LncRNA NORAD defects deteriorate the formation of age-related macular degeneration

Results

Irradiation induced AMD characteristics of retinal cells

Radiation exposure could damage the lens epithelial cells of the eye to induce cataracts [40, 41]. We thus examined whether irradiation could injure retinal cells to give rise to AMD. We used ARPE-19, a human retinal pigment epithelial cell line, for irradiation at 20 min under 100 cGy/min dosage rate (Figure 1A). Cell morphology changed obviously at 96 h after irradiation compared with control groups. SA-β-gal staining was also performed to assess cell senescence. The positively stained cells in the irradiated control group were obviously higher than those in the control group without irradiation (Figure 1B). Irradiation-induced apoptosis of ARPE-19 was detected via Annexin V–FITC/PI double staining. As shown in Figure 1C, irradiation increased cell apoptosis. A cell cycle experiment was then performed to examine the effect of irradiation on the ARPE-19 cell cycle progression. In irradiated cells, the percentage of ARPE-19 in the G2/M phase was enhanced compared with that in the control group, whereas the percentage of S-phase and G0/G1 cells was decreased, suggesting that the irradiation could lead to cell cycle arrest at the G2/M phase (Figure 1D). We performed a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) experiment to identify that complement/inflammatory and AMD/drusen transcripts were enhanced in the irradiated group compared with the control groups (Figure 1E). In examining the secretion of C3, VEGF-A, Aβ-40, and Aβ-42 (Figure 1F) from the ARPE-19 culture supernatant medium using ELISA after irradiation, we found higher levels in the irradiated group compared with the control groups. Overall, these results suggested that irradiation could induce senescence and apoptosis of ARPE-19 cells, thereby leading to subsequent AMD formation.

Figure 1. Irradiation induced AMD markers of ARPE-19. ARPE-19 cell lines were treated with a dose of irradiation and the cells were then observed for AMD phenotypes at different time periods. (A) Schematic diagram of the experimental design for AMD cell model. (B) Morphological observation of ARPE-19 cells at 96 h after irradiation. ARPE-19 was stained in 48 h after irradiation to determine the SA-β-gal staining. Irradiation increased the number of positive-stained cell. Results were representative of three separate experiments (100X, measure scar 50um) (C) Irradiation increased cell apoptosis. The apoptosis rate was detected through flow cytometry by using annexin V-FITC/PI double staining. The apoptotic rate was analyzed in 72h after irradiation in terms of the percentage of the lower and upper right quadrants. n = 3, ***P < 0.001. (D) Irradiation induced cell cycle arrest at G2/M phase. Flow cytometry was used to detect the cell cycle distribution of irradiation-treated ARPE-19 in 72h after irradiation. n = 3, ***P < 0.001. (E) RT-qPCR analysis of ARPE-19 in 96h after irradiation for AMD markers. n = 3, *P < 0.05, **P < 0.01, ***P<0.001. (F) ELISA testing of culture supernatant from normal ARPE-19 and irradiation-treated ARPE-19 obtained 96h after irradiation. n = 3, ***P<0.001.

NORAD-knockdown retinal cells were more susceptible to radiation damage to form AMD

