Cataract, a common cause of vision loss, results from the gradual opacification of the lens, mostly at an elderly age. It has been recognized that risk factors such as aging, diabetes mellitus, ultraviolet B exposure, long-term use of corticosteroids, and smoking can contribute to the development of cataract. (Truscott, 2005). Physiologically, the lens epithelial cells (LECs) residing under the anterior capsule of the lens continuously proliferate and differentiate into the elongated fiber cells to form the compacted nuclear mass of the lens throughout its lifespan. These normal activities of LECs guarantee the homeostasis and transparency of the lens. And any disturbance to the LECs activities can result in cataractogenesis. A marked reduction of the lens epithelial cell density is observed in advanced senile cataract. (Tseng et al., 1994). With the aging of the lens, the LECs demonstrate a remarked increase of apoptosis in a time-dependent manner, and the experimentally induced apoptosis of LECs can give rise to the development of cataracts. (Li et al., 1995; Zhang et al., 2010). Moreover, there is a relatively high level of apoptosis of LECs in diabetes-induced cataracts. (Takamura et al., 2003; Xie et al., 2022). However, the molecular mechanisms of cataract development are not fully elucidated.

Oxidative stress is a prominent and critical factor for cataractogenesis. Generally, the metabolically produced reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂) and hydroxyl radical, can be reduced by the antioxidants like reduced glutathione (GGH), thus maintaining a stable and balanced redox environment in LECs. (Giblin, 2000). Any disturbance to this balance, whether due to aging, ultraviolet exposure, or some other factors, will result in redundancy of free radicals, which in turn contribute to cataract development. (Yildirim et al., 2009; Pescosolido et al., 2016; Hsueh and Chen, 2022). The lens proteins, such as crystallins, are found oxidized and aggregated in the nucleus of the lens and therefore scatter the light during the development of cataract. (Cobb and Petrash, 2002; Vetter et al., 2020; Hanafy and Cave, 2021). Of note, the most metabolically-active LECs are more susceptible to oxidative radicals. Excessive ROS can incur the apoptosis of LECs by targeting the DNA, membrane proteins, and many other constitutive components (Long et al., 2004; Cui et al., 2012), while antioxidants administration can ameliorate the H₂O₂-induced apoptosis of LECs(Zhou et al., 2016; Bai et al., 2017). Nevertheless, the exact and comprehensive molecular mechanisms of LECs apoptosis and the ensuing cataract under oxidative stress are, to our knowledge, not completely elaborated.

Autophagy (here referred specifically to macroautophagy) is an evolutionally conserved, catabolic process across a variety of species, which can break down the dysfunctional or unneeded macromolecules and membrane-coated organelles in the cytoplasm to recycle the necessary building substrates. It entails the formation of autophagosomes, their fusion with lysosomes, and the degradation of the inside components, each phase involving certain critical proteins, such as BECN1, LC3B, and ATG7. Although it is controversial whether autophagy is involved in the formation of the organelle-free zone during lens development (Matsui et al., 2006; Nishida et al., 2009; Morishita et al., 2013; Tu et al., 2021; Gheyas et al., 2022), the development of cataract is closely associated with dysfunctional autophagy. (Morishita, Eguchi, Kimura, Sasaki, Sakamaki, Robinson, Sasaki and Mizushima, 2013; Ping et al., 2021). A recent study revealed that rapamycin-induced autophagy can alleviate the level of ROS in LECs cultured in high glucose. (Liu J. et al., 2020). Furthermore, an elevated autography accompanied reduced apoptosis in H₂O₂-treated LECs(Han et al., 2021), and the overexpression of ATG4a in LECs can mitigate the apoptosis of cells. (Yan et al., 2020). It may suggest a cytoprotective role of autophagy toward LECs under oxidative stress by regulating cell apoptosis, which is quite contradictory to the findings of Huang J et al., whose study revealed autophagy-facilitated apoptosis. (Huang et al., 2022). Therefore, the detailed molecular mechanisms concerning the roles of autophagy in oxidative stress need further explored.

