High-throughput sequencing and Co-expression network analysis of LncRNAs and mRNAs of PBMCs in AMD patients

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Differentially expressed transcriptomes were screened by paired sequencing PBMCs of 5 wet AMD patients, 3 dry AMD patients and 4 normal controls respectively. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were conducted on them. Real-time PCR was used to verify the expression of selected Long non-coding RNA(lncRNA). GO and KEGG enrichment analysis of transcriptomes showed that, compared with normal controls, the RNA transcription, metabolism and ribosome-related pathways were all down-regulated.In wAMD patients, T lymphocyte activation is significantly up-regulated and participates in T cell receptor signaling pathway. Metabolism are significantly down-regulated, mainly involved in the lysosomal pathway. Compared with dAMD patients, wAMD patients mainly exhibit up-regulated immune system regulation and MYD88-dependent toll-like receptor signaling pathways. Transcriptome sequencing analysis showed that, in patients with wAMD, Major Histocompatibility Complex (MHC) class I molecules bind to CD8⁺ T-cell receptor(TCR), and the high expression of linc00861 may induce T cell activation by regulating ZAP70, thereby mediating downstream TCR signaling and NF-κB signaling, leading to retinal immune inflammation. Age-related macular degeneration Peripheral blood mononuclear cell transcriptome amyloid β retinal pigment epithelium Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Age-related macular degeneration (AMD) is a common, irreversible blinding eye disease that has become the leading cause of central vision loss in individuals over 50 in Western countries [ 1 – 4 ] . With the accelerated aging of the global population, it is projected that by 2040, the number of AMD patients worldwide will reach 288 million [ 4 ] . Clinically, advanced AMD can be broadly categorized into dry AMD (non-exudative or atrophic AMD, dAMD) and wet AMD (exudative or neovascular AMD, wAMD), with dAMD accounting for 80% of all intermediate and advanced AMD [ 5 ] .The etiology of age-related macular degeneration (AMD) remains unclear; however, it is widely believed that immune-inflammatory responses, retinal pigment epithelium (RPE) cell senescence, and metabolic dysregulation are major factors contributing to the occurrence of AMD and play a critical role in its pathogenesis [ 6 ] . Although intravitreal injection of anti-VEGF drugs has revolutionized the treatment of wAMD, the short duration of the concentration of anti-VEGF drugs requires frequent intraocular injections, which impose significant social and economic burdens for ongoing treatment [ 7 ] . Additionally, around 10% of patients do not respond to anti-VEGF therapy, further underscoring the need for new therapeutic targets [ 8 ] . In contrast, gene therapy can offer a tailored long-term treatment solution. RNA sequencing (RNA-seq) analyzes the transcriptome to detect the status of differentially expressed genes in organisms, so as to gain deeper insights into the changes in their corresponding biological functions [ 9 ] . Currently, there have been reports on the whole transcriptome analysis of aqueous humor and the RPE-choroid complex, RPE cells, and rodent-induced AMD-related models using transcriptome sequencing methods. Increasing evidence indicates that lncRNAs play important roles in retinal inflammatory responses, cell apoptosis, immune cell activation, metabolic abnormalities, and neovascularization [ 10 – 14 ] . At present, transcriptomic studies on retinal-related diseases, especially AMD, mostly focus on animal or cell models, with few comparative studies involving human patient samples. Increasing evidence shows that plasma solutes and PBMCs appear to play an important role in the pathophysiology of AMD [ 15 – 18 ] , and are widely used in research on biomarkers associated with AMD [ 19 – 23 ] . In clinical practice, blood samples from patients are generally easier to obtain than aqueous humor or retinal tissue, and collecting these samples may have a lesser impact on the patients. Therefore, this study selected PBMCs from AMD patients and healthy individuals as research samples. Previous findings have identified that Aβ is the major components of drusen deposits in the subretinal space of AMD patients [ 24 ] , which can participate in the pathogenesis of AMD through multiple pathways [ 25 ] .In this study, less toxic Aβ1–40 was used to stimulate RPE cells in order to establish an inflammation model of RPE [ 26 – 27 ] . By conducting high-throughput sequencing of lncRNAs and mRNAs in PBMCs from AMD patients and healthy individuals, this study analyzes the differentially expressed genes and co-expression networks, focusing on changes in gene expression and investigating the logical structure and relationships of key biological processes and signaling pathways. This approach aims to deepen the understanding of the mechanisms underlying the occurrence and development of AMD and to identify key signaling pathways and target lncRNA. 2. Materials and methods 2. 1 Participants We recruited a total of 8 newly diagnosed, untreated AMD patients (5 with wAMD and 3 with dAMD), as well as 4 normal controls who were age-related cataract patients without any other fundus diseases, from our Hospital for blood sample collection and high-throughput sequencing. Additionally, we collected blood samples from an additional cohort of 12 newly diagnosed, untreated wAMD patients and 12 normal controls (same criteria as above) in order to validate our findings.The basic informations of the participants is presented in Table 1 . All volunteers underwent thorough screening to exclude any infectious diseases. The diagnosis of AMD was confirmed by ophthalmologists at our hospital through comprehensive ocular examinations, ruling out the presence of any other fundus disorders. Table 1 Basic informations of participants Participants n Age/year [Median (range)] Female [n(%)] Male [n(%)] Normal controls 16 64.8 (51–81) 7(43.8) 9(56.2) wAMD 17 72.9(54–86) 6(35.3) 11(64.7) dAMD 3 67.6(64–71) 1(33.3) 2(66.7) 2. 2 Sample collection and preparation PBMCs were isolated from participants using Lymphocyte separation medium according to its commercial protocol and the total RNA of the PBMC samples were extracted using TRIzol regent(invitrogen) following standard procedures as previously described. 2. 3 RNA-seq and data analysis RNA-seq was performed using Illumina Hiseq 4000 platform by Novegene(Beijing, china). Total RNAs were extracted from PBMCs and treated with Epicentre Ribo-zeroTM rRNA Removal kit(Epicentre, USA) to removed ribosomal RNA. A total amount of 3µg RNA per sample was used as input material for the RNA sample preparations. Sequcing libraries were generated using the rRNA-depleted RNA by NEBNext® Ultra™ Directional RNA Library prep kit for Illumina®(New England Balboa, USA) following the manuafacture’s recommendations. Random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH-) were used to synthesized first strand cDNA. Double-stranded cDNA were synthesized using RNase H and DNA polymerase I. In order to select cDNA fragments of preferentially 150 ~ 200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). The library quality was assessed on the Agilent analyzer 2100 system. Cluster generation system was used to cluster the index-coded samples. FPKMs of both lncRNAs and coding genes in each sample were caculated by cuffdiff(v2.1.1). Total of gene FPKMs were computed by summing the FPKMs of transcripts in every grouping. DESeq R software were used to determined the differential expression gene of AMD group and control group. DESeq provides standard statistical routines for determining differential expression data using a model based on the negative binomial distribution. P-value less than 0.05 and an absolute value of log2-fold change (FC) <2 were used as the threshold to assigned significance of differentially expressed genes. 2. 4 GO and KEGG enrichment analysis Gene Ontology (GO) enrichment analysis of differentially expressed genes or lncRNA target genes were implemented by the GOseq R package, in which gene length bias was corrected. GO terms with corrected Pvalue less than 0.05 were considered significantly enriched by differential expressed genes.We used KOBAS software to test the statistical enrichment of differential expression genes or lncRNA target genes in KEGG pathways. 2. 5 Real-time PCR validation To identify the reliability of sequencing, the selected target genes was verified by RT-PCR. As mentioned before, venous peripheral blood from 12 wAMD patients and 12 normal controls was collected to isolate PBMCs, and total RNAs were also isolated and extracted using the Trizol method. Aβ1–40 oligomers were prepared according to the method described in the literature [ 28 ] . Cell viability in the Aβ-induced RPE inflammation model is assessed using the CCK-8 assay. RPE cells were stimulated with Aβ1–40 at concentrations of 0, 0.01µM, 0.1µM, 1µM and 10.0µM for 24 hours and 48 hours respectively. Use a microplate reader to measure the optical density (O.D. value) at a wavelength of 450 nm to assess cell proliferation activity. 1 µM Aβ1–40 was used to stimulate APRE-19 cells for 24 hours to establish an RPE inflammation model. ARPE-19 cells were divided into an unstimulated group and a 1 µM concentration Aβ1–40 stimulated group. Subsequently, RNA was isolated and extracted using the Trizol method. The above RNA samples were reverse transcribed into cDNA using a reverse transcription kit and stored at -20°C for later use. During detection, cDNA was amplified and fluorescence intensity was measured on an ABI 7500 system according to the instructions of the quantitative fluorescence kit. Each sample is set up with a duplicate well, and the average Ct value of the two is calculated as the final Ct value. The data were normalized using the endogenous control β-actin. The relative quantification of target transcripts was achieved with the comparative 2-ΔΔct cycle threshold method. The detailed primer sequences is available as Table 2 . Table 2 Sequences of the primers for Real-Time PCR Gene Accession number Sequences β-actin NM_001101.3 Forward 5'-GGATGCAGAAGGAGATCACTG-3' Reverse 5'-TGTTCTGGGTATAGTTGCGAAGT-3' IL-2 NM_000586.4 Forward5’CCATTCAAAATCATCTGTAAATCCAGC-3’ Reverse 5’-GTAACCTCAACTCCTGCCACAAT-3’ IL-6 NM_000600.5 Forward 5’-TGAAAATCATCACTGGTCTTTTGGAG-3’ Reverse 5’-CTGGCTTGTTCCTCACTACTCT-3’ TNF-α NM_000594.4 Forward 5’-CTCCTCTCTGCCATCAAGAGC-3’ Reverse 5’-AGTAGACCTGCCCAGACTCG-3’ IFNG(IFN-γ) NM_000619.3 Forward 5’-TGAATGTCCAACGCAAAGCAA-3’ Reverse 5’-TTCGCTTCCCTGTTTTAGCTG-3’ linc00861 NC_000008.11 Forward 5’- TGACCTGGGACTACTTTACTCG-3’ Reverse 5’- TGTCAAGGACAGTTTAATCGC-3’ 2. 