Ischemia-reperfusion (I/R) is a common pathology when the blood supply to an organ was disrupted and then restored. During the reperfusion process, inflammation and tissue injury were triggered, which were mediated by immunocytes and cytokines. However, the mechanisms initiating I/R-induced inflammation and driving immunocytes activation remained largely unknown. In this study, we identified long non-coding RNA (lncRNA)-H19 as the key onset of I/R-induced inflammation. We found that I/R increased lncRNA-H19 expression to significantly promote NLRP3/6 inflammasome imbalance and resulted in microglial pyroptosis, cytokines overproduction, and neuronal death. These damages were effectively inhibited by lncRNA-H19 knockout. Specifically, lncRNA-H19 functioned via sponging miR-21 to facilitate PDCD4 expression and formed a competing endogenous RNA network (ceRNET) in ischemic cascade. LncRNA H19/miR-21/PDCD4 ceRNET can directly regulate I/R-induced sterile inflammation and neuronal lesion in vivo. We thus propose that lncRNA-H19 is a previously unknown danger signals in the molecular and immunological pathways of I/R injury, and pharmacological approaches to inhibit H19 seem likely to become treatment modalities for patients in the near future based on these mechanistic findings.
PURPOSE. Differentiated from adult stem cells (ASCs), transit-amplifying cells (TACs) play an important role in tissue homeostasis, development, and regeneration. This study aimed to characterize the gene expression profile of a candidate TAC population in limbal basal epithelial cells using single-cell RNA sequencing (scRNA-seq).
METHODS.Single cells isolated from the basal corneal limbus were subjected to scRNA-seq using the 10x Genomics platform. Cell types were clustered by graph-based visualization methods and unbiased computational analysis. BrdU proliferation assays, immunofluorescent staining, and real-time reverse transcription quantitative polymerase chain reaction were performed using multiple culture models of primary human limbal epithelial cells to characterize the TAC pool.
RESULTS.Single-cell transcriptomics of 16,360 limbal basal cells revealed 12 cell clusters. A unique cluster (3.21% of total cells) was identified as a TAC entity, based on its less differentiated progenitor status and enriched exclusive proliferation marker genes, with 98.1% cells in S and G2/M phases. The cell cycle-dependent genes were revealed to be largely enriched by the TAC population. The top genes were characterized morphologically and functionally at protein and mRNA levels. The specific expression patterns of RRM2, TK1, CENPF, NUSAP1, UBE2C, and CDC20 were well correlated in a time-and cycle-dependent manner with proliferation stages in the cell growth and regeneration models.
CONCLUSIONS.For the first time, to the best of our knowledge, we have identified a unique TAC entity and uncovered a group of cell cycle-dependent genes that serve as TAC signature markers. The findings provide insight into ASCs and TACs and lay the foundation for understanding corneal homeostasis and diseases.
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