Non-coding RNAs (ncRNAs) refer to a considerable amount of transcriptomes without protein-coding function, which can be simply grouped into long non-coding RNAs (lncRNAs), small RNAs like microRNAs (miRNAs), transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) based on their length and function. Over the past few years, ncRNAs, especially lncRNAs and miRNAs, are found to play a vital role in the development of a variety of diseases, including cataracts. Recent studies showed that the elevated miR-23b-3p and miR-34a expressions could promote the apoptosis of LECs under the oxidative stress state while overexpression of miR-124 reduced the apoptosis of H₂O₂-treated LECs. (Fan et al., 2017; Gu, 2018; Zhou et al., 2019; Zhang et al., 2020). Moreover, lncRNA-H19 was up-regulated in cataract tissue and its knockdown could accelerate apoptosis of LECs under oxidative stress, and lncRNA TUG1 can promote the apoptosis of H₂O₂-treated LECs via targeting the miR-196a-5p. (Liu et al., 2018; Shen and Zhou, 2021). Although there is growing evidence of ncRNAs involved in pathogenic processes during cataract development, the comprehensive profile of ncRNAs and the interaction network concerning cataractogenesis have not been reported.

Autophagy requires the elongation of the phagophore at the beginning, during which ATG7 is critical for the formation and activation of two important complexes, ATG12-ATG5 complex and LC3-PE complex (Rubinsztein et al., 2012). Ablation of ATG7 is thought to completely block the elongation of the phagophore, thus abolishing the autophagy activity. Herein, we selected the LECs knocked out of ATG7 to abolish the autophagy process during oxidative stress, and conducted detailed analyses of RNA sequencing data from the Gene Expression Omnibus (GEO) database to explore the expression profiles of mRNAs and lncRNAs of LECs under the circumstance of oxidative stress and to try to construct the possible molecular network of ATG7-associated cataractogenic mechanisms, hoping to offer some clues to the studies regarding the prevention and/or treatment of cataracts.

Age-related cataract (ARC) is the most prominent type of cataracts and oxidative stress is one of the well-established culprit factors during cataract development, since there is a loss of antioxidants, such as reduced GSH, and increased protein oxidation in the nuclear region of the lens prior to the cataract development. (Giblin, 2000; Truscott, 2005; Vinson, 2006). Based on the location of opacity, ARC can be subdivided into three main types: nuclear, cortical, and posterior subcapsular cataracts. (Liu et al., 2017). Despite their association with aging, the underlying pathological changes and mechanisms seem somewhat different. Nuclear cataract, as the most common type, is more subject to oxidative damage compared with the other two types. (Truscott, 2005). In contrast, the destruction of cell structure and precipitation of soluble proteins are more likely observed in cortical cataract, although it is also associated with some degree of oxidation. As for the less common posterior subcapsular cataract, the failed elongation of swollen fiber cells may be the main cause. When considering the risk factors, there are also differences among the three types of cataracts. Cortical cataract is closely associated with high sunlight exposure and is more commonly seen in high-latitude regions, such as northern Japan and northern China. (Sasaki et al., 2002). Wearing sunglasses is verified as an effective way for its prevention. (Taylor et al., 1988). The posterior subcapsular cataract, however, is more associated with high myopia, diabetes, steroid administration, and ionizing radiation. (Beebe et al., 2010). Such discrepancies among age-related cataracts suggest intricate or even distinct mechanisms underlying cataractogenesis. Therefore, the cell line model stimulated by H₂O₂, although commonly used to simulate the factual oxidative stress, can only partly explain the possible mechanisms with caution.

In this study, we detected several pathways and differentially expressed genes that were associated with ATG7. Among them, the intrinsic apoptotic signaling pathway was one of the most enriched pathways for the differentially expressed genes between the groups. As one of the two types of apoptosis signaling, the intrinsic apoptotic signaling pathway is usually activated by internal stimuli such as hypoxia and free radical-induced oxidative stress. It has been found involved in tumor death due to chemotherapy and thus has been investigated as the therapeutic target for drug discovery. (Carneiro and El-Deiry, 2020; Kashyap et al., 2021). With the aging of the lens, lens epithelial cells also underwent certain apoptosis induced by oxidative stress from diabetes, ultraviolet exposure, or just senescence. (Su et al., 2017; Xie et al., 2022). In our study, we detected the intrinsic apoptosis signaling pathway as the potential autophagy-associated mechanism underlying the oxidative stress-induced injury toward LECs. The involved differentially expressed genes in this enriched signaling pathway, such as PMAIP1, BBC3, ENR1, and CHAC1, are thus worthy of further investigation as potential therapeutic targets for cataract development.

