4. Results
4.3 H19 does not function via known mechanisms in endothelial cells
LncRNAs were shown to mediate histone modifications before259. To determine whether H19 regulates endothelial function via histone interactions, the subcellular localization of H19 was first assessed, alongside with the nuclear lncRNA MALAT-1 and ribosomal protein large subunit P0 (P0) mRNA, by separating HUVEC nuclei and cytoplasm and subsequent qRT-PCR. H19 was predominantly located in the cytoplasm of HUVECs, comparable to the mRNA of P0 and in contrast to the known nuclear lncRNA MALAT-1260 (Figure 18A). Physical interaction with histones was tested in RNA immunoprecipitation (RIP) experiments against histone H3, H3K27me3, and H3K9me3. H19 did not bind to any of these histones (Figure 18B).
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Figure 18: H19 is predominantly located in the cytoplasm of HUVECs and does not physically interact with histones. A:
Nuclear and cytoplasmic RNA was isolated from KLF2 overexpressing HUVECs and gene expression was analyzed with qPCR. B: RNA immunoprecipitation against histone H3 and its trimethylated forms was performed from lysates of WT HUVECs and H19 expression was analyzed with qPCR. The abundance of H19 is depicted as fold input. Statistical significance was depicted as follows: *p<0.05, n≥3.
H19 was previously described to bind miRNAs and act as a sponge 246,247. Publicly available AGO HITS CLIP data from HUVECs were analyzed for possible miRNA binding sites in the H19 transcript.
MALAT1 was used as a control for comparison150. MALAT1 showed several binding sites for AGO proteins. In contrast, the H19 transcript did not bind to AGO proteins (Figure 19A). Kallen et al.
showed that the H19 transcript harbors 4 binding sites for miRNAs of the let-7 family 246. To test whether H19 binds let-7 miRNAs in ECs, target genes of let-7a were identified using microRNA.org target gene predicting tool 261. 15 of these genes were analyzed in the microarray data from HUVECs upon H19 depletion. None of the let-7a target genes was significantly regulated upon siRNA mediated H19 depletion, although HMGA2 showed a trend towards reduced mRNA expression and SMARCAD1 mRNA showed a trend towards higher expression upon H19 depletion (Figure 19B).
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Figure 19: H19 does not interact with AGO proteins and predicted target genes of let-7a were not regulated. A: AGO HITS CLIP data from HUVECs were analyzed for binding of AGO proteins to MALAT1 and H19. MALAT1 showed a total of 9 binding sites for AGO proteins, while the H19 transcript did not bind AGO proteins. Screenshot from Integrative Genomics Viewer (IGV, Broad Institute). B: KLF2 overexpressing HUVECs were transfected with siRNA directed against H19 and subjected to microarray analysis 48 h after transfection and 15 predicted let-7a target genes were analyzed. n≥3.
H19 is known to act as a precursor for miR-675 234. To analyze if this miRNA plays a role in HUVECs, its expression upon KLF2 overexpression, H19 overexpression, and H19 depletion in KLF2 overexpressing HUVECs was analyzed. miR-675-5p showed a nonsignificant trend towards reduced expression upon KLF2 overexpression (Figure 20A). Both, miR-675-3p and -5p were upregulated upon lentiviral H19 overexpression in HUVECs (Figure 20B). Upon siRNA-mediated H19 depletion, miR-675-5p showed a nonsignificant trend towards increased expression (Figure 20C). miRNA-675 target genes were predicted with microrna.org target prediction tool and analyzed in HUVECs upon H19 depletion 261. FANCA showed a nonsignificant lower expression, while LNPEP, SYDE1, TUBB4, LRIG2, and RNF103 showed a similar nonsignificant increase in expression compared to si Ctrl (Figure 20D).
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Figure 20: miR-675 5p was upregulated upon H19 overexpression, while KLF2 overexpression and siRNA-mediated H19 depletion did not change miR-675 levels. A: WT HUVECs were transduced with lentivirus carrying human KLF2 gene or empty vector and miR-675 expression was analyzed at least 7 days after transduction with qRT-PCR. B: WT HUVECs were transduced with lentivirus carrying H19 gene or empty vector and miR-675 expression was analyzed at least 7 days after transduction with qRT-PCR. C: WT HUVECs were transfected with siRNA directed against H19 or firefly luciferase and miR-675 expression was analyzed with qRT-PCR. D: KLF2 overexpressing HUVECs were transfected with siRNA directed against H19 and subjected to microarray analysis 48 h after transfection. Predicted miR-675 target genes were analyzed.
Statistical significance was depicted as follows: *p<0.05, ***p<0.001, n.s. = not significant, n≥3.
Several lncRNAs were shown to possess a function on their neighboring genes in cis and the effect of H19 depletion or overexpression on IGF2 was analyzed in HUVECs. IGF2 was significantly higher expressed in the absence of H19 and showed a trend towards reduced expression upon H19 overexpression (Figure 21A&B). IGF2 was previously described to induce expression of VEGF and to promote tumor angiogenesis 259. To test whether IGF2 induced upregulation of VEGF upon H19 depletion plays a role in endothelial cells, expression of VEGF mRNA was analyzed upon H19 depletion. VEGF levels were not changed upon H19 depletion in HUVECs (Figure 21C).
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Figure 21: Insulin growth factor 2 (IGF2) mRNA was upregulated upon H19 depletion and downregulated upon H19 overexpression, while the IGF2 target VEGFA was not regulated. A: KLF2 overexpressing HUVECs were transfected with siRNA directed against H19 or control and IGF2 expression was analyzed by qRT-PCR 48 h after transfection. B: WT HUVECs were transduced with lentivirus carrying H19 gene or empty vector and IGF2 expression was analyzed at least 7 days after transfection by qRT-PCR. C: KLF2 overexpressing HUVECs were transfected with siRNA directed against H19 and subjected to microarray analysis 48 h after transfection. VEGFA expression was analyzed. Statistical significance was depicted as follows: ***p<0.001, n.s. = not significant, n≥3.
Taken together, despite its predominant cytoplasmic localization, H19 can be pharmacologically depleted with siRNAs and LNA GapmeRs. H19 was not bound to histones in HUVECs and its depletion did not change expression levels of target genes of let-7a miRNA, which was shown to be bound by H19. Interestingly, KLF2-mediated H19 upregulation, lentivirus-mediated H19 overexpression, and siRNA-mediated H19 depletion did change levels of the endogenously encoded miRNA miR-675, but this regulation was not consistent. Furthermore, H19 depletion did change levels of its neighboring gene IGF2, but levels of IGF2-regulated gene VEGF were not changed.