6 Statistical methods All data are presented as mean ± standard error of the mean (SEM). The results were analyzed using one-way analysis of variance (ANOVA) with GraphPad Prism 8.2.0, and a P-value of < 0.05 is considered statistically significant. 3. Results 3. 1 Differentially expressed genes (DEGs) analysis The results showed that, compared with normal controls, dAMD patients had 146 up-regulated (Figure 1.1A) and 384 down-regulated (Figure 1.1A) differentially expressed mRNAs; 1 up-regulated (Figure 1.2A) and 19 down-regulated (Figure 1.2A) differentially expressed lncRNAs. In contrast, in the comparison between wAMD patients and normal controls, 663 mRNAs were up-regulated (Figure 1.1B) and 628 mRNAs were down-regulated (Figure 1.1B); 41 lncRNAs were up-regulated (Figure 1.2B) and 33 lncRNAs were down-regulated (Figure 1.2B). When comparing wAMD patients with dAMD patients, 301 mRNAs were up-regulated (Figure 1.1C) and 149 mRNAs were down-regulated (Figure 1.1C); 16 lncRNAs were up-regulated (Figure 1.2C) and 3 lncRNAs were down-regulated (Figure 1.2C). 3. 2 dAMD patients compared to normal controls 3. 2. 1 GO enrichment analysis of DEGs in dAMD patients compared to normal controls Compared to normal controls, 47 significantly up-regulated and 165 significantly down-regulated GO terms were identified in the mRNAs of the dAMD patient group (Figure 2.1 A-B). Analysis revealed that the up-regulated biological processes (BP) were mostly related to macromolecule metabolism process and interspecies interaction between organisms, while the down-regulated biological processes were mainly involved in mRNA metabolism process and gene expression. The up-regulated cellular components (CC) were mostly localized to the nucleus, while the down-regulated ones were predominantly found in the nuclear lumen. The significantly enriched molecular functions (MF) were primarily associated with protein binding in the up-regulated terms, while the down-regulated terms were mainly related to RNA binding.In the GO enrichment analysis of the co-expressed target mRNAs of differentially expressed lncRNAs, compared to normal controls, the dAMD patient group only showed 36 significantly down-regulated GO terms (Figure 2.1 C). Analysis revealed that the significantly enriched biological processes (BP) were mainly related to intracellular transport and RNA transcription, while the cellular components (CC) were predominantly localized to intracellular organelle part. The horizontal axis is the enriched significant differences in GO Terms and the vertical axis represents the number of significant genes in the term. Red marks biological process, Blue indicates cellular component, and Green represents molecular function. The corrected p <0.05 was an absolute threshold used to select significantly enriched GO terms (adjusted p <0.05). 3. 2. 2 KEGG enrichment analysis of DEGs in dAMD patients compared to normal controls Compared to normal controls, only one significantly down-regulated KEGG pathway related to the ribosome was enriched in the differentially expressed mRNAs of dAMD patients; no significantly up-regulated pathways were identified (Figure 2.2). Additionally, no significant KEGG pathways were enriched in the co-expressed target genes of differentially expressed lncRNAs between dAMD patients and normal controls. The horizontal axis and the vertical axis indicate the log (corrected p-value) and the function descriptions of the enriched pathways, respectively. The corrected p <0.05 was an absolute threshold used to select significantly enriched pathways (adjusted p-value <0.05). 3. 3 wAMD patients compared to normal controls 3. 3. 1 GO enrichment analysis of DEGs in wAMD patients compared to normal controls Compared to normal controls, the wAMD patient group showed 316 significantly up-regulated and 69 significantly down-regulated GO terms in the differentially expressed mRNAs (Figure 3.1 A-B). Analysis revealed that the significantly up-regulated biological processes (BP) were closely related to the immune system, such as activation of lymphocytes (T cells), while the down-regulated processes were primarily involved in the organic substance catabolic process. The significantly enriched cellular components (CC) were predominantly localized to the nucleus and T cell receptors in the up-regulated terms, while the down-regulated terms were more associated with lysosomes. The significantly enriched molecular functions (MF) in the up-regulated terms were mostly related to MHC class I proteins, kinase, and cyclic compound binding, while the down-regulated terms were mostly associated with phosphate transmembrane transporter activity. In the analysis of the co-expressed target mRNAs of differentially expressed lncRNAs in the wAMD patient group, 36 significantly up-regulated and 40 significantly downregulated GO terms were identified (Figure3.1 C-D). Analysis showed that the significantly enriched biological processes (BP) in the up-regulated terms were mainly related to T cell activation, while the down-regulated processes were mostly associated with response to stress and metabolic processes. The cellular components (CC) in the up-regulated terms were mostly localized to T cell receptor complexes and the nucleoplasm, while the down-regulated terms were more associated with membrane-bound organelles. 3. 3. 2 KEGG enrichment analysis of DEGs in wAMD patients compared to normal controls Compared to normal controls, 5 KEGG pathways are significantly up-regulated in wAMD patients, including: T cell receptor signaling pathway, natural killer cell-mediated cytotoxicity, spliceosome, Parkinson’s disease, and primary immunodeficiency. Additionally, one significantly down-regulated KEGG pathway is associated with lysosomes (Figure 3.2 A-B).In the co-expressed target genes of differentially expressed lncRNAs in wAMD patients compared to normal controls, 2 significantly up-regulated KEGG pathways were identified, including the T cell receptor signaling pathway and NF-kappa B signaling pathway. Two significantly down-regulated pathways were related to lysosomes and other glycan degradation (Figure 3.2 C-D). 3. 4 wAMD patients compared to dAMD patients 3. 4. 1 GO enrichment analysis of DEGs in wAMD patients compared to dAMD patients In the control of wAMD and dAMD patients, 55 significantly up-regulated and 34 significantly down-regulated GO Terms were enriched in the differentially expressed mRNAs in the wAMD patient group (Figure 4.1 A-B). Analysis revealed that the significantly up-regulated biological processes (BP) were closely related to the regulation of immune response and MyD88-dependent Toll-like receptor signaling pathway, while the down-regulated processes were primarily enriched in multi-organism process. The significantly enriched cellular components (CC) in the up-regulated terms were mostly localized to intracellular membrane-bounded organelle, while the down-regulated terms were mainly associated with the cytoplasm, nucleus, and adhesion junctions. The significantly enriched differential molecular functions (MF) were mostly related to protein binding. No significant differences in GO terms were identified in the co-expressed target mRNAs of differentially expressed lncRNAs between wAMD and dAMD patients. 3. 4. 2 No significantly different KEGG pathways were enriched in the differential mRNAs and lncRNAs co-expressed target genes between wAMD patients and dAMD patients. 3. 5 RT-PCR validation of the differential expression gene We found that the differentially expressed mRNAs and the co-expressed target gene mRNAs of lncRNAs in wAMD patients compared to normal controls both significantly enriched in the T cell receptor signaling pathway and the NF-κB signaling pathway. Among all lncRNAs with significantly altered expression levels, the target mRNAs co-expressed with linc00861 included 6 related to the T cell receptor signaling pathway and 3 related to the NF-kappa B signaling pathway. lncRNA-mRNA co-expression prediction revealed that linc00861 co-expressed with ZAP70, which was enriched in both the T cell receptor signaling pathway and the NF-kappa B signaling pathway. Therefore, we selected linc00861 as the gene for further validation. 3. 5. 1 RT-PCR validation of linc00861 in PBMCs In the comparison of PBMCs between wAMD patients and normal controls，the expression of linc00861 was up-regulated in PBMCs from wAMD patients.(Figure 5.1). 3. 5. 2 RT-PCR validation of linc00861 in the RPE inflammation model ARPE-19 cells are treated with Aβ1-40 at concentrations of 0 μM, 0.01 μM, 0.1 μM, and 1 μM for either 24 hours or 48 hours. These treatments do not affect the proliferative capacity of the cells. However, compared to the control group (0 μM Aβ1-40), treatment with 10 μM Aβ1-40 for 24 hours does not significantly impair cell viability; yet, after 48 hours of stimulation, it significantly inhibits cell viability (Figure 5.2.1). To avoid cytotoxic effects influencing the proliferative capacity, we choose to treat ARPE-19 cells with 1 μM Aβ1-40 for 24 hours as the established condition for subsequent experiments. 3. 5. 3 Effects of Aβ1-40 stimulation on expression of linc00861 and inflammatory cytokines in ARPE-19 cells To investigate the effects of Aβ1-40 on cell apoptosis and inflammation, ARPE-19 cells were stimulated with 1 μM Aβ1-40 for 24 hours. The mRNA expression levels of IL-2, IL-6, TNF-α, and IFNG (IFN-γ), as well as the expression of linc00861, were assessed by RT- PCR. As shown in Figure 5.2.2, after stimulating ARPE-19 cells with 1 μM Aβ1-40, the expression levels of inflammatory cytokines in the cells increased, and the expression level of linc00861 also increased. 