UNASSIGNED: N6-methyladenosine (m6A) methylation is a chemical modification that occurs on RNA molecules, where the hydrogen atom of adenine (A) nucleotides is replaced by a methyl group, forming N6-methyladenosine. This modification is a dynamic and reversible process that plays a crucial role in regulating various biological processes, including RNA stability, transport, translation, and degradation. Currently, there is a lack of research on the role of m6A modifications in maintaining the characteristics of RPE cells. m6A readers play a crucial role in executing the functions of m6A modifications, which prompted our investigation into their regulatory roles in the RPE.
UNASSIGNED: Phagocytosis assays, immunofluorescence staining, flow cytometry experiments, β-galactosidase staining, and RNA sequencing (RNA-seq) were conducted to assess the functional and cellular characteristics changes in retinal pigment epithelium (RPE) cells following short-hairpin RNA-mediated knockdown of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). RNA-seq and ultraviolet crosslinking immunoprecipitation with high-throughput sequencing (HITS-CLIP) were employed to identify the target genes regulated by IGF2BP2. adeno-associated virus (AAV) subretinal injection was performed in 6- to 8-week-old C57 mice to reduce IGF2BP2 expression in the RPE, and the impact of IGF2BP2 knockdown on mouse visual function was assessed using immunofluorescence, quantitative real-time PCR, optical coherence tomography, and electroretinography.
UNASSIGNED: IGF2BP2 was found to have a pronounced effect on RPE phagocytosis. Subsequent in-depth exploration revealed that IGF2BP2 modulates the mRNA stability of PAX6 and OTX2, and the loss of IGF2BP2 induces inflammatory and aging phenotypes in RPE cells. IGF2BP2 knockdown impaired RPE function, leading to retinal dysfunction in vivo.
UNASSIGNED: Our data suggest a crucial role of IGF2BP2 as an m6A reader in maintaining RPE homeostasis by regulating the stability of PAX6 and OTX2, making it a potential target for preventing the occurrence of retinal diseases related to RPE malfunction.

BACKGROUND: Corneal injuries, often leading to severe vision loss or blindness, have traditionally been treated with the belief that limbal stem cells (LSCs) are essential for repair and homeostasis, while central corneal epithelial cells (CCECs) were thought incapable of such repair. However, our research reveals that CCECs can fully heal and maintain the homeostasis of injured corneas in rats, even without LSCs. We discovered that CXCL14, under PAX6\'s influence, significantly boosts the stemness, proliferation, and migration of CCECs, facilitating corneal wound healing and homeostasis. This finding introduces CXCL14 as a promising new drug target for corneal injury treatment.
METHODS: To investigate the PAX6/CXCL14 regulatory axis\'s role in CCECs wound healing, we cultured human corneal epithelial cell lines with either increased or decreased expression of PAX6 and CXCL14 using adenovirus transfection in vitro. Techniques such as coimmunoprecipitation, chromatin immunoprecipitation, immunofluorescence staining, western blot, real-time PCR, cell colony formation, and cell cycle analysis were employed to validate the axis\'s function. In vivo, a rat corneal epithelial injury model was developed to further confirm the PAX6/CXCL14 axis\'s mechanism in repairing corneal damage and maintaining corneal homeostasis, as well as to assess the potential of CXCL14 protein as a therapeutic agent for corneal injuries.
RESULTS: Our study reveals that CCECs naturally express high levels of CXCL14, which is significantly upregulated by PAX6 following corneal damage. We identified SDC1 as CXCL14\'s receptor, whose engagement activates the NF-κB pathway to stimulate corneal repair by enhancing the stemness, proliferative, and migratory capacities of CCECs. Moreover, our research underscores CXCL14\'s therapeutic promise for corneal injuries, showing that recombinant CXCL14 effectively accelerates corneal healing in rat models.
CONCLUSIONS: CCECs play a critical and independent role in the repair of corneal injuries and the maintenance of corneal homeostasis, distinct from that of LSCs. The PAX6/CXCL14 regulatory axis is pivotal in this process. Additionally, our research demonstrates that the important function of CXCL14 in corneal repair endows it with the potential to be developed into a novel therapeutic agent for treating corneal injuries.

