**3. Results**

miRNA-212/132 are transcribed as a single RNA transcript and subsequently processed into two di fferent mature microRNA miR-132 and miR-212. Due to the di fferent e fficiency in pre-miR processing, miR-132 is the dominant family member, as shown in Figure 1F. In most of the tissues, the expression of miR-212 are hardly detectable with the only exception being the brain. For this reason, mir-132 was used as the primary target in this study. We first examined the expression of miR-132 in relation to VHL loss-of-function and (pseudo)-hypoxic signaling. We found that endothelial cells grown in hypoxic conditions display significantly elevated levels of miR-132 expression (Figure 1A). We observe similar e ffects in HUVECs transfected with siRNA targeting *VHL* mRNA relative to those treated with non-targeting siRNA (Figure 1B). miR-212/132 are well conserved in most species, including zebrafish (Supplementary Figure S1A). To confirm this e ffect in vivo, we used a previously established zebrafish model of *VHL* deficiency [12–14]. Like our cell models, *vhl*−/− zebrafish also show an increased level of expression of miR-132 (Figure 1C). In isogenic cell lines taken from human ccRCC *VHL*−/− tumors, the expression of miR-132 is reduced upon *VHL* reconstitution with ectopic *VHL* (Figure 1E). The reduction of miR-132 expression is present in all cell lines upon VHL introduction but less pronounced in the RCC10, which might be related to a cell-line-specific genetic alteration on top of the VHL disruption that a ffects the miR-132 pathway. To assess the functional consequences of miR-212/132 loss in these cells, we examined the expression of a known target mRNA of this miRNA family. PTEN, phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase, antagonizes the activity of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), suppressing cellular proliferation, cell survival, and angiogenesis by inactivating the PI3K-driven AKT signaling pathway [15]. *PTEN* has been predicted to be a potential target of miR-212/132 in humans by targetscan (Supplemental Figure S1B), and the rat homologue of *PTEN* has been shown to be targeted by miR-212/132 in rat vascular smooth muscle cells [16]. Moreover, downregulation of PTEN has been significantly correlated with lower survival rate in ccRCC patients [2]. We reasoned that upregulated miR-212/132 upon mutation or silencing of *VHL* could result in the subsequent reduction of PTEN. We therefore examined PTEN expression in HUVEC cells transfected with miR-212/132 mimics and found that the expression of PTEN was significantly reduced in these cells (Figure 1D). In order to assess the relative e ffects of miR-212 versus miR-132, we examined the di fferential expression of these miRNA in di fferent mouse tissues. miR-212 is found at extremely low levels in the tissues tested, except brain tissue (Figure 1F). Based on this information, we examined miR-132 expression in histology slides taken from ccRCC tumors using microRNA in situ hybridization. In agreemen<sup>t</sup> with our previous qPCR results, we

observed widespread overexpression of miR-132 in tumor material from ccRCC samples with biallelic *VHL* mutations proven by sequencing (Figure 1E). These results demonstrate that miR-132 is increased in response to the pseudo-hypoxia induced by the lack of functional pVHL, which eventually leads to overexpression of miR-132.

**Figure 1.** Characterization of miR-132 expression under hypoxic and pseudo-hypoxic conditions: (**A**) The expression of miR-132 in human umbilical vascular endothelial cells (HUVECs) under normoxia and hypoxia as compared by qPCR. (**B**) The expression of von Hippel–Lindau (VHL) in HUVECs after transfection with siRNA against VHL and the expression of miR-132 in siSham and siVHL transfected HUVECs as compared by qPCR. (**C**) The expression of miR-132 in wildtype (WT) and vhl−/− mutant zebrafish as compared by qPCR. (**D**) The expression of known miR-132 target PTEN (phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase) in HUVECs treated with miR-132/212 mimics versus control as compared by qPCR. (**E**) The expression of miR-132 in established VHL−/− lines RCC10, A498, and 786-0 as well as the same lines reconstituted with ectopic VHL. The presented data is a mean of 3 in-depended PCR experiments with counting error. (**F**) Relative expression of miR-132 and 212 in different tissues in mouse. Note miR-132 expression is considerably higher than miR-212. *n* = 3. (**G**) The expression of miR-132 in healthy kidney tissue and ccRCC from two patients with known bilateral VHL mutations in their tumor as shown by miR-132 in situ hybridization. miR-132 in situ is in purple blue. Light eosin counterstaining appears pink. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001

To assess the functional consequences of miR-212/132 expression in a *VHL*-null environment, we used an in vitro coculture assay designed to gauge angiogenesis. In agreemen<sup>t</sup> with the important role of VHL in HIF1 degradation, knockdown of VHL in HUVEC/pericyte coculture shows significantly more vascular junctions, tubule number, and total tubular length as compared to siSham control treatment (Figure 2A–C). To evaluate whether the pro-angiogenic effects of *VHL* silencing are mediated by a downstream increase in miR-212/132, GFP-labelled HUVECs were treated with anti-miRs against miR-212/132 in combination with siRNA targeting *VHL*. Inhibiting the action of miR-212/132 reduced the

excessive angiogenetic response induced by the silencing of *VHL* significantly (Figure 2D), suggesting that VHL-regulated angiogenesis is at least partially mediated by the upregulation of miR-212/132.