4. Discussion AMD is a common irreversible blinding eye disease in individuals over 50 years of age, and it is clinically classified into dAMD and wAMD [ 1 – 5 ] . Currently, the treatment for wAMD mainly involves intravitreal injection of anti-VEGF drugs. However, these drugs have a short duration of action, requiring frequent injections, and there is still no effective treatment for dAMD patients to date [ 7 – 8 ] . Therefore, there is a need to continue searching for new therapeutic targets for AMD. In this study, high-throughput transcriptomic sequencing of PBMCs from AMD patients and healthy individuals was performed to screen for differentially expressed genes and related biological functions and signaling pathways. Compared with normal individuals, the significantly changed co-expression analysis of differentially expressed mRNAs and lncRNAs in the dAMD patient group were mostly down-regulated GO Terms and KEGG pathways, which are mainly involved in biological processes such as RNA transcription and metabolism, as well as ribosomes associated pathway. Therefore, we speculate that most of the genes involved in ribosomes associated pathway in dAMD patients are in a state of inhibition or disorder, which also inhibits the transcription and metabolism of mRNA. The study argued that in the initial stage of AMD, the synthesis of ribosomal proteins is abnormally altered, and the protein translation initiation factors eIF2α, eIF3η, eIF5 and elongation factor eEF2 are anomalously regulated, leading to AMD ribosomal dysfunction [ 25 ] . Our transcriptomic analysis results suggest that consistent with previous reports, ribosomal dysfunction affects RNA transcription and molecular metabolism in dAMD patients. In the comparison between wAMD patients and normal controls, the significantly up-regulated GO terms of differentially expressed mRNAs and lncRNAs co-expressed target genes, were mainly involved in the activation of T lymphocytes. These genes were primarily located on T cell receptors and were mostly associated with molecular functions related to MHC class I proteins. The significantly up-regulated pathways were primarily related to T cell receptor signaling.wAMD patients are primarily in a state of immune system overactivation. Based on the sequencing results and bioinformatics analysis of this study, we hypothesize that MHC class I molecules, by binding with CD8⁺ TCR, activate cellular immunity and cytokine secretion to regulate other immune cells, which may contribute to the pathological changes in wAMD.As many reports have indicated, the pathogenesis of AMD involves systemic and local immune inflammation. For example, macrophages and CD8⁺ T cells are present in the choroid of AMD eyes [ 29 ] . Faber et al. also reported that in different subtypes of AMD patients, the proportion of aged, differentiated memory CD8⁺ T cells was higher [ 30 ] . CD8⁺ T cells bind to MHC class I molecules and recognize specific antigen targets. Upon activation, CD8⁺ T cells produce IL-2 and IFN-γ, which regulate the effector functions of other immune cells such as macrophages and NK cells [ 31 ] . In conclusion, consistent with the enrichment analysis of this study, CD8 + T cells may be involved in the pathbiology of AMD. It is worth noting that in a transcriptomic study based on microarray chip analysis of PBMCs from patients with different clinical subtypes of advanced AMD [ 32 ] , the enrichment analysis also indicated that immune response and lymphocyte activation were involved. In the study by Grunin et al., when analyzing the transcriptome of peripheral mononuclear cells from wAMD patients, the most significant DAVID enrichment was related to leukocyte/lymphocyte activation and immune response [ 33 ] . Multiple clinical and experimental models have demonstrated the involvement of both innate and adaptive immunity in wAMD [ 34 – 45 ] . Howeverh,in the comparison between wAMD patients and healthy controls, the significantly down-regulated biological processes were equally related to metabolism, primarily occurring in the lysosome. This suggests that metabolic dysfunction also occurs in wAMD patients, but different from dAMD patients, it is not a suppression of processes at the RNA level. Instead, it primarily manifests as the inhibition of lysosome-related functions. Literature reports indicate that RPE cell degeneration, a process related to the accumulation of lysosomal lipofuscin derived from oxidative stress, impairs lysosomal degradation [ 46 – 49 ] . Previous studies have also shown that the interaction between phagocytosis and autophagy in the RPE is essential for the degradation of photoreceptor outer segments and the maintenance of retinoid levels to support vision. Disruption of genes related to lysosomal function can lead to lysosomal dysfunction and negatively impact the homeostasis regulation of phagocytosis and autophagy [ 50 – 51 ] . Autophagy is a lysosomal mechanism for the degradation of cytoplasmic proteins and damaged organelles. Through autophagy, protein aggregates are transported to the lysosome. With aging and the senescence of RPE cells, lysosomal enzymatic activity decreases. Autophagy is inhibited to eliminate mitochondria and protein aggregates from the intercellular space, thus accelerating lipofuscin accumulation and promoting the progression of AMD [ 52 ] . In comparison to dAMD patients, the significantly upregulated biological processes enriched in the differential genes of wAMD patients are closely related to the regulation of the immune system and the MyD88-dependent Toll-like receptor signaling pathway. These sequencing results suggest that wAMD patients have more immune system-related gene changes than dAMD patients, along with higher levels of inflammation. This indicates that the formation of neovascularization in wAMD is more closely associated with immune inflammatory responses [ 53 – 54 ] . As previously mentioned, we identified the key gene linc00861, which showed increased expression in both PBMCs from wAMD patients and in an inflammation model from Aβ-stimulated RPE cells. lncRNA-mRNA co-expression prediction revealed that linc00861 co-expressed with ZAP70, and both were enriched in the T cell receptor signaling pathway and NF-kappa B signaling pathway. This suggests that the abnormal activation of these two pathways is closely associated with the pathogenesis of AMD, with ZAP70 acting as a key upstream factor in these signaling pathways. Its abnormal activation may be a potential trigger for the development of AMD.ZAP70, a 70Kd protein tyrosine kinase associated with the ζ chain of T cell receptor, belongs to the syk tyrosine kinase family and plays a key role in inducing T cell activation after binding to T cell antigen receptor (TCR) [ 55 ] . The activation of ZAP70 is the first step in the activation of all other signaling pathways. Activated ZAP70 phosphorylates downstream effector molecules, such as the linker for activation of T cell(LAT), the SH2 domain-containing leukocyte protein 76 kDa (SLP-76), and mitogen-activated protein kinase (MAPK) P38, initiating the PLC-γ activation pathway, MAPK pathway, and NF-κB signaling pathway [ 56 ] .Currently, there is limited research on ZAP70 in ophthalmic diseases. Some studies have shown that in a spontaneous autoimmune uveitis mouse model (R161H), after retinal aldehyde-binding proteins are presented by dendritic cells, ZAP70 mediates the T cell receptor signaling pathway to activate intraocular CD4⁺ T lymphocytes, triggering an immune response in the eye and leading to ocular inflammation and histopathological lesions [ 57 ] . In this model, the expression of phosphorylated ZAP70 in intraocular lymphoid follicles was significantly increased, resulting in the increased secretion of downstream inflammatory cytokines such as IL-2, IL-4, and IL-5, which induced ocular inflammatory responses [ 58 ] .A recent study showed that the CD3ζ-Hck-Syk/Zap70 signaling pathway protects retinal ganglion cells in retinal excitotoxicity induced by the glutamate receptor agonist (NMDA) [ 59 ] . In recent years, with the deepening of research into the molecular structure and function of ZAP70, it has been confirmed that ZAP70 is a key point linking inflammation and immune signaling. ZAP70 not only participates in the transduction of T cell receptor signaling pathways but also plays a role in the regulation of NF-κB signaling pathways, NK cell cytotoxic responses, and the modulation of inflammation and immune responses. Existing reports on linc00861 are mostly related to cancer, including cervical cancer, prostate cancer, ovarian cancer, hepatocellular carcinoma, and breast cancer [ 60 – 65 ] . Notably, linc00861 found in other diseases is also largely involved in immune system-related changes, such as in eczema [ 66 ] , Parkinson's disease [ 67 ] , and sepsis [ 68 ] .These reports suggest that linc00861 plays an important role in immune system-related pathological changes in diseases. Based on this, we hypothesize that in wAMD patients, MHC class I molecules bind to CD8⁺ TCR, and the high expression of linc00861 induces T cell activation by regulating ZAP70, thereby mediating downstream T cell receptor signaling and NF-κB signaling, leading to retinal immune inflammatory responses. However, this research has certain limitations. Firstly, the sample size is relatively small, the dAMD group in this study includes early, intermediate, and late-stage geographic atrophy AMD which affects the macular fovea. The distinct pathological features at different stages of dAMD make it challenging to accurately identify differential genes, biological processes, and signaling pathway changes specific to each phase. This limitation hinders the precise identification of key nodes in the onset and progression of AMD and the effective prediction of disease progression, as well as the formulation of targeted intervention strategies. Future research should classify AMD into early, intermediate, and late stages and increase the sample size for transcriptomic analyses to enhance the accuracy of the results. At the same time, we will verify whether the key gene linc00861 screened in this study can activate T cells through ZAP70, regulate the transduction factors NF-Κb and ZAP70-P38-nFAT signaling pathway and participate in the regulation of retinal inflammation, so as to further clarify the specific mechanism of linc00861 participating in the intraocular immune inflammation. It provides a new target and evidence for the diagnosis and treatment of wAMD. 5. Conclusion Through high-throughput sequencing and co-expression network analysis of lncRNAs and mRNAs in PBMCs from AMD patients, we found that the development and progression of AMD are associated with metabolic disorders, dysfunction of the ribosome and lysosomes, and excessive activation of the immune system. Notably, in wAMD patients, the expression of linc00861 was up-regulated, and high expression of linc00861 may regulate ZAP70-mediated cell-mediated immunityof the T cell receptor signaling pathway, leading to retinal immune-inflammatory responses and contributing to the pathogenesis of wAMD. Declarations Author Contributions: Conceptualization, Wenxi XIE; Data curation, Wenxi XIE; Formal analysis, Wenxi XIE; Project administration, Hui PENG; Resources, Hui PENG; Supervision, Xing WANG and Hui PENG; Validation, Wenxi XIE; Writing – original draft, Wenxi XIE; Writing – review & editing, Xing WANG. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: The study was conducted in accordance with the Declaration 114 of Helsinki, and approved by the Institutional Review Board of the First Affiliated Hospital of Chongqing Medical University(2021 Research Ethics 2021-292). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: All data generated during this study are included in the manuscript. Acknowledgments: We would like to express our gratitude to the Department of Ophthalmology, the First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory for the Prevention and Treatment of Major Blinding Eye Diseases, Chongqing Eye Institute for their dedicated care and support throughout the course of this study. We also would like to thank AMD patients, cataract patientstheir families, and their physicians for their consent to be involved in this research. Conflicts of Interest: The authors declare no conflicts of interest. References W, Meng YF, Xing Q, et al. Identification of lncRNAs involved in biological regulation in early age-relate Friedman DS, O’Colmain BJ, Muñoz B, et al. Prevalence of age-related macular degeneration in the United States[J]. 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Identification of an lncRNA-miRNA-mRNA interaction mechanism in breast cancer based on bioinformatic analysis[J]. Mol Med Rep. 2017 Oct, 16(4):5113-5120. Grosche S, Marenholz I, Esparza-Gordillo J, et al. Rare variant analysis in eczema identifies exonic variants in DUSP1, NOTCH4 and SLC9A4[J]. Nat Commun. 2021 Nov 16, 12(1):6618. Lei K, Zhang L, He Y, et al. Immune-associated biomarkers for early diagnosis of Parkinson's disease based on hematological lncRNA-mRNA co-expression[J]. Biosci Rep. 2020 Nov 27, 40(12):BSR20202921. Cheng Y, Cao X, Zhang J, et al. Dysregulated lncRNAs are Involved in the Progress of Sepsis by Constructing Regulatory Networks in Whole Blood Cells[J]. Front Pharmacol. 2021 Aug 17, 12:6782 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6639890","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":467497436,"identity":"22b62f96-0616-4128-bcc5-014e28501d21","order_by":0,"name":"Wenxi XIE","email":"","orcid":"","institution":"The First Affiliated Hospital of Chongqing Medical University, Chongqing Eye Institute","correspondingAuthor":false,"prefix":"","firstName":"Wenxi","middleName":"","lastName":"XIE","suffix":""},{"id":467497437,"identity":"2f781b3e-20d8-442d-8757-541bca19e852","order_by":1,"name":"xing wang","email":"","orcid":"","institution":"The First Affiliated Hospital of Chongqing Medical University, Chongqing Eye Institute","correspondingAuthor":false,"prefix":"","firstName":"xing","middleName":"","lastName":"wang","suffix":""},{"id":467497438,"identity":"b042e286-0da0-4c54-a773-0e925059441a","order_by":2,"name":"Hui PENG","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAs0lEQVRIiWNgGAWjYJACZoYKGwY2ErWcSSNVC2PbYRKUm7P3Hn5d2Hbenk+6+QHDj4pthLVY9pxLs55x7nZim8wxA8aeM7cJazG4kWNmzFN2O4FNIsEA6EJitNx/A9TCds6eTSL9A5FabvAYP+ZpO8DYJpFDpC2WPTlmzDxnkhOBWgoOEuUXc/Yzxp95Kuzs5Wekb3zwo4IYhzEwsEnAOAcIq4doYf5AlMpRMApGwSgYuQAAkyM4Dh8TRHIAAAAASUVORK5CYII=","orcid":"","institution":"The First Affiliated Hospital of Chongqing Medical University, Chongqing Eye Institute","correspondingAuthor":true,"prefix":"","firstName":"Hui","middleName":"","lastName":"PENG","suffix":""}],"badges":[],"createdAt":"2025-05-11 13:38:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6639890/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6639890/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84365751,"identity":"17489059-7205-41ab-8d95-73839f69ed41","added_by":"auto","created_at":"2025-06-11 06:04:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":106345,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 1.1 Volcanic plot of differential expressed mRNAs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the volcano plots, the horizontal axis is log 2-fold change (FC), and the vertical axis represents the p-value after the negative logarithm conversion. The red dots indicate the up-regulated genes, whereas the green dots represent the down-regulated genes. The corrected p\u0026lt;0.05 was an absolute threshold used to select DEGs. (A): Differentially expressed mRNAs in dAMD patients and normal controls; (B) Differentially expressed mRNAs in wAMD patients and normal controls; (C): Differentially expressed mRNAs in wAMD patients and dAMD patients.\u003c/p\u003e","description":"","filename":"1.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6639890/v1/a5dda6ca4d5a4aeebd0b98d6.png"},{"id":84365750,"identity":"dda3d695-99ae-4a5a-89cd-6e2d607ab480","added_by":"auto","created_at":"2025-06-11 06:04:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":79888,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 1.2 Volcanic plot of differential expressed lncRNAs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe legend is illustrated in figure 1.1 volcano plots. (A): Differentially expressed lncRNAs in dAMD patients and normal controls; (B) Differentially expressed lncRNAs in wAMD patients and normal controls; (C): Differentially expressed lncRNAs in wAMD patients and dAMD patients.\u003c/p\u003e","description":"","filename":"1.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6639890/v1/c08a9369570632843ed61620.png"},{"id":84366843,"identity":"3a25d672-0b85-4ef3-9f99-99eaca4bbe30","added_by":"auto","created_at":"2025-06-11 06:12:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":263756,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 2.1 Gene ontology (GO) enrichment of differentially expressed genes in dAMD patients compared with normal controls.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A): The up-regulated GO enriched Terms of mRNAs;(B): The down-regulated GO enriched Terms of mRNAs; (C): The down-regulated GO enriched Terms of Co-expression of lncRNAs.\u003c/p\u003e","description":"","filename":"2.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6639890/v1/fa635657c14d24dd7dfca773.png"},{"id":84365753,"identity":"cbcb0400-5d57-4aec-98d8-b12a96d3428b","added_by":"auto","created_at":"2025-06-11 06:04:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":105894,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 2.2 KEGG enrichment of down-regulated mRNAs in dAMD patients compared with normal controls.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6639890/v1/f6e9d9e82133410b95d41b1b.png"},{"id":84367773,"identity":"af952d8e-3f73-4021-a90c-93a424c7ceb9","added_by":"auto","created_at":"2025-06-11 06:28:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":201818,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 3.1 Gene ontology (GO) enrichment of differentially expressed genes in wAMD patients compared with normal controls.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A): The up-regulated GO enriched Terms of mRNAs; (B): The down-regulated GO enriched Terms of mRNAs. (C): The up-regulated GO enriched Terms of Co-expression of lncRNAs;(D): The down-regulated GO enriched Terms of Co-expression of lncRNAs.\u003c/p\u003e\n\u003cp\u003eThe legend is illustrated in figure 2.1\u003c/p\u003e","description":"","filename":"3.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6639890/v1/b674f836f050de23459811ad.png"},{"id":84367192,"identity":"9a1961d9-e4ab-45b9-8312-3c0a23d47cee","added_by":"auto","created_at":"2025-06-11 06:20:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":187403,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 3.2 KEGG enrichment of differentially expressed genes in wAMD patients compared with normal controls.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A): Significant differences KEGG pathways of the up-regulated mRNA;(B): Significant differences KEGG pathways of the down-regulated mRNA. The legend is illustrated in figure 3.1. (C): The up-regulated KEGG pathways of Co-expression of lncRNAs;(D): The down-regulated KEGG pathways of Co-expression of lncRNAs.\u003c/p\u003e\n\u003cp\u003eThe legend is illustrated in figure 2.2.\u003c/p\u003e","description":"","filename":"3.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6639890/v1/35d1a7a6e86ba304b2084e82.png"},{"id":84365762,"identity":"86606599-d50e-4b51-9e62-7b520497a264","added_by":"auto","created_at":"2025-06-11 06:04:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":184825,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 4. 1 Gene ontology (GO) enrichment of differentially expressed mRNAs in wAMD patients compared with dAMD patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A): The up-regulated GO enriched Terms of mRNAs;(B): The down-regulated GO enriched Terms of mRNAs.\u003c/p\u003e\n\u003cp\u003eThe legend is illustrated in figure 2.1.\u003c/p\u003e","description":"","filename":"4.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6639890/v1/92b3db215524187853fc9f72.png"},{"id":84366845,"identity":"9fedfff3-9207-44fa-a19a-41dfe5b16925","added_by":"auto","created_at":"2025-06-11 06:12:46","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":14160,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 5.1 Validation of the expression of linc00861 in PBMCs with RT-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expressions of linc00861 in PBMCs of wAMD patients was significantly higher than that of normal controls (CN). The endogenous reference gene was β-actin (p \u0026lt;0.05, n =6).\u003c/p\u003e","description":"","filename":"5.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6639890/v1/884eff2f0bf119fd1f7edea3.png"},{"id":84365777,"identity":"ed79e0a9-3372-49e1-ab5d-0fe55be01208","added_by":"auto","created_at":"2025-06-11 06:04:47","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":110532,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 5.2.1: CCK-8 detects the effects of Aβ1-40 at concentrations of 0,0.01μmol / L,0.1μmol / L,1μmol / L, and 10μmol / L on the cell viability of ARPE-19 after 24 and 48 hours of stimulation.(P\u0026lt;0.0001 vs unstimulated group, n=6)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.2.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6639890/v1/a727a40c44722da90862fd3b.png"},{"id":84365764,"identity":"52c9914d-306c-4b42-b807-88948e815212","added_by":"auto","created_at":"2025-06-11 06:04:47","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":246802,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 5.2.2 RT-PCR detected the expression of inflammatory cytokines such as IL-2, IL-6, TNF, IFNG and linc00861 after ARPE-19 cells stimulated by Aβ1-40 for 24h（P\u0026lt;0.05,n=3）\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.2.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6639890/v1/af6dfeb70cdbcf6712414009.png"},{"id":84368098,"identity":"56e3dd46-56fd-44e6-8080-e10031975ccb","added_by":"auto","created_at":"2025-06-11 06:36:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2598058,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6639890/v1/50c24c75-ac3c-4d41-826b-054ba3d6c4db.