We next explored whether NORAD knockdown in the ARPE-19 cell line leads to the eventual worsening of AMD development after irradiation at a certain incubation time (Figure 2A). Small interfering RNA-targeting NORAD (siNORAD) was transfected into ARPE-19 to knock down the NORAD expression. The NORAD knockdown was verified and assessed through RT-qPCR. The NORAD expression in siNORAD cells was knocked down by 70% compared with the counterpart without small interfering RNA (siNC) cells (Figure 2B). Subsequently, we investigated whether NORAD knockdown increased the sensitivity of ARPE-19 to irradiation stress. Figure 2C shows that the cell morphology of the irradiated siNORAD group was significantly changed compared with that of the irradiated siNC group. In irradiation treatment cells, the percentage of SA-β-gal-positive cells in the siNORAD group was obviously enhanced compared with that in the siNC group (Figure 2C). The effect of NORAD-knockdown on the irradiation-induced apoptosis of ARPE-19 was detected via Annexin V–FITC/PI double staining. As shown in Figure 2D, NORAD knockdown intensively increased irradiation-induced cell apoptosis. In irradiated cells, the percentage of ARPE-19 in the G0/G1 and S phase was decreased in the siNORAD group compared with that in the siNC group, but the percentage of G2/M phase cells was enhanced significantly, indicating a G2/M phase arrest (Figure 2E). Then, we examined the mRNA level of AMD-related markers including complement/inflammatory and AMD/drusen transcripts using RT-qPCR and verified that the outcome for the irradiated siNORAD group was higher than that for the irradiated siNC group (Figure 2F). In analyzing the protein level of AMD-related markers ARPE-19 secreted, we used ELISA and found that NORAD-knockdown significantly accelerated the levels of C3, VEGF-A, Aβ-40, and Aβ-42 in culture supernatants (Figure 2G). Correspondingly, the levels of the aging-related proteins, p-P53 and P21, were significantly increased in irradiation-induced ARPE-19 cell lines. Surprisingly, NORAD knockdown accelerated the expression of p-P53 and P21 (Figure 2H). Taken together, these findings indicate that the irradiated siNORAD group presented more severe G2/M arrest, senescence, and apoptosis of ARPE-19 than the irradiated siNC group, thereby NORAD-knockdown in ARPE-19 promoted the sensitivity of ARPE-19 to irradiation damage.

Figure 2. NORAD-knockdown aggravates irradiation-induced AMD markers of ARPE-19. ARPE-19 cell lines were transfected with siRNA of NORAD, after 24h, the cells were treated with irradiation, then observed for AMD phenotypes at different time periods. (A) Schematic diagram of experiment design to test the effect of NORAD on the irradiation-induced AMD model. (B) ARPE-19 were transfected with siNORAD or siNC. NORAD levels were analyzed through RT-qPCR. n = 3, ***P < 0.001. (C) ARPE-19 was observed cell morphology and stained to determine SA-β-gal. NORAD knockdown increased the number of positive-stained cells after they were treated with irradiation. Results were representative of three separate experiments. (100X, measure scar 50um) (D) NORAD-knockdown increased irradiation-induced cell apoptosis. The apoptosis rate was detected through flow cytometry by using annexin V-FITC/PI double staining. The apoptotic rate was analyzed in terms of the percentage of the lower and upper right quadrants. n = 3, ***P < 0.001. (E) NORAD silencing induced irradiation-treated cell cycle arrest at G2/M phase. Flow cytometry was used to detect the cell cycle distribution of irradiation-treated ARPE-19. n = 3, *P < 0.05, ***P < 0.001. (F) NORAD-knockdown increased mRNA expression of AMD markers in irradiation-treated ARPE-19 through qRT-PCR. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. (G) NORAD knockdown increased AMD markers of culture supernatant from irradiation-treated ARPE-19. n = 3, *P<0.05, ***P<0.001. (H) NORAD-knockdown increased the p-P53 and P21 expression observed through western blot. The ratio of p-P53 and P21 to GAPDH was analyzed with ImageJ. Values were shown as mean ± SD, n = 3, *P<0.05, ***P<0.001.