**Figure 2.** Reduced levels of VHL enhances endothelial cell neovascularization capacity and can be inhibited by blocking miR-132 or miR-212. (**A**) Schematic outline of the coculture experiment with HUVECs and pericytes. (**B**) Representative images showing the analysis process of tubular structures in the endothelial cells and pericytes coculture assay. (**C**) VHL siRNA knockdown in HUVECs enhances endothelial cell neovascularization capacity. (**D**) Blocking miR-132/212 inhibits neovascularization enhancement induced by VHL knockdown. Cell images are used to produce skeletonized 2D images which can be analyzed automatically. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001

*vhl*−/− zebrafish embryos display a phenotype of post-vascularization branching/sprouting around the intersomitic vessels in the tails. Counting these sprouts is known as a quantitative measure of angiogenesis in zebrafish [13]. *vhl*−/− zebrafish were injected at a one-cell stage with anti-miRs directed against miR-132 or miR-212, and four days later, tails of the living fish were imaged with a confocal microscope (Figure 3A). The cloaca of the zebrafish is placed in the center of the image, and the branches sprouting from the inter-somitic vessels were counted for the four vessels anterior and the four vessels posterior to the cloaca (Figure 3B). Anti-miR injections against miR-212/132 significantly reduced the extent of intersomitic vessel sprouting in *vhl*−/− fish (Figure 3C,D). In addition, injecting wild-type zebrafish with miR-212/132 mimics partially recapitulated the *vhl* mutant vessel sprouting phenotype (Supplemental Figure S1C,D). In light of the fact that miR-212/132 expression is therefore linked to vessel sprouting in *vhl*−/− zebrafish, we proceeded to look at the expression of PTEN in our VHL-null models, as we had previously done in HUVECs. Zebrafish, as opposed to mammals, have

two copies of the *pten* gene: *ptena* and *ptenb.* Zebrafish with loss of both *ptena* and *ptenb* [17] also display a vessel sprouting phenotype that phenocopies the one found in zebrafish injected with miR-212/132 mimics. *ptenb* is a predicted target of miR-212/132 in targetscan but not *ptena* (Supplemental Figure S1E). Accordingly, we found significantly reduced *ptenb* expression in *vhl*−/− zebrafish with no significant changes observed in *ptena* (Figure 4E).

**Figure 3.** Inhibition of miR-132 or miR-212 suppresses VHL loss of function-induced vasculature outgrowth in zebrafish. (**A**) Schematic outline of the zebrafish embryo microinjection experiment. microRNA mimics and anti-miRs are injected into the yolk of the eggs on day 0 and imaged with a confocal microscope on day 5. (**B**) Schematic cartoon showing the area of the zebrafish embryo that is imaged after microinjection. The cloaca is marked with a red arrow. The imaging area is shown with a red box. The vessels of the tail are shown in green. (**C**) Representative images of zebrafish tail vascular structures in vhl+/− and vhl−/− zebrafish after injection with scrambled or miR-132 and miR-212 inhibitors. White arrows designate examples of structures which have been scored as branches. (**D**) Quantification of vascular branching in zebrafish tail structures after injection with scrambled control inhibitors, miR-132 inhibitors, or miR-212 inhibitors. (**E**) The expression levels of ptena and ptenb in WT and vhl−/− zebrafish determined by qPCR. \* *p* < 0.05; \*\* *p* < 0.01.

**Figure 4.** Proposed mechanism of miR-132/212 in modulation of the VHL/phosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K)/Protein kinase B(AKT)pathways. (**A**) During normoxia, hypoxia-inducible transcription factor 1 (HIF1) is ubiquitinated by the VHL-ubiquinition complex, targeting it for degradation. Some effactors, such as *PTEN*, antagonizes PI3k to prevent AKT from being activated. (**B**) Upon hypoxia, HIF1 can no longer be hydroxylated, which prohibits VHL-regulated degradation, and allows stabilized HIF1 to translocate to the nucleus, upregulating its downstream targets such as *vascular endothelial growth factor* (*VEGF*). VEGF in turn activates the PI3k-AKT pathway and upregulates miR-132/212 expression as well. Upregulated miR-132/212 inhibits effector (e.g., *PTEN)* expression, which in turn prolongs AKT activity. (**C**) *VHL* loss-of-function phenocopies hypoxic conditions even in the presence of oxygen (pseudo-hypoxia).