BACKGROUND: Neuroendocrine prostate cancer (NEPC) is a lethal subset of prostate cancer which is characterized by neuroendocrine differentiation and loss of androgen receptor (AR) signaling. Growing evidence reveals that cell lineage plasticity is crucial in the failure of NEPC therapies. Although studies suggest the involvement of the neural transcription factor PAX6 in drug resistance, its specific role in NEPC remains unclear.
METHODS: The expression of PAX6 in NEPC was identified via bioinformatics and immunohistochemistry. CCK8 assay, colony formation assay, tumorsphere formation assay and apoptosis assay were used to illustrate the key role of PAX6 in the progression of in vitro. ChIP and Dual-luciferase reporter assays were conducted to confirm the binding sequences of AR in the promoter region of PAX6, as well as the binding sequences of PAX6 in the promoter regions of STAT5A and MET. For in vivo validation, the xenograft model representing NEPC subtype underwent pathological analysis to verify the significant role of PAX6 in disease progression. Complementary diagnoses were established through public clinical datasets and transcriptome sequencing of specific cell lines. ATAC-seq was used to detect the chromatin accessibility of specific cell lines.
RESULTS: PAX6 expression was significantly elevated in NEPC and negatively regulated by AR signaling. Activation of PAX6 in non-NEPC cells led to NE trans-differentiation, while knock-down of PAX6 in NEPC cells inhibited the development and progression of NEPC. Importantly, loss of AR resulted in an enhanced expression of PAX6, which reprogramed the lineage plasticity of prostate cancer cells to develop NE phenotypes through the MET/STAT5A signaling pathway. Through ATAC-seq, we found that a high expression level of PAX6 elicited enhanced chromatin accessibility, mainly through attenuation of H4K20me3, which typically causes chromatin silence in cancer cells.
CONCLUSIONS: This study reveals a novel neural transcription factor PAX6 could drive NEPC progression and suggest that it might serve as a potential therapeutic target for the management of NEPC.

Impaired brain glucose metabolism is an early indicator of Alzheimer\'s disease (AD); however, the fundamental mechanism is unknown. In this study, we found a substantial decline in isocitrate dehydrogenase 3β (IDH3β) levels, a critical tricarboxylic acid cycle enzyme, in AD patients and AD-transgenic mice\'s brains. Further investigations demonstrated that the knockdown of IDH3β induced oxidation-phosphorylation uncoupling, leading to reduced energy metabolism and lactate accumulation. The resulting increased lactate, a source of lactyl, was found to promote histone lactylation, thereby enhancing the expression of paired-box gene 6 (PAX6). As an inhibitory transcription factor of IDH3β, the elevated PAX6 in turn inhibited the expression of IDH3β, leading to tau hyperphosphorylation, synapse impairment, and learning and memory deficits resembling those seen in AD. In AD-transgenic mice, upregulating IDH3β and downregulating PAX6 were found to improve cognitive functioning and reverse AD-like pathologies. Collectively, our data suggest that impaired oxidative phosphorylation accelerates AD progression via a positive feedback inhibition loop of IDH3β-lactate-PAX6-IDH3β. Breaking this loop by upregulating IDH3β or downregulating PAX6 attenuates AD neurodegeneration and cognitive impairments.

BACKGROUND: Aniridia is a rare eye disorder with a high incidence of glaucoma, and surgical intervention is often needed to control the intraocular pressure (IOP). Here, we reported a case of illuminated microcatheter-assisted circumferential trabeculotomy (MAT) performed on an aniridic glaucoma patient following a previous failed angle surgery. The surgical procedures for aniridic glaucoma were also reviewed.
METHODS: A 21-year-old man, diagnosed with aniridic glaucoma, came to our hospital consulting for the poor control of left eye\'s IOP despite receiving goniotomy surgery 3 years ago. The IOP was 26 mmHg with maximum topical antiglaucoma eyedrops. The central cornea was opaque and the majority of iris was absent. The gonioscopy and ultrasound biomicroscopy (UBM) demonstrated that 360° anterior chamber angle was closed. The whole exome sequencing of peripheral blood confirmed a 13.39 Mb copy number loss at chromosome 11p15.1p13, containing PAX6 and WT1 gene. The 360° MAT surgery was performed on his left eye. At 1-year follow-up, the IOP was 19mmHg with 2 kinds of topical antiglaucoma medications, and the postoperative UBM demonstrated the successful incision of the anterior chamber angle.
CONCLUSIONS: The case presented here exhibited a case of aniridic glaucoma treated by MAT surgery. The MAT surgery may be an effective option for IOP control in aniridic glaucoma patients following a previous failed angle surgery.

