MicroRNA-21 inhibits p57Kip2 expression in prostate cancer
© Mishra et al.; licensee BioMed Central Ltd. 2014
Received: 5 June 2014
Accepted: 9 September 2014
Published: 12 September 2014
p57Kip2, a cyclin-dependent kinase inhibitor, is considered to be a candidate tumor suppressor gene that has been implicated in Beckwith-Wiedemann syndrome and sporadic cancers. In addition, decreased expression of p57Kip2 protein has been frequently observed in pancreatic, lung, breast, bladder, gastrointestinal tract and prostate cancers. However, p57Kip2 gene mutations are rare in these cancers suggesting that other unknown mechanisms might be at play in reducing its expression. The aim of this study was to investigate the molecular mechanism of down-regulation of p57Kip2 in prostate cancer.
We observed a significant negative correlation between the expression of p57Kip2 and microRNA-21 (miR-21) in prostate cancer samples and after androgen deprivation with castration in the CWR22 human prostate cancer xenograft model. We report that miR-21 targeted the coding region and decreased p57Kip2 mRNA and protein levels in prostate cancer cells. Conversely, inhibition of endogenous miR-21 by an anti-miR-21 inhibitor strongly induced p57Kip2 expression. Furthermore, we found that knockdown of p57Kip2 reversed the effects of the anti-miR-21 inhibitor on cell migration and anchorage-independent cell growth.
Our results indicate that miR-21 is able to downregulate p57Kip2 expression by targeting the coding region of the gene and is also able to attenuate p57Kip2 mediated functional responses. This is the first report demonstrating that p57Kip2 is a novel target of miR-21 in prostate cancer and revealing a novel oncogenic function of this microRNA.
Uncontrolled cell proliferation due to aberrant regulation of cell cycle control can lead to the development of cancer. Cyclin dependent kinase inhibitors (CKIs) are the common inhibitors of cell cycle which consists of two families: INK4 family (consisting of p16INK4a, p15INK4b, p18INK4c and p19INK4d) and Cip/Kip family (p21CIP1/WAF1, p27Kip2 and p57Kip2) . Human p57Kip2 gene is maternally expressed and paternally imprinted and is located on chromosome 11p15.5 , which is implicated in Beckwith-Wiedemann syndrome  and in sporadic cancers. p57Kip2 is also required for normal development as p57Kip2 null mice die at 2 weeks of age and show increased apoptosis and delayed differentiation during mouse development . Because of its chromosomal location, imprinting status and functional activities, p57Kip2 is considered to be a candidate tumor suppressor gene. p57Kip2 overexpression in LNCaP prostate cancer cells resulted in the conversion of adenocarcinoma to a more differentiated squamous tumor in nude mice, with reduced cell proliferation and tumor invasion . p57Kip2 is considered to be a tumor suppressor gene since it functions to block cell proliferation by inhibiting cell cycle progression, promotes apoptosis and cell differentiation, inhibits tissue invasion and metastasis and also inhibits angiogenesis . Hence, cancer cells frequently down-regulate p57Kip2 in order to gain a proliferative advantage.
Loss of or reduced p57Kip2 expression occurs in carcinomas of the prostate, bladder, liver, pancreas, breast and others. The absence of p57Kip2 gene mutations in a wide variety of cancers suggests that other transcriptional or post-transcriptional mechanisms might be involved in its down-regulated protein expression. Inactivation of p57Kip2 gene due to promoter DNA methylation was observed in non-small cell lung cancer and in lymphoid malignancies of B-cells [7, 8]. Yang et al. found that p57Kip2 expression in breast cancer cells was repressed due to Polycomb protein EZH2-mediated H3K27me3 chromatin mark. Increased p57Kip2 degradation due to ubiquitylation by E3 ligase Skp1/ Cul1/ F-box (SCF complex) was observed in non-small cell lung carcinoma and hepatocellular carcinoma [9, 10].
Mature miRNAs are ~22 nucleotides long non-coding single-stranded RNAs, which upon binding to the 3’-UTR region of target mRNAs can result in mRNA cleavage, or translational repression. p57Kip2 has been reported to be targeted by miR-221/222 cluster in gastric carcinoma, ovarian cancer and hepatocellular carcinoma . miR-92b and miR-25 have also been reported to down-regulate p57Kip2 expression in human embryonic stem cells and in gastric cancer respectively [12, 13]. Jin et al. demonstrated that expression of p57Kip2 is significantly decreased in human prostate cancer and overexpression of p57Kip2 in prostate cancer cells decreased cell proliferation and reduced invasiveness . However, the mechanism behind p57Kip2 down-regulation in prostate cancer cells has not been investigated. Unexpectedly, we discovered that p57Kip2 is one of the novel downstream target genes of miR-21 in prostate cancer. We observed a very strong negative correlation between p57Kip2 and miR-21 expression in human prostate tumor samples and in CWR22, a human prostate cancer xenograft model. Our results for the first time show that miR-21 can down-regulate p57Kip2 mRNA and protein expression by targeting its coding region to attenuate its activity in prostate cancer cells. Thus, therapeutic approaches aimed at restoring p57Kip2 expression might be beneficial for prostate cancer prevention and therapy.
Findings and discussion
MicroRNA-21 targets p57Kip2 gene
MicroRNA-21 downregulates p57Kip2 expression
MicroRNA-21 abrogates p57Kip2-mediated functional responses in prostate cancer cells
In summary, we discovered p57Kip2 to be a novel target gene of microRNA-21 in prostate cancer. Our findings provide a novel mechanism of p57Kip2 downregulation in prostate cancer. These findings warrant further research to test if p57Kip2 is also a novel target gene of miR-21 in other cancer types as well. In our previous study, we found miR-21 to be an oncogenic regulator in prostate cancer by targeting the tumor suppressive effects of TGF-beta signaling pathway in cancer cells . Given, the regulation of p57Kip2 by microRNA-21 in the current study, we provide a strong rationale to perform preclinical testing of microRNA-21 inhibitor as a novel therapeutic drug for prostate cancer.
We thank Dr. Majeet Rao for the pMIR luciferase construct and Dr. Clifford G. Tepper for the CWR22 xenograft tissue. This work was supported in part by funding from NIH grants RO1CA079683, RO1CA172886, and P30CA054174.
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