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"High-throughput sequencing and Co-expression network analysis of LncRNAs and mRNAs of PBMCs in AMD patients","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAge-related macular degeneration (AMD) is a common, irreversible blinding eye disease that has become the leading cause of central vision loss in individuals over 50 in Western countries \u003csup\u003e[\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. With the accelerated aging of the global population, it is projected that by 2040, the number of AMD patients worldwide will reach 288\u0026nbsp;million \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Clinically, advanced AMD can be broadly categorized into dry AMD (non-exudative or atrophic AMD, dAMD) and wet AMD (exudative or neovascular AMD, wAMD), with dAMD accounting for 80% of all intermediate and advanced AMD\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.The etiology of age-related macular degeneration (AMD) remains unclear; however, it is widely believed that immune-inflammatory responses, retinal pigment epithelium (RPE) cell senescence, and metabolic dysregulation are major factors contributing to the occurrence of AMD and play a critical role in its pathogenesis\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Although intravitreal injection of anti-VEGF drugs has revolutionized the treatment of wAMD, the short duration of the concentration of anti-VEGF drugs requires frequent intraocular injections, which impose significant social and economic burdens for ongoing treatment\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Additionally, around 10% of patients do not respond to anti-VEGF therapy, further underscoring the need for new therapeutic targets \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. In contrast, gene therapy can offer a tailored long-term treatment solution.\u003c/p\u003e \u003cp\u003eRNA sequencing (RNA-seq) analyzes the transcriptome to detect the status of differentially expressed genes in organisms, so as to gain deeper insights into the changes in their corresponding biological functions\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Currently, there have been reports on the whole transcriptome analysis of aqueous humor and the RPE-choroid complex, RPE cells, and rodent-induced AMD-related models using transcriptome sequencing methods. Increasing evidence indicates that lncRNAs play important roles in retinal inflammatory responses, cell apoptosis, immune cell activation, metabolic abnormalities, and neovascularization\u003csup\u003e[\u003cspan additionalcitationids=\"CR11 CR12 CR13\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. At present, transcriptomic studies on retinal-related diseases, especially AMD, mostly focus on animal or cell models, with few comparative studies involving human patient samples. Increasing evidence shows that plasma solutes and PBMCs appear to play an important role in the pathophysiology of AMD \u003csup\u003e[\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, and are widely used in research on biomarkers associated with AMD \u003csup\u003e[\u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. In clinical practice, blood samples from patients are generally easier to obtain than aqueous humor or retinal tissue, and collecting these samples may have a lesser impact on the patients. Therefore, this study selected PBMCs from AMD patients and healthy individuals as research samples.\u003c/p\u003e \u003cp\u003ePrevious findings have identified that Aβ is the major components of drusen deposits in the subretinal space of AMD patients\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, which can participate in the pathogenesis of AMD through multiple pathways \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e.In this study, less toxic Aβ1\u0026ndash;40 was used to stimulate RPE cells in order to establish an inflammation model of RPE \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBy conducting high-throughput sequencing of lncRNAs and mRNAs in PBMCs from AMD patients and healthy individuals, this study analyzes the differentially expressed genes and co-expression networks, focusing on changes in gene expression and investigating the logical structure and relationships of key biological processes and signaling pathways. This approach aims to deepen the understanding of the mechanisms underlying the occurrence and development of AMD and to identify key signaling pathways and target lncRNA.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\n\u003ch3\u003e2. 1 Participants\u003c/h3\u003e\n\u003cp\u003eWe recruited a total of 8 newly diagnosed, untreated AMD patients (5 with wAMD and 3 with dAMD), as well as 4 normal controls who were age-related cataract patients without any other fundus diseases, from our Hospital for blood sample collection and high-throughput sequencing. Additionally, we collected blood samples from an additional cohort of 12 newly diagnosed, untreated wAMD patients and 12 normal controls (same criteria as above) in order to validate our findings.The basic informations of the participants is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. All volunteers underwent thorough screening to exclude any infectious diseases. The diagnosis of AMD was confirmed by ophthalmologists at our hospital through comprehensive ocular examinations, ruling out the presence of any other fundus disorders.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBasic informations of participants\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParticipants\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAge/year\u003c/p\u003e \u003cp\u003e[Median (range)]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003cp\u003e[n(%)]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003cp\u003e[n(%)]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNormal controls\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64.8 (51\u0026ndash;81)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7(43.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9(56.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ewAMD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e72.9(54\u0026ndash;86)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6(35.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11(64.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003edAMD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e67.6(64\u0026ndash;71)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1(33.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2(66.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003e2. 2 Sample collection and preparation\u003c/h3\u003e\n\u003cp\u003ePBMCs were isolated from participants using Lymphocyte separation medium according to its commercial protocol and the total RNA of the PBMC samples were extracted using TRIzol regent(invitrogen) following standard procedures as previously described.\u003c/p\u003e\n\u003ch3\u003e2. 3 RNA-seq and data analysis\u003c/h3\u003e\n\u003cp\u003eRNA-seq was performed using Illumina Hiseq 4000 platform by Novegene(Beijing, china). Total RNAs were extracted from PBMCs and treated with Epicentre Ribo-zeroTM rRNA Removal kit(Epicentre, USA) to removed ribosomal RNA. A total amount of 3\u0026micro;g RNA per sample was used as input material for the RNA sample preparations. Sequcing libraries were generated using the rRNA-depleted RNA by NEBNext\u0026reg; Ultra\u0026trade; Directional RNA Library prep kit for Illumina\u0026reg;(New England Balboa, USA) following the manuafacture\u0026rsquo;s recommendations. Random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH-) were used to synthesized first strand cDNA. Double-stranded cDNA were synthesized using RNase H and DNA polymerase I. In order to select cDNA fragments of preferentially 150\u0026thinsp;~\u0026thinsp;200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). The library quality was assessed on the Agilent analyzer 2100 system. Cluster generation system was used to cluster the index-coded samples. FPKMs of both lncRNAs and coding genes in each sample were caculated by cuffdiff(v2.1.1). Total of gene FPKMs were computed by summing the FPKMs of transcripts in every grouping. DESeq R software were used to determined the differential expression gene of AMD group and control group. DESeq provides standard statistical routines for determining differential expression data using a model based on the negative binomial distribution. P-value less than 0.05 and an absolute value of log2-fold change (FC) \u0026lt;2 were used as the threshold to assigned significance of differentially expressed genes.\u003c/p\u003e\n\u003ch3\u003e2. 4 GO and KEGG enrichment analysis\u003c/h3\u003e\n\u003cp\u003eGene Ontology (GO) enrichment analysis of differentially expressed genes or lncRNA target genes were implemented by the GOseq R package, in which gene length bias was corrected. GO terms with corrected Pvalue less than 0.05 were considered significantly enriched by differential expressed genes.We used KOBAS software to test the statistical enrichment of differential expression genes or lncRNA target genes in KEGG pathways.\u003c/p\u003e\n\u003ch3\u003e2. 5 Real-time PCR validation\u003c/h3\u003e\n\u003cp\u003eTo identify the reliability of sequencing, the selected target genes was verified by RT-PCR. As mentioned before, venous peripheral blood from 12 wAMD patients and 12 normal controls was collected to isolate PBMCs, and total RNAs were also isolated and extracted using the Trizol method.\u003c/p\u003e \u003cp\u003eAβ1\u0026ndash;40 oligomers were prepared according to the method described in the literature \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Cell viability in the Aβ-induced RPE inflammation model is assessed using the CCK-8 assay. RPE cells were stimulated with Aβ1\u0026ndash;40 at concentrations of 0, 0.01\u0026micro;M, 0.1\u0026micro;M, 1\u0026micro;M and 10.0\u0026micro;M for 24 hours and 48 hours respectively. Use a microplate reader to measure the optical density (O.D. value) at a wavelength of 450 nm to assess cell proliferation activity. 1 \u0026micro;M Aβ1\u0026ndash;40 was used to stimulate APRE-19 cells for 24 hours to establish an RPE inflammation model. ARPE-19 cells were divided into an unstimulated group and a 1 \u0026micro;M concentration Aβ1\u0026ndash;40 stimulated group. Subsequently, RNA was isolated and extracted using the Trizol method.\u003c/p\u003e \u003cp\u003eThe above RNA samples were reverse transcribed into cDNA using a reverse transcription kit and stored at -20\u0026deg;C for later use. During detection, cDNA was amplified and fluorescence intensity was measured on an ABI 7500 system according to the instructions of the quantitative fluorescence kit. Each sample is set up with a duplicate well, and the average Ct value of the two is calculated as the final Ct value. The data were normalized using the endogenous control β-actin. The relative quantification of target transcripts was achieved with the comparative 2-ΔΔct cycle threshold method. The detailed primer sequences is available as Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSequences of the primers for Real-Time PCR\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAccession number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSequences\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eβ-actin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNM_001101.