Remifentanil‑induced hyperalgesia (RIH) is characterized by the emergence of stimulation‑induced pain, including phenomena such as allodynia and thermal hyperalgesia following remifentanil infusion. As a sequence‑specific DNA binding transcription factor, PAX6 positively and negatively regulates transcription and is expressed in multiple cell types in the developing and adult central nervous system. It was hypothesized that puerarin could relieve RIH via targeting PAX6 to regulate transcription of transient receptor potential cation channel subfamily V Member 1 (TRPV1). A total of 32 rats were randomly divided into five groups, namely control group, RI group, RI + 10 mg/kg puerarin group (RI + puerarin10), RI + 20 mg/kg puerarin group (RI + puerarin20), and RI + 40 mg/kg puerarin group (RI + puerarin40). Mechanical and thermal hyperalgesia were tested at ‑24, 2, 6, 24 and 48 h after remifentanil infusion. Following the sacrifice of rats after the last behavioral test, western blot was used to detect the expression levels of TRPV1 in the tissues; Immunofluorescence staining and western blotting were used to detect the expression of PAX6 in the spinal cord. PharmMapper and JASPAR were used to predict the binding sites of puerarin/PAX6/TRPV1. Chromatin immunoprecipitation‑PCR and dual luciferase reporter assay were used to verify the targeting relationship between PAX6 and TRPV1. Immunofluorescence was used to detect the expression levels of TRPV1 and p‑NR2B. The results revealed that puerarin (10, 20, 40 mg/kg) dose‑dependently reduced thermal and mechanical hyperalgesia from 2 to 48 h after remifentanil infusion. Remifentanil infusion remarkably stimulated the expression of phosphorylated (p‑)NR2B. Nevertheless, the increased amount of p‑NR2B by RIH was dose‑dependently suppressed by puerarin in rats. In conclusion, puerarin was revealed to attenuate postoperative RIH via targeting PAX6 to regulate the transcription of TRPV1.

Colorectal cancer is a cancer that arises from the abnormal growth of cells in the colon or rectum. Osteosarcoma (OS) is a common primary bone tumor with high degree of malignancy. The configuration files for colorectal cancer dataset GSE142279 and OS datasets GSE197158 and GSE206448 were downloaded from Gene Expression Omnibus database using the platforms GPL20795, GPL20301, and GPL24676. Differentially expressed genes (DEGs) were screened and weighted gene co-expression network analysis (WGCNA) was performed. Construction and analysis of protein-protein interactions (PPI) network. Functional enrichment analysis, gene set enrichment analysis (GSEA) were performed. A heat map of gene expression was drawn. The Comparative Toxicogenomics Database (CTD) was used to find the diseases most associated with the core genes. TargetScan was used to screen miRNAs regulating DEGs. According to the Gene Ontology (GO) analysis, DEGs are mainly enriched in acetylcholine binding receptor activity involved in Wnt signaling pathway, cell polarity pathway, PI3K-Akt signaling pathway, receptor regulator activity, cytokine-cytokine receptor interaction, transcriptional misregulation in cancer, and inflammation-mediated regulation of tryptophan transport. In the Metascape enrichment analysis, GO enrichment items related to the regulation of Wnt signaling pathway, regulation of muscle system process, and regulation of actin filament-based movement. Eight core genes (CUX1, NES, BCL11B, PAX6, EMX1, MCOLN2, TRPA1, TRPC4) were identified. CTD showed that 4 genes (CUX1, EMX1, TRPA1, BCL11B) were associated with colorectal neoplasms, colorectal tumors, colonic diseases, multiple myeloma, OS, and inflammation. PAX6, TRPA1, BCL11B, MCOLN2, CUX1, and EMX1 are highly expressed in colorectal cancer and OS, and the higher the expression level, the worse the prognosis.

