MicroRNA-7, a homeobox D10 target, inhibits p21-activated kinase 1 and regulates its functions

Abstract

MicroRNAs are noncoding RNAs that inhibit the expression of their targets in a sequence-specific manner and play crucial roles during oncogenesis. Here we show that microRNA-7 (miR-7) inhibits p21-activated kinase 1 (Pak1) expression, a widely up-regulated signaling kinase in multiple human cancers, by targeting the 3'-untranslated region (UTR) of Pak1 mRNA. We noticed an inverse correlation between the levels of endogenous miR-7 and Pak1 expression in human cancer cells. We discovered that endogenous miR-7 expression is positively regulated by a homeodomain transcription factor, HoxD10, the loss of which leads to an increased invasiveness. HoxD10 directly interacts with the miR-7 chromatin. Accordingly, the levels of Pak1 protein are progressively up-regulated whereas those of miR-7 and its upstream activator HoxD10 are progressively down-regulated in a cellular model of breast cancer progression from low to highly invasive phenotypes. Furthermore, HoxD10 expression in highly invasive breast cancer cells resulted in an increased miR-7 expression but reduced Pak1 3'-UTR-luciferase activity and reduced Pak1 protein. Finally, we show that miR-7 introduction inhibits the motility, invasiveness, anchorage-independent growth, and tumorigenic potential of highly invasive breast cancer cells. Collectively, these findings establish for the first time that Pak1 is a target of miR-7 and that HoxD10 plays a regulatory role in modifying the expression of miR-7 and, consequently, the functions of the miR-7-Pak1 pathway in human cancer cells.

Fig. 1. miRNA-7 inhibits Pak1 expression

A, Sites of miRNA-7 seed matches in the Pak1 3′UTR. B, Western blot of Pak1, Pak2 and actin in HeLa, ZR-75 and MDA-231 cells after 48 hr of transfection with miR-7 or miR-con. C, normalized Pak1 3′UTR-luc reporter activity in HeLa, ZR-75 and HEK-293 cells after 48 hr of transfection of miR-7 or miR-con. Mean of three experiments; bars, SD. D, Western blot for Pak1 expression and relative expression of miR-7 by q- PCR in the indicated cell lines. Mean of three experiments; bars, SD.

Fig. 2. HoxD10 regulation of the miR-7 promoter

A, Schematic representation of miR-7 putative promoter with the binding sites for HoxD10 and Sp1. B, Normalized pGL-miR-7-luc activity in the HeLa cells. Mean of three experiments; bars, SD. C, Normalized pGL-miR-7-luc activity in the indicated cells transfected with HoxD10 or pCMV control vector. Mean of three experiments; bars, SD. D, Recruitment of Myc-HoxD10 onto the miR-7 gene chromatin (240 bp, -1139 to − 899) by ChIP in the HEK-293 and MCF10-AT cells.

Fig. 3. Dynamic correlation of Pak1, miR-7 and HoxD10 in breast cancer progression model

A, Western blot analysis of Pak1 and HoxD10, and q-PCR analysis of the endogenous miR-7 and HoxD10 is MCF10A model system. B, Effect of HoxD10 expression on the levels of miR-7 in MCF-10DICS and HEK-293 cells. Mean of three experiments; bars, SD. C, Normalized Pak1 3′UTR-luc activity in MCF10DCIS and HEK-293 cells transfected with HoxD10 or control vector. Mean of three experiments; bars, SD. D, Western blot analysis of Pak1 and vinculin in MCF10-DCIS and HEK-293 cells transfected with HoxD10 or control vector.

Fig. 4. Biologic effects of miR-7 in breast cancer cells

A, Confocal analysis of Pak1 expression in miR-7 transfected MDA-231 cells. B, Effect of miR-7 or miR-con on the motility, invasiveness, and anchorage-independence of the MDA-231 cells. C, Effect of miR-7 on the tumorigenic potential of MDAMB-231 cells in nude mice. D, An integrated working model of the findings presented here.