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward 5'-GGATGCAGAAGGAGATCACTG-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse 5'-TGTTCTGGGTATAGTTGCGAAGT-3'\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIL-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNM_000586.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward5\u0026rsquo;CCATTCAAAATCATCTGTAAATCCAGC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse 5\u0026rsquo;-GTAACCTCAACTCCTGCCACAAT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIL-6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNM_000600.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward 5\u0026rsquo;-TGAAAATCATCACTGGTCTTTTGGAG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse 5\u0026rsquo;-CTGGCTTGTTCCTCACTACTCT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTNF-α\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNM_000594.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward 5\u0026rsquo;-CTCCTCTCTGCCATCAAGAGC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse 5\u0026rsquo;-AGTAGACCTGCCCAGACTCG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIFNG(IFN-γ)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNM_000619.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward 5\u0026rsquo;-TGAATGTCCAACGCAAAGCAA-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse 5\u0026rsquo;-TTCGCTTCCCTGTTTTAGCTG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003elinc00861\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNC_000008.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward 5\u0026rsquo;- TGACCTGGGACTACTTTACTCG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse 5\u0026rsquo;- TGTCAAGGACAGTTTAATCGC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003e2. 6 Statistical methods\u003c/h3\u003e\n\u003cp\u003eAll data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). The results were analyzed using one-way analysis of variance (ANOVA) with GraphPad Prism 8.2.0, and a P-value of \u0026lt;\u0026thinsp;0.05 is considered statistically significant.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3. 1\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eDifferentially expressed genes (DEGs) analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results showed that, compared with normal controls, dAMD patients had 146 up-regulated (Figure 1.1A) and 384 down-regulated (Figure 1.1A) differentially expressed mRNAs; 1 up-regulated (Figure 1.2A) and 19 down-regulated (Figure 1.2A) differentially expressed lncRNAs. In contrast, in the comparison between wAMD patients and normal controls, 663 mRNAs were up-regulated (Figure 1.1B) and 628 mRNAs were down-regulated (Figure 1.1B); 41 lncRNAs were up-regulated (Figure 1.2B) and 33 lncRNAs were down-regulated (Figure 1.2B). When comparing wAMD patients with dAMD patients, 301 mRNAs were up-regulated (Figure 1.1C) and 149 mRNAs were down-regulated (Figure 1.1C); 16 lncRNAs were up-regulated (Figure 1.2C) and 3 lncRNAs were down-regulated (Figure 1.2C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. 2\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003edAMD patients compared to normal controls\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. 2. 1\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eGO enrichment analysis of DEGs in dAMD patients compared to normal controls\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompared to normal controls, 47 significantly up-regulated and 165 significantly down-regulated GO terms were identified in the mRNAs of the dAMD patient group (Figure 2.1 A-B). Analysis revealed that the up-regulated biological processes (BP) were mostly related to macromolecule metabolism process and interspecies interaction between organisms, while the down-regulated biological processes were mainly involved in mRNA metabolism process and gene expression. The up-regulated cellular components (CC) were mostly localized to the nucleus, while the down-regulated ones were predominantly found in the nuclear lumen. The significantly enriched molecular functions (MF) were primarily associated with protein binding in the up-regulated terms, while the down-regulated terms were mainly related to RNA binding.In the GO enrichment analysis of the co-expressed target mRNAs of differentially expressed lncRNAs, compared to normal controls, the dAMD patient group only showed 36 significantly down-regulated GO terms (Figure 2.1 C). Analysis revealed that the significantly enriched biological processes (BP) were mainly related to intracellular transport and RNA transcription, while the cellular components (CC) were predominantly localized to intracellular organelle part.\u003c/p\u003e\n\u003cp\u003eThe horizontal axis is the enriched significant differences in GO Terms and the vertical axis represents the number of significant genes in the term.\u0026nbsp;Red marks biological process, Blue indicates cellular component, and Green represents molecular function. The corrected p \u0026lt;0.05 was an absolute threshold used to select significantly enriched GO terms (adjusted p \u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. 2. 2\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eKEGG enrichment analysis of DEGs in dAMD patients compared to normal controls\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompared to normal controls, only one significantly down-regulated KEGG pathway related to the ribosome was enriched in the differentially expressed mRNAs of dAMD patients; no significantly up-regulated pathways were identified (Figure 2.2). Additionally, no significant KEGG pathways were enriched in the co-expressed target genes of differentially expressed lncRNAs between dAMD patients and normal controls.\u003c/p\u003e\n\u003cp\u003eThe horizontal axis and the vertical axis indicate the log (corrected p-value) and the function descriptions of the enriched pathways, respectively. The corrected p \u0026lt;0.05 was an absolute threshold used to select significantly enriched pathways (adjusted p-value \u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. 3\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ewAMD patients compared to normal controls \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. 3. 1\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eGO enrichment analysis of DEGs in wAMD patients compared to normal controls\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompared to normal controls, the wAMD patient group showed 316 significantly up-regulated and 69 significantly down-regulated GO terms in the differentially expressed mRNAs (Figure 3.1 A-B). Analysis revealed that the significantly up-regulated biological processes (BP) were closely related to the immune system, such as activation of lymphocytes (T cells), while the down-regulated processes were primarily involved in the organic substance catabolic process. The significantly enriched cellular components (CC) were predominantly localized to the nucleus and T cell receptors in the up-regulated terms, while the down-regulated terms were more associated with lysosomes. The significantly enriched molecular functions (MF) in the up-regulated terms were mostly related to MHC class I proteins, kinase, and cyclic compound binding, while the down-regulated terms were mostly associated with phosphate transmembrane transporter activity. In the analysis of the co-expressed target mRNAs of differentially expressed lncRNAs in the wAMD patient group, 36 significantly up-regulated and 40 significantly downregulated GO terms were identified (Figure3.1 C-D). Analysis showed that the significantly enriched biological processes (BP) in the up-regulated terms were mainly related to T cell activation, while the down-regulated processes were mostly associated with response to stress and metabolic processes. The cellular components (CC) in the up-regulated terms were mostly localized to T cell receptor complexes and the nucleoplasm, while the down-regulated terms were more associated with membrane-bound organelles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. 3. 2\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eKEGG enrichment analysis of DEGs in wAMD patients compared to normal controls\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompared to normal controls, 5 KEGG pathways are significantly up-regulated in wAMD patients, including: T cell receptor signaling pathway, natural killer cell-mediated cytotoxicity, spliceosome, Parkinson\u0026rsquo;s disease, and primary immunodeficiency. Additionally, one significantly down-regulated KEGG pathway is associated with lysosomes (Figure 3.2 A-B).In the co-expressed target genes of differentially expressed lncRNAs in wAMD patients compared to normal controls, 2 significantly up-regulated KEGG pathways were identified, including the T cell receptor signaling pathway and NF-kappa B signaling pathway. Two significantly down-regulated pathways were related to lysosomes and other glycan degradation (Figure 3.2 C-D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. 4\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ewAMD patients compared to dAMD patients\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. 4. 1\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eGO enrichment analysis of DEGs in wAMD patients compared to dAMD patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the control of wAMD and dAMD patients, 55 significantly up-regulated and 34 significantly down-regulated GO Terms were enriched in the differentially expressed mRNAs in the wAMD patient group (Figure 4.1 A-B). Analysis revealed that the significantly up-regulated biological processes (BP) were closely related to the regulation of immune response and MyD88-dependent Toll-like receptor signaling pathway, while the down-regulated processes were primarily enriched in multi-organism process. The significantly enriched cellular components (CC) in the up-regulated terms were mostly localized to intracellular membrane-bounded organelle, while the down-regulated terms were mainly associated with the cytoplasm, nucleus, and adhesion junctions. The significantly enriched differential molecular functions (MF) were mostly related to protein binding. No significant differences in GO terms were identified in the co-expressed target mRNAs of differentially expressed lncRNAs between wAMD and dAMD patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. 