HnRNPU regulates intra- and intercellular microRNA trafficking in a sequence specific manner

Introduction The transfer of microRNAs (miRs) through extracellular vesicles (EVs) is a well-established mechanism of vascular intercellular communication. In a previous study, we have identified the heterogeneous nuclear ribonucleoprotein U (hnRNPU) as a potent regulator of the vesicular export of a specific set of miRs in endothelial cells (ECs). Downregulation of hnRNPU caused an increased export of miRs into large EVs and lead to a reduction of the migratory capacity in EV up-taking ECs. The underlying mechanism, how hnRNPU recognizes only certain miRs and regulates their export, however, remained unclear. Methods and results Increasingly exported microRNAs into EVs after siRNA-mediated downregulation of hnRNPU were identified in a miR array and used to perform a sequence analysis with the Multiple Em for Motif Elicitation tool (MEME). The software identified the motif AAMRUGCU to be significantly enriched within the regulated miRs. Binding of hnRNPU to miR-30c-5p, which was one of the most significantly increased miRs in EVs upon downregulation of hnRNPU, was confirmed in an immunoprecipitation and an RNA-pulldown experiment. In order to investigate, if the in silico motif AARUGCU was the binding site of miR-30c-5p and hnRNPU an electrophoretic mobility shift assay (EMSA) was conducted with native miR-30c-5p, mutated miR-30c-5p as well as miR-125a-3p as a negative control. The EMSA showed that a partial mutation, which conserves the purine / pyrimidine structure in the motif of miR-30c-5p does not affect binding, while a complete disruption of the motif structure abolishes binding of hnRNPU and miR-30c-5p. Furthermore, we found that hnRNPU protects miR-30c-5p from rapid degradation after transcriptional blockage by Actinomycin D. Binding of miR-30c-5p to the predominantly nuclearly expressed hnRNPU retains miR-30c-5p in the nucleus. HnRNPU knockdown leads to a redistribution of miR-30c-5p from the nucleus to the cytoplasm. The regulating effect of hnRNPU expression on vesicular miR export was confirmed by overexpression of hnRNPU through plasmid transfection. In contrast to siRNA-mediated downregulation, overexpression of hnRNPU lead to a decrease of vesicular miR-30c-5p levels. In order to extent our findings to other cell types with high vascular relevance, siRNA mediated downregulation of hnRNPU was performed in human cardiac fibroblasts (HCF). Similar to our findings in ECs, downregulation of hnRNPU in HCFs lead to an increase in vesicular miR-30c-5p levels. Conclusion In conclusion, we show that hnRNPU binds miRs, which contain the motif AAMRUGCU. This leads to nuclear retainment, stabilization and inhibits vesicular export, which can be further decreased by overexpression of hnRNPU and enhanced by downregulation of hnRNPU. The effect is not restricted to ECs but applies also to HCFs. hnRNPU may therefore be an important target to influence intra- and intercellular trafficking of a specific set of miRs including miR-30c-5p. Funding Acknowledgement Type of funding source: Public hospital(s). Main funding source(s): Medical faculty of the University of Bonn, German Cardiac Society

Sdccag3 Promotes Implant Osseointegration during Experimental Hyperlipidemia

Hyperlipidemia adversely affects bone metabolism, often resulting in compromised osseointegration and implant loss. In addition, genetic networks associated with osseointegration have been proposed. Serologically defined colon cancer antigen 3 (Sdccag3) is a novel endosomal protein that functions in actin cytoskeleton remodeling, protein trafficking and secretion, cytokinesis, and apoptosis, but its roles in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and in implant osseointegration under hyperlipidemic conditions have not been uncovered. Here, we performed microarray and RNA sequencing analysis to determine the differential expression of the Sdccag3 gene and related noncoding RNAs (ncRNAs) and to assess the long noncoding RNA (lncRNA) MSTRG.97162.4-miR-193a-3p-Sdccag3 coexpression network in bone tissues within the region 0.5 mm around implants in hyperlipidemic rats. In this experiment, we found that Sdccag3 and the previously uncharacterized lncRNA-MSTRG.97162.4 were downregulated during hyperlipidemia, while miR-193a-3p was upregulated. Sdccag3 overexpression increased new trabecular formation, the bone volume/total volume (BV/TV) (1.24-fold), and bone-implant combination ratio (BIC%) (1.26-fold). An RNA pulldown experiment revealed that Sdccag3 protein targeted lncRNA-MSTRG.97162.4 nucleotides 361 to 389. In addition, lncRNA-MSTRG.97162.4 overexpression significantly enhanced Sdccag3 (2.78-fold) expression and increased BV/TV (1.45-fold) and BIC% (1.07-fold) at the bone-implant interface. Taken together, these findings indicate that Sdccag3 overexpression enhances implant osseointegration under hyperlipidemic conditions by binding to lncRNA-MSTRG.97162.4. Furthermore, miR-193a-3p overexpression inhibited lncRNA-MSTRG.97162.4 (0.63-fold) and Sdccag3 (0.88-fold) expression and induced poor implant osseointegration (BV/TV, 0.86-fold; BIC%, 0.82-fold), while miR-193a-3p downregulation produced the opposite results (lncRNA-MSTRG.97162.4, 10.69-fold; Sdccag3, 6.96-fold; BV/TV, 1.20-fold; BIC%, 1.26-fold). Therefore, our findings show that Sdccag3 promotes implant osseointegration, and its related lncRNA-MSTRG.97162.4 and miR-193a-3p play an important role in osseointegration during hyperlipidemia, which might be a promising therapeutic target for improving dental implantation success rates.