Huiqing Cao

Many long noncoding RNAs (lncRNAs) have been identified through siRNA-based screening as essential regulators of embryonic stem cell (ESC) pluripotency. However, the biological and molecular functions of most lncRNAs remain unclear. Here, we employed CRISPR/Cas9-mediated knockout technology to explore the functions of 8 lncRNAs previously reported to promote pluripotency in mouse ESCs. Unexpectedly, all of these lncRNAs were dispensable for pluripotency maintenance and proliferation in mouse ESCs when disrupted individually or in combination. Single-cell transcriptomic analysis also showed that the knockout of these lncRNAs has a minimal impact on pluripotency gene expression and cell identity. We further showed that several small hairpin RNAs (shRNAs) previously used to knock down lncRNAs caused the downregulation of pluripotency genes in the corresponding lncRNA-knockout ESCs, indicating that off-target effects likely responsible for the pluripotency defects caused by these shRNAs. Interestingly, linc1343-knockout and linc1343-knockdown ESCs failed to form cystic structures and exhibited high expression of pluripotency genes during embryoid body (EB) differentiation. By reintroducing RNA products generated from the linc1343 locus, we found that two snoRNAs, Snora73a and Snora73b, but not lncRNAs, could rescue pluripotency silencing defects during EB differentiation of linc1343 knockout ESCs. Our results suggest that the 8 previously annotated pluripotency-regulating lncRNAs have no overt functions in conventional ESC culture; however, we identified snoRNA products derived from an annotated lncRNA locus as essential regulators for silencing pluripotency genes.

Background: The high morbidity and mortality of calcific aortic valve disease (CAVD) represents an unmet clinical need to investigate the molecular mechanisms involved. Evidence suggests that long non-coding RNAs (lncRNAs) can act as competitive endogenous RNAs (ceRNAs) by binding to microRNAs and regulating target genes in cardiovascular diseases. Nevertheless, the role of lncRNAs related ceRNA regulation in CAVD remains unclear. Methods: RNAseq data of human diseased aortic valves were downloaded from GEO data sets (GSE153555, GSE199718), and differentially expressed lncRNAs (DElncRNAs), mRNAs (DEmRNAs) between CAVD and non-calcific aortic valve tissues with limma R package. Gene Ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Set Enrichment analysis (GSEA) were performed with clusterProfiler and gesaplot2 R package. The pivotal microRNAs were predicted by three databases intersection including TargetScan, MiRwalk, miRDB according to the genes related to the crucial pathways. ENCORI was used to predict targeted lncRNAs of hub microRNAs. We constructed lncRNA-miRNA-mRNA ceRNA network with Cytoscape software. The lncRNAs in ceRNA network were verified by RT-qPCR in human 30 calcific and 20 noncalcified aortic valve tissues. Results: In total, 1739 DEmRNAs and 266 DElncRNAs were identified in CAVD. GO, KEGG pathway, GSEA annotations suggested that most of these genes are enriched in extracellular matrix (ECM)-reporter interaction pathways. The ceRNA networks associated with ECM-reporter interaction are constructed and related lncRNAs including H19, SNHG3 and ZNF436-AS1 were significant upregulated in human calcific aortic valve tissues, which might be potential therapeutic targets for CAVD. Conclusions: In this study, we proposed a novel lncRNA-miRNA-mRNA ceRNA network related to ECM-reporter interaction pathways, which potentially regulates CAVD progression.

The pluripotent state of embryonic stem cells (ESCs) is regulated by a sophisticated network of transcription factors. High expression of KLF17 has recently been identified as a hallmark of naive state of human ESCs (hESCs). However, the functional role of KLF17 in naive state is not clear. Here, by employing various gain and loss-of-function approaches, we demonstrate that KLF17 is essential for the maintenance of naive state and promotes the primed to naive state transition in hESCs. Mechanistically, we identify MAPK3 and ZIC2 as two direct targets repressed by KLF17. Overexpression of MAPK3 or ZIC2 partially blocks KLF17 from promoting the naive pluripotency. Furthermore, we find that human and mouse homologs of KLF17 retain conserved functions in promoting naive pluripotency of both species. Finally, we show that Klf17 may be essential for early embryo development in mouse. These findings demonstrate the important and conserved function of KLF17 in promoting naive pluripotency and reveal two essential transcriptional targets of KLF17 that underlie its function.

The DiGeorge syndrome critical region gene 8 (Dgcr8) knockout strategy has been widely used to study the function of canonical microRNAs (miRNAs) in vitro and in vivo. However, primary miRNA (pri-miRNA) transcripts are accumulated in Dgcr8 knockout cells due to interrupted processing. Whether abnormally accumulated pri-miRNAs have any function is unknown. Here, using clustered regularly interspaced short palindromic repeats system/CRISPR-associated protein 9 (CRISPR/Cas9), we successfully knocked out the primary microRNA-290~295 (pri-miR-290~295) cluster, the most highly expressed miRNA cluster in mouse embryonic stem cells (ESCs), in Dgcr8 knockout background. We found that the major defects associated with Dgcr8 knockout in mouse ESCs, including higher expression of epithelial-to-mesenchymal transition (EMT) markers, slower proliferation, G1 accumulation, and defects in silencing self-renewal, were not affected by the deletion of pri-miR-290~290 cluster. Interestingly, the transcription of neighboring gene nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 12(Nlrp12) was upregulated upon the deletion of the pri-miR-290~295 cluster. Together, our results suggested that the major defects in Dgcr8 knockout ESCs were not due to the accumulation of pri-miR-290~295, and the deletion of miRNA genes could affect the transcription of neighboring DNA elements.

Heart failure (HF) is associated with mitochondrial dysfunction and energy metabolism impairment. MicroRNAs are implicated in the development of heart failure. However, the mitochondria enriched microRNA during heart failure remains elusive. Here, we generated a pressure overload-induced early and late stage heart failure model at 4 weeks and 8 weeks following transverse aortic constriction (TAC) in mice. We found that expression of mitochondrion protein COX4 was highly enriched in isolated mitochondria from cardiac tissues while GAPDH could hardly be detected. Furthermore, small RNA sequencing for mitochondria RNAs from failing hearts was performed. It was found that 69 microRNAs were upregulated and 2 were downregulated in early heart failure, while 16 microRNAs were upregulated and 6 were downregulated in late heart failure. 15 microRNA candidates were measured in both mitochondria and total cardiac tissues of heart failure by real-time PCR. MiR-696, miR-532, miR-690, and miR-345-3p were enriched in mitochondria from the failing heart at early stage. Bioinformatics analysis showed that mitochondria enriched microRNAs in HF were associated with energy metabolism and oxidative stress pathway. For the first time, we demonstrated microRNAs were enriched in mitochondria during heart failure, which established a link between microRNA and mitochondrion in heart failure.