It is well recognized that autophagy can contribute to the degradation of damaged or wasted molecules and organelles, including oxidized proteins and lipids, which, if gradually accumulated, could result in cell apoptosis or even necrosis. (Truscott, 2005). The overexpression of ATG4 can activate autophagy and meanwhile inhibit apoptosis of the HLE-B3 cell line under H₂O₂-challenged circumstances. (Yan, Zhao, Qin, Zhao, Ji, and Zhang, 2020). And the rapamycin-induced autophagy can alleviate the ROS production in mice LECs cultured in high glucose. (Liu X. et al, 2020). However, some studies revealed the opposite results. The suppression of autophagy by EphA2 can attenuate the apoptosis of SRA01/04 cells induced by H₂O₂. (Han, Wang, Lv, Liu, Dong, Shi and Ji, 2021). Huang J et al. also found that autophagy facilitated the apoptosis of HLE-B3 cells under H₂O₂ stimulation. (Huang, Yu, He, He, Yang, Chen and Han, 2022). By mining the RNA sequencing data, our results showed that the differentially down-regulated genes in autophagy-deficient cells are mostly enriched in GO terms related to cell proliferation and differentiation, such as regulation of hemopoiesis, muscle organ development, and fat cell differentiation, which may suggest a pro-apoptotic function of autophagy. The controversial functions of autophagy toward cell apoptosis under oxidative stress may reside in the distinct cell lines and different concentrations and times of H₂O₂ stimulation. Further studies are warranted to clarify the relationship between autophagy and apoptosis in a detailed manner.

ATG7, as one of the autophagy-related proteins, is a ubiquitin-activating enzyme E1-like protein and participates, along with ATG3, in the conjugation of ATG8 family proteins to phosphatidylethanolamine (PE) during the phagophore expansion. (Dooley et al., 2014). Although ATG7 is an essential mediator in the canonical autophagy pathway, Atg7-independent autophagy was found in Atg7-modified mice, where knockout of Atg7 did not affect the formation of autophagosome and the subsequent bulk degradation. (Nishida, Arakawa, Fujitani, Yamaguchi, Mizuta, Kanaseki, Komatsu, Otsu, Tsujimoto and Shimizu, 2009). The loss of Atg7 in Drosophila did not prevent the occurrence of autophagy and the accompanied cell size reduction. (Chang et al., 2013). However, such ATG7-independent autophagy has not yet been reported in human cells. Furthermore, ATG components, including ATG7, have found to engage in non-autophagic activities, such as phagocytosis (Sanjuan et al., 2007), osteoclastic bone resorption (DeSelm et al., 2011) and antiviral activity of IFNγ (Hwang et al., 2012). In this study, we found that the DEGs shared by the two types of HLE-B3 cells (KO and WT) under oxidative stress are enriched in apoptotic signaling pathways (Figure 3C), which may suggest an ATG7-independent autophagy involved in cell apoptosis.

miRNAs, the highly conserved small ncRNAs across species, have long been found regulating substantial gene expressions by targeting the microRNA response elements (MREs) of mRNAs. (Thomas et al., 2010). Each miRNA can regulate many mRNAs and one mRNA can be regulated by a number of miRNAs. (Friedman et al., 2009). And lncRNA, a group of ncRNAs larger than 200 nucleotides in length, has emerged as an important regulator in a myriad of diseases, including cataract. (Liu, Liu, Shan, Zhang, Lu, Yan, and Luo, 2018; Liu X. et al., 2020; Tu et al., 2020; Shen and Zhou, 2021). On the one hand, they can directly target miRNAs to regulate certain mRNA expressions (Ye and Ma, 2020) while on the other, they can also be targeted by the miRNAs (Chi et al., 2009). Such complicated interactions within the ncRNAs and mRNAs suggest the mechanism of ceRNA. (Salmena et al., 2011). In this article, we constructed a predicated lncRNA-miRNA-mRNA ceRNA network based on the differentially expressed lncRNAs and mRNAs and the reported cataract-related miRNAs. Such a ceRNA network may propose an ATG7-associated potential regulatory mechanism underlying cataractogenesis, which warrants further verification.

In all, this study reveals the differentially expressed gene profiles of HLE-B3 cells with or without ATG7 knockout subjected to oxidative stress. And by comparing the gene expressions in these two types of cells under oxidative stimulation, we eventually detected differentially expressed 292 mRNAs and 24 lncRNAs that also interacted with the 10 cataract-associated miRNAs. Thus a competing endogenous lncRNA-miRNA-mRNA network was finally constructed based on such interactions, which warrants further investigations.