**4. Discussion**

In this study, we used patient material, human cells, and zebrafish to examine the role of the miRNA-212/132 family in ccRCC tumor neovascularization caused by the loss-of-functional pVHL. We observed that miR-212/132 is upregulated in response to *VHL* mutation both in zebrafish model systems and in human patient ccRCC tumor material carrying biallelic inactivating *VHL* mutations. We demonstrated that the excessive angiogenesis attributable to *VHL* mutation is strongly affected by miR-212/132. Indeed, targeting these miRNAs with anti-miRs can significantly reduce angiogenesis in both in vitro and in vivo models of *VHL* deficiency. We identified the tumor suppressor PTEN as one of the targets affected by miRNA-212/132 in *VHL*-null models. Taken together, our results implicate miRNA-212/132 as an important intermediate in angiogenesis after loss-of-functional pVHL or tissue hypoxia.

The miR-212/132 family is clustered in the genome and is highly conserved in vertebrates. miR-212/132 is initially expressed as one primary miRNA and then processed into two mature miRNA with the same target-defining "seed" sequence [18]. This miRNA family plays a number of roles in the promotion of angiogenesis. Mice without functional miR-212/132 show impaired arteriogenesis response after hindlimb ischemia [19]. The pro-angiogenic potential of miR-132 has been used to increase angiogenesis in endothelial cell grafts and after ischemic injury [20]. miR-212/132 frequently act as a promoter of cell proliferation, and increases in their expression levels have also been suggested as contributors to tumorigenesis in addition to their angiogenic role. miR-132 has previously been shown to induce neovascularization in the endothelium by targeting p120 Ras GTPase-activating protein [5]. In addition, anti-miR-132 has also been shown to reduce tumor burden in a mouse xenograft model of human breast carcinoma [5].

This study supports the previously reported role of miR-212/132 in angiogenesis and expands upon its role in the context of VHL-regulated hypoxia signaling. When VHL is mutated or downregulated, miR-212/132 is consequently upregulated. miR-212/132 targeting of mRNA such as *PTEN* ensures that downstream effectors are upregulated. For example, in the case of *PTEN*, PI3K may activate AKT, leading to an increase in proliferation. Indeed, cysts taken from VHL patients display hyperactivation of PI3K signaling [21]. Due to technical reasons, we were not able to detect this increased AKT signaling directly in our zebrafish model. In addition, *Pten* −/− *Vhl* −/− double mutant mice develop benign squamous metaplasia and cystadenoma [21] and display kidney cysts that are very similar to those taken from the kidneys of human VHL patients, while mouse models with *Vhl* mutations only do not develop renal tumors [22]. Uncontrolled proliferation and angiogenesis are hallmarks of cancer, and many tumors contain mutations leading to hyperactivation of signaling networks which act to promote these processes [23–25]. Di fferential expression of miRNA has been previously reported in tumors including ccRCC [26] and is widely believed to be an important player in tumorigenesis [27–29]. A group of miRs, termed hypoxamiRs, has been shown to be upregulated in hypoxia and play a role in the modulation of cellular responses to a lack of oxygen. The hypoxamiRs include miR-21; 23; 24; 26; 103/107; 373; and, most well studied, miR-210. Hypoxia is also considered a hallmark of the microenvironment of solid tumors, and a number of hypoxamiRs have been implicated in tumorigenesis [30,31]. Thus, the action of miRNA may play an important role in tumorigenesis and therefore presents an interesting potential target for the treatment of cancer.

One of the hallmarks of ccRCC is resistance to cytotoxic treatment. Antiapoptotic signaling is upregulated after HIF1 α hyper-stabilization in ccRCC tumors. Many experimental treatments focus on inhibiting the action of downstream antiapoptosis proteins such as mammalian target of rapamycin (mTOR), an important pro-survival protein induced by activated AKT signaling [32]. Our results and the results of other studies sugges<sup>t</sup> that miR-212/132 may act as a promoter of tumorigenesis by targeting inhibitors of proliferation, survival, and angiogenesis, presenting an interesting opportunity for pharmaceutical intervention. Currently, medications which target miRNA are largely unexplored as an avenue by which to target cancer. Here, we have shown that antagonizing the activity of this miRNA family can reduce angiogenesis in relevant models of *VHL* deficiency. *PTEN*, which we have confirmed to be a target of the miR-212/132 family in these models, is frequently mutated in cancer. Another miRNA, miRNA-21, has previously been shown to target PTEN in multiple cancer types and has been implicated in tumorigenesis [33]. Loss of PTEN leads to upregulation of mTOR signaling, and mTOR inhibitors have been used to treat multiple cancer types. Therefore, we envision that treatments designed to antagonize the e ffect of miRNA-212/132 or other miRNA might be able to reduce the angiogenic burden of tumors in patients, to reduce tumor resistance to other chemotherapeutic agents, or to slow or halt tumor growth.

Conversely, treatments with miR-212/132 might also be a useful method to promote angiogenesis and to increase neovascularization in the cases where neovascularization would be helpful, such as certain ischemic tissues or newly transplanted engineered tissue constructs. In fact, ex vivo transfection of mir-132 into endothelial cells has been shown to be beneficial for transplantation and vascularization of transplanted endothelial cells [34]. Based on our results and the results of other studies, further work is warranted in this area.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4409/9/4/1017/s1, Figure S1: Upreguation of miR-132/212 improve neovascularization in WT Zebrafish.

**Author Contributions:** Z.L., T.D.K., J.P.G.S., and R.H.G. conceived and designed the experiments. T.D.K., Z.L., M.M.B., G.v.d.H. and I.L. performed the experiments. C.C., P.A.D., J.P.G.S. and R.H.G supervise the study. T.D.K., Z.L., and M.M.B. analysed the data. T.D.K., Z.L., P.A.D., C.C., J.P.G.S., and R.H.G. wrote the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research forms part of the Project P1.05 LUST of the research program of the BioMedical Materials institute, cofunded by the Dutch Ministry of Economic Affairs. We furthermore acknowledge the financial support of the European Community's Seventh Framework Programme FP7 under gran<sup>t</sup> agreemen<sup>t</sup> numbers 305608 (EURONOMICS) and 241955 (SYSCILIA); the Dutch Kidney Foundation Consortium CP11.18 "KOUNCIL"; and the Netherlands CardioVascular Research Initiative (CVON): the Dutch Heart Foundation, Dutch Federation of University Medical Centers, the Netherlands Organization for Health Research and Development, and the Royal Netherlands Academy of Sciences, CUREPLaN foundation Leducq (P.D.). This project was supported by the project EVICARE (No.725229) of the European Research Council (ERC) to J.P.G.S; by NOW-CAS gran<sup>t</sup> (116006102) to P.D. and J.S.

**Conflicts of Interest:** The authors declare no conflict of interest.
