Meanwhile, several lines of evidence indicate that SIRT4, which is localized in mitochondria, acts as a tumor suppressor [
19‐
23]. From their results, SIRT4 participated in DNA damage-mediated metabolic responses and repressed mitochondrial glutamine metabolism [
19]. Notably, it has been shown that
SIRT4-knockout mice spontaneously develop several types of tumors and SIRT4 expression is significantly down-regulated in certain human cancers [
19]. Together with these findings, our results suggest that miR-424-mediated decrease in the expressions of tumor suppressor
UNC5B and
SIRT4 play a pivotal role in the acquisition and/or the maintenance of CDDP resistance in bladder cancer.
In accordance with the previous observations [
9‐
12], our present observations demonstrated that HIF-1α is required for CDDP-mediated transactivation of miR-424 in bladder cancer cells. CDDP treatment in bladder cancer 5637 cells induced miR-424 expression in a HIF-1α-dependent manner under normoxia, which was consistent with the recent study describing that miR-424 is implicated in the resistance to doxorubicin and etoposide of colon cancer HCT116 cells and malignant melanoma A375 cells under normoxia [
15]. It has been also shown that certain anti-cancer drugs contribute to the acquisition of the chemo-resistance through up-regulation of HIF-1α under normoxia [
9‐
12]. Indeed, our present results revealed that CDDP treatment increases HIF-1α protein level, its recruitment onto the putative HIF-1α-binding site of miR-424 and enhances its transcriptional activity in bladder cancer cells under normoxia. Therefore, it is suggestive that HIF-1α/miR-424/UNC5B/SIRT4 regulatory axis contributes to the acquisition and/or the maintenance of CDDP resistance of bladder cancer cells irrespective of hypoxia. In other words, current study suggests that combination chemotherapy with HIF-1a inhibition would target the normoxic bladder cancer cells, which may help to optimize conventional chemotherapy with HIF-1 inhibitors. However, due to the important function of HIF signaling pathway in stimulating red blood cell proliferation, a common side effect of HIF inhibitors is anemia, which will also affect their safety in the treatment of cancer patients. Therefore, it is necessary to carry out further clinical exploration and safety assessment.