BACKGROUND: To identify the disease-causing gene in a Chinese family affected with congenital aniridia.
METHODS: Patients underwent systematic ophthalmic examinations such as anterior segment photography, fundus photography, optical coherence tomography, and fundus fluorescein angiography. The proband was screened for pathogenic variants by whole exome sequencing (WES) and copy number variant (CNV) analysis. Real-time quantitative PCR (RT-qPCR) was applied to confirm the CNV results. Breakpoints were identified by long-range PCR followed by Sanger sequencing.
RESULTS: All seven members of this Chinese family, including four patients and three normal individuals, were recruited for this study. All patients showed bilateral congenital aniridia with nystagmus, except the son of the proband, who presented with bilateral partial coloboma of the iris. A novel heterozygous deletion (chr11:31,139,019-31,655,997) containing the 3\' regulatory enhancers of the PAX6 gene was detected in this family. We also reviewed the reported microdeletions downstream of PAX6 in patients with aniridia.
CONCLUSIONS: We identified a novel microdeletion, 517 kb in size located about 133 kb downstream of the PAX6 gene, responsible for congenital aniridia in this Chinese family, which expands the spectrum of aniridia-associated mutations in PAX6.

OBJECTIVE: Paired box gene 6 (PAX6) plays a major role in the regulation of embryonic development. Abnormal expression of PAX6 is associated with the development of various tumors. PAX6 can play a role in promoting or suppressing cancer in different tumors. This study aim to observe the effect of overexpression of PAX6 on the growth of hepatocellular carcinoma cells, and the killing of hepatocellular carcinoma cells via natural killer (NK) cell and the possible mechanism.
METHODS: The protein levels of PAX6, soluble major histocompatibility complex class I-like protein A (sMICA) and soluble UL16 binding protein 2 (sULBP2) in peripheral blood from 68 cases of hepatocellular carcinoma (HCC) patients and 10 healthy volunteers were detected by ELISA. Hepatocellular carcinoma cell line (HepG2, LM3) and human normal liver cells (LO2) were cultured at 37 ℃ and 5% CO2 condition in vitro. The PAX6 overexpressed plasmid (PAX6-OE) and empty vector (NC) were transferred into HepG2 and LM3 cells to construct stable cell lines. The mRNA and protein expression levels of PAX6 in HepG2 and LM3 cells were detected by real-time PCR, Western blotting and immunofluorescence, respectively. PAX6 was overexpressed in HepG2 and LM3 cells, the cell growth and migration ability were detected by CCK-8 method and cell scratch assay, and the levels of sMICA and sULBP2 in the supernatant were detected by ELISA. Matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9) and disintegrin and metalloproteinase 10 (ADAM10) in HepG2 and LM3 cells were detected by Western blotting. The killing ability of NK cells against these 2 HCC cells was detected by flow cytometry.
RESULTS: Compared with the healthy volunteers, the expressions of PAX6 in the HCC patients were significantly decreased (P=0.002), while the expression of sMICA and sULBP2 were significantly increased (P=0.004 and P<0.001, respectively). Real-time PCR and Western blotting results showed that compared with LO2 cells, mRNA and protein expressions of PAX6 in HepG2 and LM3 cells were significantly decreased (all P<0.05). Immunofluorescence results also showed that the expressions of PAX6 in HepG2 and LM3 were lower than those of LO2 cells. Compared with the NC group, the ability of proliferation and migration of HepG2 and LM3 cells were decreased (both P<0.05). The protein expressions of MMP2, MMP9 and ADAM10 in HepG2 and LM3 cells in the PAX6-OE group were significantly decreased, and the levels of sMICA and sULBP2 in superneant of HepG2 and LM3 cells in the PAX6-OE group were significantly lower than those in the NC group (all P<0.05). Flow cytometry results showed that compared with the NC group, the proportion of NK cells killing HepG2 and LM3 cells in PAX6-OE group was significantly increased (both P<0.05).
CONCLUSIONS: The expression of PAX6 is decreased in serum of HCC patients and hepatocellular carcinoma cell lines. Overexpression of PAX6 can inhibit the growth of hepatocellular carcinoma cells, enhance the killing efficiency of NK cells against hepatoma cells. The mechanism is related to the inhibition of the expression of metalloproteinase via PAX6 and the decrease of the secretion levels of sMICA and sULBP2.
目的: 配对盒基因6(paired box gene 6，PAX6)主要在胚胎发育中发挥调控作用，其异常表达与多种肿瘤的发生、发展有关，在不同肿瘤中可发挥促癌或抑癌的作用。本研究旨在观察过表达PAX6对肝癌细胞生长及自然杀伤(natural killer，NK)细胞对肝癌细胞杀伤能力的影响及其分子机制。方法: 采用ELISA技术检测68例肝细胞癌(hepatocellular carcinoma，HCC)患者和10例健康人外周血的PAX6、可溶性主要组织相容性复合体I类样蛋白A(soluble major histocompatibility complex class I-like protein A，sMICA)和可溶性UL16结合蛋白2(soluble UL16 binding protein 2，sULBP2)的表达水平；体外培养肝癌细胞HepG2和LM3及人正常肝细胞LO2，将PAX6过表达质粒(PAX6-OE)和空载体(NC)转入HepG2和LM3细胞中构建稳定细胞株，分别采用real-time PCR、蛋白质印迹法、免疫荧光等方法检测HepG2和LM3细胞中PAX6的表达水平；在HepG2和LM3细胞中过表达PAX6，采用CCK-8法和细胞划痕实验检测细胞生长和迁移能力，ELISA法检测其上清液中sMICA和sULBP2的分泌水平，蛋白质印迹法检测基质金属蛋白酶2(matrix metalloproteinase 2，MMP2)、基质金属蛋白酶9(matrix metalloproteinase 9，MMP9)、去整合素样金属蛋白酶10(disintegrin and metalloproteinase 10，ADAM10)的蛋白质表达水平；采用流式细胞术检测NK细胞对2种肝癌细胞的杀伤能力。结果: 与健康人相比较，HCC患者血清中PAX6表达水平显著降低(P=0.002)，而sMICA和sULBP2的表达水平显著增高(P=0.004和P<0.001)。Real-time PCR、蛋白质印迹法结果显示：与正常肝细胞LO2相比，HepG2和LM3细胞PAX6的mRNA和蛋白质表达水平均显著降低(均P<0.05)。免疫荧光结果亦可见肝癌细胞HepG2和LM3中PAX6的表达均低于正常肝细胞LO2。与NC组比较，PAX6-OE组肝癌细胞HepG2和LM3的增殖及迁移能力下降(均P<0.05)；PAX6-OE组的HepG2和LM3细胞中MMP2、MMP9、ADAM10的蛋白质表达水平均显著降低(均P<0.05)，且PAX6-OE组的HepG2和LM3细胞上清液中sMICA和sULBP2的分泌水平均显著低于NC组(均P<0.05)。流式细胞术显示：与NC组比较，PAX6-OE组NK细胞对HepG2和LM3细胞的杀伤率显著增高(均P<0.05)。结论: PAX6在HCC患者血清及肝癌细胞系中表达降低，过表达PAX6可抑制肝癌细胞生长，增强NK细胞对肝癌细胞的杀伤效率，其机制与PAX6抑制金属蛋白酶的表达、降低sMICA和sULBP2的分泌水平有关。.