The retinas of NORAD knockout mice were more susceptible to sodium iodate damage

To prove that NORAD-knockout could accelerate the outside damage to the retina in vivo, we used NORAD-knockout mice injected with NaIO₃ in the tail vein. After 7 days of NaIO₃ treatment, OCT was employed to observe the thickness of the entire retina (Figure 3A). Little difference was observed in retinal thickness between NORAD- knockout mice and normal wild mice (Figure 3C). When we injected the mice with NaIO₃, retinal thickness decreased in both wild and NORAD-knockout mice (Figure 3C), but the thickness of the retinal damage in NORAD-knockout mice was greater than that in WT mice (Figure 3D). To validate the results, we performed a funduscopic examination to evaluate the retinal pigment loss (Figure 3B). Before the NaIO₃ injection, WT and NORAD-knockout mice had similar retinas (Figure 3E). After the NaIO₃ injection, the fundus images of WT mice showed yellow spots on the retina, representing RPE loss by NaIO₃. By contrast, the RPE loss area of NORAD-knockout mice was beyond the WT mice with NaIO₃ (Figure 3F). Next, we assessed the functional integrity of the retina using electroretinography (Figure 3G). The amplitudes of the a- and b-waves of WT and NORAD-knockout mice are similar before the NaIO₃ injection (Figures 3H, 3J). Moreover, the amplitudes of the a- and b-waves decreased in NaIO₃-injected WT mice compared with those of the WT group without NaIO₃, thereby demonstrating functional damage of the photoreceptors and dysfunction in the bipolar cells of NaIO₃-injected WT mice. The amplitudes of the a- and b-waves of NORAD-out mice also decreased more than that of the wild mice (Figures 3I, 3K). We then measured the expression of p-P53 and P21 proteins in the eyes of the mice. No significant change in p53 and p21 between WT mice and NORAD-knockout mice. Conversely, the increase in p-P53 and P21 in NORAD-knockout mice was greater than that in wild mice under sodium iodate injection induced AMD model. (Figure 3L). These findings indicate that the retinas of NORAD-knockout mice were more vulnerable to injury than those of WT mice, an outcome that was consistent with in vitro results.

Figure 3. Injury effects of NORAD knockout on the retina of NaIO₃-treated mice. Different techniques were used to detect retinal damage in wild-type mice and NORAD knockout mice treated with sodium iodate. (A) Optical coherence tomography (OCT) was performed seven days after NaIO₃ treatment for all three study groups. (B) Funduscopic examinations were performed seven days after SI injection. Representative OCT and fundus fluorescence shows the RPE of the mice in each experimental group. (C) Statistical graph of retinal thickness. (D) Statistical graph of retinal damage between WT mouse and NORAD-KO mouse after SI injection. (E) Statistical graph of retinal pigment loss area. (F) Statistical graph of retinal pigment loss degree between WT mouse and NORAD-knockout mouse after SI injection. (G) Electroretinography was performed to investigate the function of the retina in response to light. (H) Statistical graph of b wave amplitude change. (I) Statistical graph of the degree of change in b wave amplitude between WT mouse and NORAD-knockout mouse in eight days after SI injection. (J) Statistical graph of a wave amplitude change. (K) Statistical graph of the degree of change in a wave amplitude between WT mouse and NORAD-knockout mouse in eight days after SI injection. n≥3, *P < 0.05, **P < 0.01, ***P < 0.001. (L) Western blot was used to detect the p-P53 and P21 expression levels. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

NORAD knockdown increased mitochondrial ROS production and complement 3 expressions in the eyes of mice

Considering that mitochondrial ROS elevation is a reason for the increase of p-P53 and P21, we tested the mitochondrial ROS production in multiple organs of mice. One week after NaIO₃ injection, the mitochondrial ROS in the eyes, heart, liver, lung, brain, and kidney of wild mice increased significantly, and the expression of mitochondrial ROS in the eyes, heart, and liver of NORAD-knockout mice was higher than that of wild mice (Figure 4A–4F). Next, we detected AMD markers in the eyes of mice and found that the mRNA levels of C3 and ICAM-1 were significantly increased in the eyes one week after NaIO₃ injection, and the expression of C3 was increased in NORAD-knockout mice (Figure 4G). These results suggest that NORAD-knockout exacerbates the increase of mitochondrial ROS and C3 in the eye.

Figure 4. NORAD-knockout aggravates SI-induced mitochondrial ROS and C3 expression level in vivo. Mitochondrial ROS and AMD markers were detected in wild-type mice and NORAD knockout mice treated with sodium iodate. (A–F) Flow cytometry was used to detect the mitochondrial ROS levels in the eyes, heart, lungs, liver, brain, and kidneys of mice on day 8 after SI injection. n=3, *P < 0.05, **P < 0.01. (G) mRNA expression of AMD markers in eyes of the mouse through qRT-PCR. n = 3, *P < 0.05, **P < 0.01.

