Autophagy-prominent cell clusters among human lens epithelial cells: integrated single-cell RNA-sequencing analysis

Abstract

Background: Autophagy is an important process that maintains the quality of intracellular proteins and organelles. There is extensive evidence that autophagy has an important role in the lens. Human lens epithelial cells (HLECs) play a key role in the internal homeostasis of the lens. HLEC subtypes have been identified, but autophagy-prominent cell clusters among HLECs have not been characterized.

Purpose: To explore the existence of autophagy-prominent cell clusters in HLECs.

Methods: Three donated lenses (HLECs from two whole lenses and HLECs from one lens without the anterior central 6-mm zone) were used for single-cell RNA sequencing (scRNA-seq). AUCell and AddModuleScore analysis were used to identify potential autophagy-prominent cell clusters. Transmission electron microscopy (TEM) was used to confirm the results.

Results: High-quality transcripts from 18,120 cells were acquired by scRNA-seq of the two intact lenses. Unsupervised clustering classified the cells into four clusters. AUCell and AddModuleScore analysis revealed cluster 1 is autophagy-prominent. scRNA-seq analysis of HLECs from the lens capsule lacking the central zone confirmed the cluster 1 HLECs was located in the central capsule zone. The TEM result showed that greater autophagy activity was observed in the HLECs in central capsule zone, which further supported the above conclusions based on scRNA-seq analysis that autophagy was prominent in the central zone where the cluster 1 HLECs located.

Conclusions: We identified an autophagy-prominent cell cluster among HLECs and revealed that it was localized in the central zone of the lens capsule. Our findings will aid investigations of autophagy in HLECs and provide insights to guide related research.

Keywords: Autophagy; Human lens epithelial cells; Single-cell RNA-sequencing; Transmission electron microscopy.

Fig. 1

A: UMAP of HLECs from two donor tissues generated single-cell transcriptomic profiles of total 18,120 cells with 4 differential cell clusters .B: The distribution of two samples in the cluster. C: correspond to the top 30 differential genes in the respective HLEC clusters 0 to 3

Fig. 2

Autophagy scores. A: Autophagy score using Human Autophagy Database. B: Autophagy score using HAMdb. C: AUCell scores of autophagy signaling pathways in each of the four cell clusters. ****adj. p-value < 0.0001

Fig. 3

A: Violin plot of the top 10 differential genes in cluster 1. B: Expression of C8orf4 in HLECs from the lens capsule lacking the central zone and HLECs from the whole lens capsule. C: Map the dataset of HLECs from the lens capsule lacking the central zone to the dataset of HLECs from the whole lens capsule. D: Autophagy score using Human Autophagy Database. E: Autophagy score using HAMdb. F: AUCell scores of autophagy signaling pathways. ****adj. p-value < 0.0001

Fig. 4

Autophagic vacuoles in central and non-central zones of HLECs (n ≥ 70 cells from seven lenses). A: 67Y, central zone. B: 67Y, non-central zone. Red arrows: autophagic vacuoles. Scale bars in A and B = 0.5 μm. Data in the graph are shown as means ± standard deviations; p-values < 0.05 were considered statistically significant