To investigate the RNA interference (RNAi) effect induced by vector-derived small interfering RNA (siRNA) targeting the three gatekeeper genes (Rad52, Ku70, Ku80) and screen the more effective target sites from candidates for further research, by using siRNA design tools online, we selected 2 candidate sequences directed to every gatekeeper gene. According to the sequences, six vector-derived siRNAs (denoted psiRNA1-6) and one mocking psiRNA7 were constructed. Among them, psiRNA1 and psiRNA2 targeted Rad52, psiRNA3 and psiRNA4 to Ku70, psiRNA5 and psiRNA6 to Ku80. The mocking psiRNA7 was used as control. After sequence identification, the seven plasmids were transfected into HepG2 cell line. siRNA-induced silencing of gatekeeper genes was determined by using RT-PCR at RNA level and Western Blot at protein level. The results showed that the six plasmids specifically targeting the coding region of gatekeeper genes were successfully designed and constructed. To some extent, the six plasmids could reduce the expression of target gene. Comparatively, the plasmid-derived siRNA psiRNA1, psiRNA4 and psiRNA5 were more effective than their counterparts. The results suggest that the gene silencing efficiency of siRNA is different, depending on their targeted region, and siRNA may provide us with practical tools for further study on the three gatekeeper genes, i.e. Rad52, Ku70, Ku80.

Objective To observe the features of temporal macular thinning and its value for the diagnosis of Alport syndrome (AS) in young patients.Methods Eighty-one young patients with AS (81 eyes) from Peking University First Hospital during January 2016 and July 2017 were included in this study.There were 67 males (67 eyes) and 14 females (14 eyes),the aged from 3 to 17 years,with the mean age of 9.6 years.Among 81 patients (81 eyes),there were 64 patients with X-linked AS (XLAS,including 53 males and 11 females),17 patients with autosomal recessive AS (ARAS,including 14 males and 3 females).One hundred healthy subjects aged 4 to 17 years were included as controls.Clinical data were retrospectively evaluated,including visual acuity,slit-lamp microscopy,dilated fundus photography,and OCT.Retinal thickness was measured with an OCT scan and the temporal thinning index (TTI) was calculated as stated in a previous study.The TTI values of each group was compared by One-way ANOVA or independent sample t test.The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic effectiveness for AS.Results The TTI of the control group,XLAS and ARAS patients were 6.46 ± 1.58,10.93 ± 3.77,12.14± 4.05,respectively.Compared with the control group,the TTI value of males were larger in the XLAS and ARAS group (F=45.056,P＜0.001),the TTI value of females were larger in the ARAS group (F=26.541,P＜0.001).The difference of TTI value in females was significant between the XLAS and ARAS groups (F=26.541,P＜0.001).In males,the area under the ROC curve was 0.896 (95％CI 0.837-0.955,P＜0.001).The optimal cutoff value of the TTI was determined as 9.47,with a sensitivity of 73.1％ and a specificity of 100％.Conclusions TTI is a common ocular finding in young patients with AS.In males,a TTI ＞ 9.47 may differentiate AS from normal males.

To investigate the RNA interference (RNAi) effect induced by vector-derived small interfering RNA (siRNA) targeting the three gatekeeper genes (Rad52, Ku70, Ku80) and screen the more effective target sites from candidates for further research, by using siRNA design tools online,we selected 2 candidate sequences directed to every gatekeeper gene. According to the sequences, six vector-derived siRNAs (denoted psiRNA1-6) and one mocking psiRNA7 were constructed. Among them, psiRNA1 and psiRNA2 targeted Rad52, psiRNA3 and psiRNA4 to Ku70, psiRNA5 and psiRNA6 to Ku80. The mocking psiRNA7 was used as control. After sequence identification, the seven plasmids were transfected into HepG2 cell line. siRNA-induced silencing of gatekeeper genes was determined by using RT-PCR at RNA level and Western Blot at protein level. The results showed that the six plasmids specifically targeting the coding region of gatekeeper genes were successfully designed and constructed. To some extent, the six plasmids could reduce the expression of target gene.Comparatively, the plasmid-derived siRNA psiRNA1, psiRNA4 and psiRNA5 were more effective than their counterparts. The results suggest that the gene silencing efficiency of siRNA is different,depending on their targeted region, and siRNA may provide us with practical tools for further study on the three gatekeeper genes, i.e. Rad52, Ku70, Ku80.