NORAD knockdown increased mitochondrial damage and mitochondrial ROS and PGC-1α acetylation

To verify the results of increased ocular mitochondrial ROS in vivo, we examined mitochondrial damage and mitochondrial ROS production in vitro using an irradiation-induced AMD model. To explore the effect of NORAD-knockdown on mitochondrial genes under irradiation, we conducted an RT-qPCR experiment and determined that irradiation decreased the mitochondrial homeostasis factors TFAM and POLG and mitochondrial respiratory chain complex genes ND1 and ND5 compared to a control group. However, NORAD-knockdown exacerbated the decrease of these mitochondrial genes (Figure 5A). MitoSOX staining revealed that irradiation increased the mitochondrial ROS compared with the control group. By contrast, the increase in mitochondrial ROS production was further aggravated upon NORAD-knockdown treatment (Figure 5B). The results were validated by fluorescence microscope photography (Figure 5C). As only non-acetylated PGC-1α can activate mitochondrial oxidative processes and inhibit the production of mitochondrial ROS, we examined PGC-1α acetylation under conditions of pathological damage. We found that NaIO₃ and irradiation did not increase PGC-1α acetylation. Conversely, NORAD knockdown increased PGC-1α acetylation under both pathological and non-pathological conditions (Figures 5D, 5E). To validate the effect of NORAD on PGC-1α, we detected PGC-1α acetylation in various organs of NORAD-knockout mice. NORAD knockout increased PGC-1α acetylation in the heart, liver, lung, and eye, but was less explicit in the kidney and brain (Figure 5F). These findings indicate that the effect of NaIO₃ and irradiation on PGC-1α acetylation is not obvious; nevertheless, NORAD knockdown could promote PGC-1α acetylation.

Figure 5. NORAD knockdown increased the PGC-1α acetylation and mitochondrial ROS. ARPE-19 cell lines were transfected with siRNA of siNORAD or siNC, after 96 hours, the cells were observed for mitochondrial damage, mitochondrial ROS, PGC-1α. (A) NORAD knockdown decreased the level of mRNA of mitochondrial transcription factor A (TFAM), mitochondrial DNA polymerase catalytic subunit (POLG), mitochondrially encoded NADH dehydrogenase 1 (ND1), and mitochondrially encoded NADH dehydrogenase 5 (ND5) in irradiation treated ARPE-19. (B) Flow cytometry was used to detect the mitochondrial ROS levels. Irradiation increased the mitochondrial ROS and NORAD knockdown aggravates irradiation-induced the rise of mitochondrial ROS. Data were from three separate experiments and described as mean ± SD. **P < 0.01. (C) NORAD knockdown aggravated irradiation-induced ROS production. The ROS levels were observed under an inverted fluorescence microscope. Images were representative of three separate experiments. n = 3, **P < 0.01. (40X, measure scar 20um) (D) Immunoprecipitation and western blot analysis of acetyl-PGC-1α levels in ARPE-19 transfected with siNORAD and exposed to sodium iodate. (E) Immunoprecipitation and western blot analysis of acetyl-PGC-1α levels in ARPE-19 transfected with siNORAD and exposed to irradiation. (F) Immunoprecipitation and western blot analysis of acetyl-PGC-1α levels in heart, liver, lung, kidney, brain, and eye of NORAD knockout mouse.