4. 2\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eNo significantly different KEGG pathways were enriched in the differential mRNAs and lncRNAs co-expressed target genes between wAMD patients and dAMD patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. 5\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eRT-PCR validation of the differential expression gene\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe found that the differentially expressed mRNAs and the co-expressed target gene mRNAs of lncRNAs in wAMD patients compared to normal controls both significantly enriched in the T cell receptor signaling pathway and the NF-\u0026kappa;B signaling pathway. Among all lncRNAs with significantly altered expression levels, the target mRNAs co-expressed with linc00861 included 6 related to the T cell receptor signaling pathway and 3 related to the NF-kappa B signaling pathway. lncRNA-mRNA co-expression prediction revealed that linc00861 co-expressed with ZAP70, which was enriched in both the T cell receptor signaling pathway and the NF-kappa B signaling pathway. Therefore, we selected linc00861 as the gene for further validation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. 5. 1\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eRT-PCR validation of linc00861 in PBMCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the comparison of PBMCs between wAMD patients and normal controls，the expression of linc00861 was up-regulated in PBMCs from wAMD patients.(Figure 5.1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. 5. 2\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eRT-PCR validation of linc00861 in the RPE inflammation model\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eARPE-19 cells are treated with A\u0026beta;1-40 at concentrations of 0 \u0026mu;M, 0.01 \u0026mu;M, 0.1 \u0026mu;M, and 1 \u0026mu;M for either 24 hours or 48 hours. These treatments do not affect the proliferative capacity of the cells. However, compared to the control group (0 \u0026mu;M A\u0026beta;1-40), treatment with 10 \u0026mu;M A\u0026beta;1-40 for 24 hours does not significantly impair cell viability; yet, after 48 hours of stimulation, it significantly inhibits cell viability (Figure 5.2.1). To avoid cytotoxic effects influencing the proliferative capacity, we choose to treat ARPE-19 cells with 1 \u0026mu;M A\u0026beta;1-40 for 24 hours as the established condition for subsequent experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. 5. 3\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEffects of A\u0026beta;1-40 stimulation on expression of linc00861 and inflammatory cytokines in ARPE-19 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the effects of A\u0026beta;1-40 on cell apoptosis and inflammation, ARPE-19 cells were stimulated with 1 \u0026mu;M A\u0026beta;1-40 for 24 hours. The mRNA expression levels of IL-2, IL-6, TNF-\u0026alpha;, and IFNG (IFN-\u0026gamma;), as well as the expression of linc00861, were assessed by RT- PCR. As shown in Figure 5.2.2, after stimulating ARPE-19 cells with 1 \u0026mu;M A\u0026beta;1-40, the expression levels of inflammatory cytokines in the cells increased, and the expression level of linc00861 also increased.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAMD is a common irreversible blinding eye disease in individuals over 50 years of age, and it is clinically classified into dAMD and wAMD \u003csup\u003e[\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Currently, the treatment for wAMD mainly involves intravitreal injection of anti-VEGF drugs. However, these drugs have a short duration of action, requiring frequent injections, and there is still no effective treatment for dAMD patients to date \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Therefore, there is a need to continue searching for new therapeutic targets for AMD. In this study, high-throughput transcriptomic sequencing of PBMCs from AMD patients and healthy individuals was performed to screen for differentially expressed genes and related biological functions and signaling pathways.\u003c/p\u003e \u003cp\u003eCompared with normal individuals, the significantly changed co-expression analysis of differentially expressed mRNAs and lncRNAs in the dAMD patient group were mostly down-regulated GO Terms and KEGG pathways, which are mainly involved in biological processes such as RNA transcription and metabolism, as well as ribosomes associated pathway. Therefore, we speculate that most of the genes involved in ribosomes associated pathway in dAMD patients are in a state of inhibition or disorder, which also inhibits the transcription and metabolism of mRNA. The study argued that in the initial stage of AMD, the synthesis of ribosomal proteins is abnormally altered, and the protein translation initiation factors eIF2α, eIF3η, eIF5 and elongation factor eEF2 are anomalously regulated, leading to AMD ribosomal dysfunction \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Our transcriptomic analysis results suggest that consistent with previous reports, ribosomal dysfunction affects RNA transcription and molecular metabolism in dAMD patients.\u003c/p\u003e \u003cp\u003eIn the comparison between wAMD patients and normal controls, the significantly up-regulated GO terms of differentially expressed mRNAs and lncRNAs co-expressed target genes, were mainly involved in the activation of T lymphocytes. These genes were primarily located on T cell receptors and were mostly associated with molecular functions related to MHC class I proteins. The significantly up-regulated pathways were primarily related to T cell receptor signaling.wAMD patients are primarily in a state of immune system overactivation. Based on the sequencing results and bioinformatics analysis of this study, we hypothesize that MHC class I molecules, by binding with CD8⁺ TCR, activate cellular immunity and cytokine secretion to regulate other immune cells, which may contribute to the pathological changes in wAMD.As many reports have indicated, the pathogenesis of AMD involves systemic and local immune inflammation. For example, macrophages and CD8⁺ T cells are present in the choroid of AMD eyes \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Faber et al. also reported that in different subtypes of AMD patients, the proportion of aged, differentiated memory CD8⁺ T cells was higher \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. CD8⁺ T cells bind to MHC class I molecules and recognize specific antigen targets. Upon activation, CD8⁺ T cells produce IL-2 and IFN-γ, which regulate the effector functions of other immune cells such as macrophages and NK cells \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. In conclusion, consistent with the enrichment analysis of this study, CD8\u0026thinsp;+\u0026thinsp;T cells may be involved in the pathbiology of AMD. It is worth noting that in a transcriptomic study based on microarray chip analysis of PBMCs from patients with different clinical subtypes of advanced AMD \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e, the enrichment analysis also indicated that immune response and lymphocyte activation were involved. In the study by Grunin et al., when analyzing the transcriptome of peripheral mononuclear cells from wAMD patients, the most significant DAVID enrichment was related to leukocyte/lymphocyte activation and immune response \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Multiple clinical and experimental models have demonstrated the involvement of both innate and adaptive immunity in wAMD \u003csup\u003e[\u003cspan additionalcitationids=\"CR35 CR36 CR37 CR38 CR39 CR40 CR41 CR42 CR43 CR44\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. Howeverh,in the comparison between wAMD patients and healthy controls, the significantly down-regulated biological processes were equally related to metabolism, primarily occurring in the lysosome. This suggests that metabolic dysfunction also occurs in wAMD patients, but different from dAMD patients, it is not a suppression of processes at the RNA level. Instead, it primarily manifests as the inhibition of lysosome-related functions. Literature reports indicate that RPE cell degeneration, a process related to the accumulation of lysosomal lipofuscin derived from oxidative stress, impairs lysosomal degradation \u003csup\u003e[\u003cspan additionalcitationids=\"CR47 CR48\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. Previous studies have also shown that the interaction between phagocytosis and autophagy in the RPE is essential for the degradation of photoreceptor outer segments and the maintenance of retinoid levels to support vision. Disruption of genes related to lysosomal function can lead to lysosomal dysfunction and negatively impact the homeostasis regulation of phagocytosis and autophagy \u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. Autophagy is a lysosomal mechanism for the degradation of cytoplasmic proteins and damaged organelles. Through autophagy, protein aggregates are transported to the lysosome. With aging and the senescence of RPE cells, lysosomal enzymatic activity decreases. Autophagy is inhibited to eliminate mitochondria and protein aggregates from the intercellular space, thus accelerating lipofuscin accumulation and promoting the progression of AMD \u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn comparison to dAMD patients, the significantly upregulated biological processes enriched in the differential genes of wAMD patients are closely related to the regulation of the immune system and the MyD88-dependent Toll-like receptor signaling pathway. These sequencing results suggest that wAMD patients have more immune system-related gene changes than dAMD patients, along with higher levels of inflammation. This indicates that the formation of neovascularization in wAMD is more closely associated with immune inflammatory responses \u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs previously mentioned, we identified the key gene linc00861, which showed increased expression in both PBMCs from wAMD patients and in an inflammation model from Aβ-stimulated RPE cells. lncRNA-mRNA co-expression prediction revealed that linc00861 co-expressed with ZAP70, and both were enriched in the T cell receptor signaling pathway and NF-kappa B signaling pathway. This suggests that the abnormal activation of these two pathways is closely associated with the pathogenesis of AMD, with ZAP70 acting as a key upstream factor in these signaling pathways. Its abnormal activation may be a potential trigger for the development of AMD.ZAP70, a 70Kd protein tyrosine kinase associated with the ζ chain of T cell receptor, belongs to the syk tyrosine