BACKGROUND: According to previous reports, PAX6-associated foveal hypoplasia (FH) could usually be accompanied by various anterior segment anomalies including variable iris changes. This study aims to exhibit unusual phenotypes of a novel missense variant of PAX6 from a Chinese pedigree.
METHODS: Ophthalmic examinations including slit-lamp biomicroscopy, gonioscopy, ophthalmic ultrasound, ultrasonic biomicroscopy, optical coherence tomography, wide-field fundus imaging, and visual field test were performed to evaluate the clinical manifestations. Whole-exome sequencing (WES) and bioinformatics analysis were conducted in eight members from this pedigree to identify the causative mutation.
RESULTS: WES revealed a novel heterozygous substitution of PAX6 (NM_000280.5:c.157G > A, p.(Val53Met) (chr11:31823309 C > T, hg19)), which cosegregated with the phenotype of this pedigree. All the three patients (a pair of fraternal twins and their mother) exhibited bilateral FH and anterior segment dysgenesis (ASD) including microcornea, sclerocornea, obvious symmetrical corectopia, iris stromal dysplasia, goniodysgenesis, and abnormal distribution of fundus blood vessels. The girl of the fraternal twins also demonstrated bilateral temporal deviation of lenses and abnormal tissue membrane connecting anterior chamber angle and lens anterior capsule in the right eye. The mother additionally showed apparent cataract bilaterally and cupping of the optic disc in her left eye.
CONCLUSIONS: A novel missense variant in PAX6 gene was detected in a Chinese pedigree demonstrating bilateral FH and ASD. It is really distinctive that the ASD involves almost all parts of the anterior segment, and bilateral symmetrical corectopia is the most perceptible sign. This study expands the phenotypic and genotypic spectrum of PAX6-associated ocular diseases, and facilitates the understanding of the crucial role that PAX6 plays in the development of the eye. Meanwhile, PAX6 could be considered as a candidate pathogenic gene of bilateral symmetrical corectopia.