NORAD knockdown promoted the binding of PGC-1α and SIRT1

According to the literature, PGC-1α, SIRT1, and NORAD interact with one another, so we performed RNA binding protein immunoprecipitation assay experiments (RIP) to determine whether both PGC-1α and SIRT1 interact with NORAD (Figures 6A, 6B). On the premise that the degree of binding between SIRT1 and PGC-1α influenced PGC-1α acetylation, we then detected PGC-1α acetylation after SIRT1 knockdown, NORAD knockdown, and combined SIRT1 and NORAD knockdown using an immunoprecipitation (IP) experiment. SIRT1 knockdown was confirmed by WB (Figure 6C). We verified that both SIRT1 knockdown and NORAD knockdown increased PGC-1α acetylation with similar effects (Figure 6D). The simultaneous knockdown of SIRT1 and NORAD had a cumulative effect on PGC-1α acetylation (Figure 6D). The presence of both SIRT1 and NORAD, therefore, exerts an effect on PGC-1α acetylation. We also hypothesized that the presence of NORAD facilitates SIRT1 and PGC-1α binding. Therefore, we detected SIRT 1 and PGC-1α binding through co-immunoprecipitation (co-IP) experiments. When we captured SIRT1 with antibodies against PGC-1α, we were able to drag SIRT1 down (Figure 6E); conversely, we were also able to drag down PGC-1α with antibodies against SIRT1 (Figure 6F). Thus, SIRT1 and PGC-1α were bound to each other. Surprisingly, when NORAD was knocked down, the amount of SIRT1 dragged down by the PGC-1α antibody and the amount of PGC-1α dragged down by the SIRT1 antibody both decreased (Figure 6E, 6F). These outcomes indicate that the presence of NORAD is conducive to the binding of SIRT1 and PGC-1α, thus promoting PGC-1α deacetylation by SIRT1.

Figure 6. NORAD knockdown increased the binding of PGC-1α and SIRT1. ARPE-19 cell lines were transfected with siNORAD or siNC, after 96 hours, the interaction of NORAD with PGC-1α and SIRT1 was detected. (A) RIP assay confirmed the interaction of NORAD and PGC-1α. All groups of ARPE-9 were incubated with the anti-PGC-1α or anti-IgG antibody, and the precipitated complexes were analyzed with RT-qPCR to investigate the expression of NORAD. n = 3, ***P < 0.001. (B) RIP assay confirmed the interaction of NORAD and SIRT1. All groups of ARPE-9 were incubated with the anti-SIRT1 or anti-IgG antibody, and the precipitated complexes were analyzed with RT-qPCR to investigate the expression of NORAD. n = 3, ***P < 0.001. (C) ARPE-19 was transfected with siSIRT1 or normal contrast siNC. SIRT1 levels were analyzed through western blot. n = 3, ***P < 0.001 vs. siNC. (D) Immunoprecipitation and western blot analysis of acetyl-PGC-1α levels in ARPE-19 transfected with siNORAD, siSIRT1, or normal contrast siNC. (E) IP experiment confirmed the interaction of SIRT1 and PGC-1α. All groups of ARPE-9 were incubated with the anti-PGC-1α or anti-IgG antibody, and the precipitated complexes were analyzed with anti-SIRT1 by western blot. n = 3. (F) IP experiment confirmed the interaction of PGC-1α and SIRT1. All groups of ARPE-9 were incubated with the anti-SIRT1 or anti-IgG antibody, and the precipitated complexes were analyzed with anti-PGC-1α by western blot. n = 3.

Discussion

A specific dose of irradiation can cause retinal cells to produce AMD characterization that promoted aging and apoptosis and increased AMD-related gene makers. Using an AMD cell model, we investigated the effect of NORAD knockdown on retinal cells. We confirmed that NORAD-knockdown retinal cells were more susceptible to radiation damage, thereby generating higher AMD signs, and were more likely to age into AMD. The mitochondria of NORAD-knockdown retinal cells are also more vulnerable to radiation damage, causing the production of more mitochondrial ROS and activation of more p-P53 and P21, which, in turn, lead to the aging and apoptosis of retinal cells. We induced a mouse model of AMD with NaIO₃ and found that the retina of the NORAD knockout mouse suffered more serious damage, consistent with the results of cell experiments. More importantly, NORAD-knockdown upregulated PGC-1α acetylation, thereby suggesting that NORAD may increase PGC-1α and SIRT1 interaction and promote the deacetylation of SIRT1 to PGC-1α. Furthermore, NORAD could interact with SIRT1 and PGC-1α. Together, these findings shed light on the molecular mechanism whereby NORAD-knockdown retinal cells may increase the risk of AMD and provide a potential target for AMD treatment.