kinase family and plays a key role in inducing T cell activation after binding to T cell antigen receptor (TCR) \u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e. The activation of ZAP70 is the first step in the activation of all other signaling pathways. Activated ZAP70 phosphorylates downstream effector molecules, such as the linker for activation of T cell(LAT), the SH2 domain-containing leukocyte protein 76 kDa (SLP-76), and mitogen-activated protein kinase (MAPK) P38, initiating the PLC-γ activation pathway, MAPK pathway, and NF-κB signaling pathway \u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e.Currently, there is limited research on ZAP70 in ophthalmic diseases. Some studies have shown that in a spontaneous autoimmune uveitis mouse model (R161H), after retinal aldehyde-binding proteins are presented by dendritic cells, ZAP70 mediates the T cell receptor signaling pathway to activate intraocular CD4⁺ T lymphocytes, triggering an immune response in the eye and leading to ocular inflammation and histopathological lesions \u003csup\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e. In this model, the expression of phosphorylated ZAP70 in intraocular lymphoid follicles was significantly increased, resulting in the increased secretion of downstream inflammatory cytokines such as IL-2, IL-4, and IL-5, which induced ocular inflammatory responses \u003csup\u003e[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e.A recent study showed that the CD3ζ-Hck-Syk/Zap70 signaling pathway protects retinal ganglion cells in retinal excitotoxicity induced by the glutamate receptor agonist (NMDA) \u003csup\u003e[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]\u003c/sup\u003e. In recent years, with the deepening of research into the molecular structure and function of ZAP70, it has been confirmed that ZAP70 is a key point linking inflammation and immune signaling. ZAP70 not only participates in the transduction of T cell receptor signaling pathways but also plays a role in the regulation of NF-κB signaling pathways, NK cell cytotoxic responses, and the modulation of inflammation and immune responses. Existing reports on linc00861 are mostly related to cancer, including cervical cancer, prostate cancer, ovarian cancer, hepatocellular carcinoma, and breast cancer \u003csup\u003e[\u003cspan additionalcitationids=\"CR61 CR62 CR63 CR64\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]\u003c/sup\u003e. Notably, linc00861 found in other diseases is also largely involved in immune system-related changes, such as in eczema \u003csup\u003e[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]\u003c/sup\u003e, Parkinson's disease \u003csup\u003e[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]\u003c/sup\u003e, and sepsis \u003csup\u003e[\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]\u003c/sup\u003e.These reports suggest that linc00861 plays an important role in immune system-related pathological changes in diseases. Based on this, we hypothesize that in wAMD patients, MHC class I molecules bind to CD8⁺ TCR, and the high expression of linc00861 induces T cell activation by regulating ZAP70, thereby mediating downstream T cell receptor signaling and NF-κB signaling, leading to retinal immune inflammatory responses.\u003c/p\u003e \u003cp\u003eHowever, this research has certain limitations. Firstly, the sample size is relatively small, the dAMD group in this study includes early, intermediate, and late-stage geographic atrophy AMD which affects the macular fovea. The distinct pathological features at different stages of dAMD make it challenging to accurately identify differential genes, biological processes, and signaling pathway changes specific to each phase. This limitation hinders the precise identification of key nodes in the onset and progression of AMD and the effective prediction of disease progression, as well as the formulation of targeted intervention strategies. Future research should classify AMD into early, intermediate, and late stages and increase the sample size for transcriptomic analyses to enhance the accuracy of the results. At the same time, we will verify whether the key gene linc00861 screened in this study can activate T cells through ZAP70, regulate the transduction factors NF-Κb and ZAP70-P38-nFAT signaling pathway and participate in the regulation of retinal inflammation, so as to further clarify the specific mechanism of linc00861 participating in the intraocular immune inflammation. It provides a new target and evidence for the diagnosis and treatment of wAMD.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThrough high-throughput sequencing and co-expression network analysis of lncRNAs and mRNAs in PBMCs from AMD patients, we found that the development and progression of AMD are associated with metabolic disorders, dysfunction of the ribosome and lysosomes, and excessive activation of the immune system. Notably, in wAMD patients, the expression of linc00861 was up-regulated, and high expression of linc00861 may regulate ZAP70-mediated cell-mediated immunityof the T cell receptor signaling pathway, leading to retinal immune-inflammatory responses and contributing to the pathogenesis of wAMD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eConceptualization, Wenxi XIE; Data curation, Wenxi XIE; Formal analysis, Wenxi XIE; Project administration, Hui PENG; Resources, Hui PENG; Supervision, Xing WANG and Hui PENG; Validation, Wenxi XIE; Writing – original draft, Wenxi XIE; Writing – review \u0026amp; editing, Xing WANG. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research received no external funding.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eThe study was conducted in accordance with the Declaration 114 of Helsinki, and approved by the Institutional Review Board of the First Affiliated Hospital of Chongqing Medical University(2021 Research Ethics 2021-292).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u003c/strong\u003e Informed consent was obtained from all subjects involved in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eAll data generated during this study are included in the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We would like to express our gratitude to the Department of Ophthalmology, the First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory for the Prevention and Treatment of Major Blinding Eye Diseases, Chongqing Eye Institute for their dedicated care and support throughout the course of this study. We also would like to thank AMD patients, cataract patientstheir families, and their physicians for their consent to be involved in this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eW, Meng YF, Xing Q, et al. Identification of lncRNAs involved in biological regulation in early age-relate Friedman DS, O\u0026rsquo;Colmain BJ, Muñoz B, et al. Prevalence of age-related macular degeneration in the United States[J]. Arch Ophthalmol. 2004, 122(4): 564\u0026ndash;572. \u003c/li\u003e\n\u003cli\u003eKlein R, Klein BE, Cruickshanks KJ. The prevalence of age-related maculopathy by geographic region and ethnicity[J]. Prog Retin Eye Res. 1999, 18(3):371\u0026ndash;389. \u003c/li\u003e\n\u003cli\u003eMitchell P, Smith W, Attebo K, et al. 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Front Pharmacol. 2021 Aug 17, 12:6782 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-ophthalmology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"boph","sideBox":"Learn more about [BMC Ophthalmology](http://bmcophthalmol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/boph","title":"BMC Ophthalmology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Age-related macular degeneration, Peripheral blood mononuclear cell, transcriptome, amyloid β, retinal pigment epithelium","lastPublishedDoi":"10.21203/rs.3.rs-6639890/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6639890/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo identify differentially expressed transcriptomes through high-throughput sequencing of Peripheral blood mononuclear cells(PBMCs) from Age-related macular degeneration (AMD) patients and normal controls, and the related biological functions and signaling pathways were analyzed. Differentially expressed transcriptomes were screened by paired sequencing PBMCs of 5 wet AMD patients, 3 dry AMD patients and 4 normal controls respectively. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were conducted on them. Real-time PCR was used to verify the expression of selected Long non-coding RNA(lncRNA).\u003cstrong\u003e \u003c/strong\u003eGO and KEGG enrichment analysis of transcriptomes showed that, compared with normal controls, the RNA transcription, metabolism and ribosome-related pathways were all down-regulated.In wAMD patients, T lymphocyte activation is significantly up-regulated and participates in T cell receptor signaling pathway. Metabolism are significantly down-regulated, mainly involved in the lysosomal pathway. Compared with dAMD patients, wAMD patients mainly exhibit up-regulated immune system regulation and MYD88-dependent toll-like receptor signaling pathways. Transcriptome sequencing analysis showed that, in patients with wAMD, Major Histocompatibility Complex (MHC) class I molecules bind to CD8⁺ T-cell receptor(TCR), and the high expression of linc00861 may induce T cell activation by regulating ZAP70, thereby mediating downstream TCR signaling and NF-κB signaling, leading to retinal immune inflammation.\u003c/p\u003e","manuscriptTitle":"High-throughput sequencing and Co-expression network analysis of LncRNAs and mRNAs of PBMCs in AMD patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-11 06:04:41","doi":"10.21203/rs.3.rs-6639890/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2025-06-06T10:35:57+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-14T06:38:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-12T15:06:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-12T15:05:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Ophthalmology","date":"2025-05-11T13:32:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-ophthalmology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"boph","sideBox":"Learn more about [BMC Ophthalmology](http://bmcophthalmol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/boph","title":"BMC Ophthalmology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6860522c-d3af-412f-9bc5-05971b179f76","owner":[],"postedDate":"June 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-06-11T06:04:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-11 06:04:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6639890","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6639890","identity":"rs-6639890","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}