Objective:To investigate the risk factors of type 1 gastric neuroendocrine tumor (g-NET) in patients with autoimmune gastritis(AIG).Methods:From September 1, 2016 to February 28, 2022, 123 patients with AIG visited the First Affiliated Hospital of Zhengzhou University were retrospectively enrolled, including 37 cases with type 1 g-NET and 86 cases without type 1g-NET. The clinical data, serological indicators, and endoscopic manifestation of all the patients were analyzed, including the age at the time of AIG diagnosis (hereinafter referred to as the age at diagnosis), levels of gastrin 17 and pepsinogen Ⅰ (PGⅠ), presence or absence of gastric fundus and gastric body polyps, etc. The independent risk factors of type 1 g-NET in AIG patients were analyzed by univariate and multivariate logistic regression. The receiver operating characteristic curve (ROC) was plotted to analyze the optimal cut-off value, sensitivity and specificity of the independent risk factors in predicting type 1 g-NET in AIG patients. Independent sample t test, Mann-Whitney U test and chi-square test were used for statistical analysis. Results:Compared with those of the AIG patients without type 1 g-NET, the age at diagnosis of AIG patients with type 1 g-NET was younger ((57.49±11.16) years old vs. (48.49±10.96) years old), the level of gastrin 17 was higher (200.21 ng/L, 121.85 ng/L to 244.40 ng/L vs. 244.40 ng/L, 182.50 ng/L to 248.02 ng/L), and the proportion of patients with gastric fundus and gastric body polyps was higher(18.6%, 16/86 vs. 56.8%, 21/37), and the differences were statistically significant( t=-4.13, Z=-3.06, χ2=17.90; P<0.001, =0.002 and <0.001). The results of univariate logistic analysis showed that the age at diagnosis ( OR=0.931, 95% confidence interval (95% CI)0.895 to 0.967), gastrin 17( OR=1.012, 95% CI 1.005 to 1.019), PGⅠ( OR=0.974, 95% CI 0.950 to 0.998)and gastric fundus and gastric body polyps( OR=5.742, 95% CI 2.461 to 13.399)were the influencing factors of type 1 g-NET in AIG patients ( P<0.001, =0.001, =0.033 and <0.001). The results of multivariate logistic regression analysis indicated that the age at diagnosis( OR=0.921, 95% CI 0.881 to 0.964), gastrin 17( OR=1.011, 95% CI 1.001 to 1.020), gastric fundus and gastric body polyps( OR=7.696, 95% CI 2.710 to 21.857)were the independent risk factors of type 1 g-NET in AIG patients ( P<0.001, =0.024 and <0.001). The results of ROC analysis demonstrated that the optimal cut-off values for the age at diagnosis and gastrin 17 in predicting type 1 g-NET were 56.50 years old and 206.40 ng/L, respectively; with sensitivity of 83.8% and 70.3%, respectively, and specificity of 54.7% for both ( P<0.001 and=0.003). Conclusion:The age at diagnosis< 56.50 years old, gastrin 17>206.40 ng/L and the presence of gastric fundus and gastric body polyps are independent risk factors of type 1 g-NET in AIG patients.

Objective:To observe the healing morphology, macular microstructure and visual function of idiopathic macular hole (IMH) after pars plana vitrectomy (PPV) combined with internal limiting membrane (ILM) flap.Methods:Retrospective case study. From 2016 to 2018, 39 eyes of 39 patients with IMH diagnosed in Tianjin Eye Hospital were included in the study. Among them, there were 4 eyes in 4 males and 35 eyes in 35 females, with an average age of 64.56±7.2 years. BCVA, OCT, OCT angiography (OCTA) and MAIA microperimetry examination were performed in all patients. BCVA examination was performed with the international standard visual acuity chart, which was converted to logMAR visual acuity when recording. All patients underwent PPV combined with ILM flap covering and air tamponade. According to the characteristics of OCT images postoperatively, the eyes were divided into U-shaped closed group, V-shaped closed group, irregular closed group and flat closed group, with 26, 5, 7 and 1 eyes respectively. There was a significant difference in the minimum hole diameter ( F=5.118, P=0.005) and macular hole classification ( F=3.608, P=0.024). The shallow capillary layer (SCP) blood flow density in the U-shaped closure group was significantly higher than that in the V-shaped closure group, the irregular closure group and the flat closure group (t=2.079, 2.368; P=0.047, 0.025). At 1, 3, 6 months after the operation, the same equipment and methods were used for relevant examination. The blood flow density of BCVA, SCP, perimeter of foveal avascular zone (PERIM) and mean sensitivity (MS) were compared before and after operation. Independent sample t-test was used for quantitative data comparison between different groups, and χ2 test was used for counting data comparison. Results:Six months after operation, the logMAR of the eyes in the U-shaped closure group was -0.75±0.29 higher than that before operation, and was better than that in the V-shaped closure group, the irregular closure group and the flat closure group ( t=-2.974, -2.518; P=0.006, 0.018). The integrity of external limiting membrane (ELM) and ellipsoid in U-shaped closed group was significantly higher than that in V-shaped closed group, irregular closed group and flat closed group ( χ2=15.229, 10.809; P=0.020, 0.013). The percentage of macular central fovea reflex mass in the U-shaped closed group was significantly lower than that in the V-shaped closed group, irregular closed group and flat closed group ( χ2=20.107, P=0.000). PERIM in U-shaped closure group was smaller than that in V-shaped closure group, irregular closure group and flat closure group, and the difference was statistically significant ( t=-3.391, -2.427; P=0.002, 0.022). The total MS of macular area 10° in the U-shaped closure group was significantly higher than that in the other V-shaped closure group, irregular closure group and flat closure group ( t=2.939, 2.811; P=0.001, 0.001). Conclusion:After IMH operation, the U-shaped closure showed better BCVA and macular light sensitivity, the proportion of ELM and ellipsoid to restore structural integrity are higher, PERIM is smaller, and there are fewer macular fovea strong reflex masses.