AMD is a complex disease caused by a variety of environmental and genetic factors [42]. Many in vitro models of AMD have been established. As we know, the approaches of exposing A2E-laden ARPE-19 cells to blue light [36], the NaIO₃-induced AMD cell model [35], and the dox-induced AMD cell model [32] all take advantage of a drug’s effect on cell damage, causing RPE cell death. Transmitochondrial cybrid cell lines that contain identical nuclei but possess mitochondria from AMD patients exploit the mitochondrial damage in the pathogenesis of AMD [43]. Given the aging-inducing properties of X-radiation [39], we irradiated the ARPE-19 cell line for some time to accelerate its senescence and eventually develop AMD-related markers. We irradiated the ARPE-19 cell line for some time to accelerate its senescence and eventually develop AMD-related markers. Our model is simple and close to the real environment of eye damage.

In the initial studies, NORAD was associated with genomic instability, and most subsequent research linked NORAD dysregulation to many cancers [18, 44]. Downregulation of NORAD promotes the production of apoptosis-related proteins by increasing PARP, caspase-3, and Bax and inhibits the proliferation and invasion of gastric cancer cells [45]. NORAD knockdown suppressed the proliferation and invasion of osteosarcoma cells because NORAD acted as a sponge for miR-199a-3p [46]. Next, removing NORAD drives premature aging in mice [22]. Our laboratory determined immediately that NORAD knockdown induces cell cycle arrest and aggravates ox-LDL-induced cell senescence of HUVECs, which promotes Ox-LDL-induced vascular endothelial cell injury and atherosclerosis [47]. Recently, the increased m6A modification level of NORAD reduced the sealing effect of PUMILIO protein, inhibited the expression of E2F3, and led to the aging of nucleus pulposus cells and intervertebral disc degeneration. These results all proved the significance of NORAD in aging. Similarly, we determined that NORAD knockdown accelerates the senescence of irradiation-induced ARPE-19 and exacerbates the AMD phenotype, thereby further proving the close relationship between NORAD and aging degenerative diseases. Therefore, NORAD may be a good target for the treatment of degenerative diseases.

Aging, sun exposure, low antioxidant intake, and other factors can cause mitochondrial damage that affects genes encoding the mitochondrial electron transport chain resulting in ROS overproduction [48]. Such changes will eventually lead to senescence and damage of RPE cells, resulting in retinal degeneration and AMD [48]. We confirmed that NORAD-knockdown damages the genes on the respiratory chain NADH1 and NADH5 and the mitochondria replication and transcription genes TFAM and POLG, which induce higher mitochondrial ROS production and a higher level of senescence-related factors p-P53 and P21. TFAM decreases ROS production by controlling the expression of mitochondrial ROS-generating enzymes, such as nicotinamide adenine dinucleotide phosphate oxidases [49]. In addition, considering that loss of PGC-1α or repression of the pathway of PGC-1α could induce impairment of RPE such as increasing mitochondrial ROS [50], promotion of retinal degeneration [51], metabolic dysfunction [52–54], and AMD-like phenotypes in mice [55, 56]. We further examined the PGC-1α level and PGC-1α acetylation activation. Interestingly, only the acetylated PGC-1α increased in NORAD-knockdown RPE while the total level of PGC-1α is unchangeable. As Rodgers had demonstrated that PGC-1α deacetylation is accomplished by an increased transcription level of SIRT1 [57], and, then we detected the level of SIRT1 after the NORAD knockdown, while the expression level of SIRT 1 is still unchangeable. Note that NORAD can inhibit PUM by the formation of phase-separated PUM condensates, thereby revealing a mechanism by which RNA-driven phase separation can regulate RNA-binding-protein activity and which may be a widely used mechanism of lncRNA-mediated regulation [19]. One prior work reported that LINC00842 binds to and prevents acetylated PGC-1α from deacetylation by deacetylase SIRT1 to form PGC-1α and consequently enhance the malignant phenotypes of PDAC cells [58]. Thus, we examined whether the absence of NORAD affected the binding of SIRT1 and PGC-1α using RIP and IP experiments. Surprisingly, we observed decreased binding between SIRT1 and PGC-1α after the NORAD knockdown. In contrast to LINC00842, NORAD promotes the deacetylation of PGC-1α by SIRT1 to activate PGC-1α. Our findings identify one mechanism by which lncRNA regulates the activity between proteins through binding proteins. Given the important role of PGC-1α in aging, NORAD and PGC-1α may also play an important role in other age-related degenerative diseases.

We acknowledge some limitations and confusion in our current study. Although we focused simply on the effect of NORAD knockdown in retinal cells on the formation and development of AMD with exposure to irradiation, the effect of NORAD overexpression on AMD is worth investigating. We tried to examine that effect but were unable to find a way to overexpress NORAD in retinal cells without impairing the state of the cell itself. In addition, better irradiation conditions and conditions for AMD formation need to be further explored. Irradiation mouse models of AMD are also unavailable, so we used NaIO₃-induced AMD on mice. Finally, we found that after NORAD knockout, the PGC-1α acetylation level was increased in the eyes, heart, liver, and lungs, but ROS was decreased in the lung. No increased acetylation was observed in the kidney and brain. These results suggest that the role of NORAD in different organs may vary, along with different mechanisms involved.

In summary, these results shed light on the important role of lncRNA NORAD in AMD development and progression (Figure 7) and provide a foundation for developing more targeted AMD therapies and predicting whether low NORAD expression is likely linked to the development of AMD or other diseases of aging.

Figure 7. Diagram of mechanism. Environmental damage factors such as light exposure and irradiation could damage the retina and retinal pigment epithelial (RPE). NORAD-knockdown accelerates retinal cell senescence, apoptosis via PGC-1α acetylation, mitochondrial ROS, and the p-P53–P21signaling pathway, which resulting in worsening AMD. NORAD-mediated effect on PGC-1α acetylation might occur through the direct interaction with PGC-1α and SIRT1.

Abbreviations

NORAD: non-coding RNA activated by DNA damage; NORAD-KO or -/-: NORAD-knockout; SA-β-gal: senescence associated-beta-galactosidase; ROS: reactive oxygen species; siNORAD: small interfering RNA-targeting NORAD; siSIRT1: small interfering RNA-targeting SIRT1; siNC: small interfering RNA-targeting negative control; PGC-1α: peroxisome proliferator-activated receptor gamma coactivator 1-alpha; SIRT1: sirtuin 1; C3: complement C3; ICAM-1: intercellular adhesion molecule 1; VEGFA: Vascular Endothelial Growth Factor A; APOE: apolipoprotein E; APP: amyloid beta precursor protein; Aβ-40: amyloid β-40; Aβ-42: amyloid β-42; CRYAA: crystallin alpha A.

Author Contributions

Jing Jiang designed and directed experiments. Jinfeng Zhang performed most experiments, orchestrated data analysis, and write the draft manuscript. Shenjun Li, and Lifu Hu contributed to the establishment of the AMD animal model and performed OCT, funduscopic examination, and electroretinography experiments. Hongyu Zhou and Weihua Bian performed parts of the RT-qPCR experiments, provided data, and performed preliminary analysis. Daolai Zhang, Cong Xu, and Yeying Sun supported administrative, technical, and material or supervised some of the experiments. Jing Jiang orchestrated data interpretation and analysis and graphic presentation and finalized the manuscript. All authors have approved the final paper.

Acknowledgments

We thank Jianhui Pang, and Mengyi Ma for help with establishing the AMD animal model setup and Enping Yu for assisting in the experiment. We thank the non-clinical research department, RemeGen Co., Ltd for providing OCT, funduscopic examination, and electroretinography instruments. This project was supported by Binzhou Medical University.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Ethical Statement

This study was approved by the Ethics Committee of Binzhou Medical University.

Funding

This work funding was supported by the Shandong Provincial Natural Foundation (NO. ZR2021MH220); Yantai Science and Technology Plan (NO. 2021XDHZ083).

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