Objective:To screening differentially expressed genes (DEGs) in proliferative diabetic retinopathy (DR) patients to provide new biological therapeutic targets for proliferative DR (PDR) therapy.Methods:A basic research. A total of 3 PDR patients (group PDR) and 3 non-diabetic patients (control group) were enrolled in the study in Tianjin Medical University Eye Hospital in October 2020. In addition, 40 cases of PDR and non-diabetic patients were selected and divided into PDR validation group and control validation group. Peripheral blood validation test was performed in PDR validation group and control validation group; RNA sequencing was performed in PDR group and control group. Transcriptomics (RNAseq) sequencing technology was used to screen DEG in PDR group and control group. The selected DEGs were analyzed by gene ontology (GO) function enrichment analysis, signal pathway enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction network (PPI). The gene expression database was used to find the high-throughput data related to PDR, and multi queue comparison analysis was carried out. The target genes of differentially expressed miRNAs were predicted through targetscan platform, so as to clearly screen the correlation between DEG and PDR. Reverse transcription polymerase chain reaction and Western blot were used to verify the expression of DEG mRNA and protein related to PDR. The relative expression of PDR related DEG mRNA and protein between PDR validation group and control validation group were compared by paired t-test. Results:A total of 1 337 DEGs were screened by RNAseq sequencing in the peripheral blood of patients with PDR, of which 419 genes were up-regulated and 918 down-regulated. Among them, direct inhibitor of apoptosis protein-binding protein with low isoelectric point ( DIABLO), zinc finger and BTB domain containing 10 ( ZBTB10), polo-like kinases 3 ( PLK3), regulatory subunit 1 ( PIK3R1) and B cell translocation gene 3 (BTG3) were differentially expressed in PDR patients. The function of GO was enriched from the analysis of molecular function, biological process and cellular composition. The results showed that DIABLO, ZBTB10, PLK3, PIK3R1, BTG3 were involved in the pathological process related to PDR. KEGG enrichment analysis showed that glucose metabolic pathways such as extracellular matrix receptors, cytokine regulatory pathway, p53 signal pathway and galactose metabolism may be involved in the process of differential genes. The analysis of PPI protein interaction network showed that the larger the DEG-associated protein node, the greater the number of associated nodes. Among them, DIABLO, ZBTB10, PLK3, PIK3R1 and BTG3 played significant roles in the formation of the action network. By comparing and analyzing the existing high-throughput data related to diabetic retinopathy in Gene Expression Omnibus database and predicting by Targetscan platform, it was found that some significant differences in miRNA reported in aqueous humor, vitreous fluid and plasma of DR patients can be regulated by the differential genes found in this study. Compared with the control verification group, the relative expressions of DIABLO, ZBTB10, PLK3, PIK3R1 mRNA and protein in peripheral blood of the PDR verification group were up-regulated, and the relative expression of BTG3 mRNA and protein was down-regulated. Conclusion:DIABLO, ZBTB10, PLK3, PIK3R1 and BTG3 are DEGs in patients with PDR, and they can participate in the disease process by regulating the biological processes of cell proliferation, fibrosis and oxidative stress.

Objective:To observe the effect of bone morphogenetic protein 4 (BMP4) on the proliferation and migration of human retinal microvascular endothelial cells (hRMEC) under oxidative stress.Methods:The hRMEC cultured in vitro were divided into control group, 4-hydroxynonenal (HNE) treatment group (4-HNE group), 4-HNE+BMP4 group (BMP4 group). Cell culture medium of 4-HNE treatment group was added with 10 μmmol/L 4-HNE; cell culture of BMP4 group was cultured with 10 μmmol/L 4-HNE, and after stimulation for 6 h, 100 ng/ml recombinant human BMP4 was added. The effects of 4-HNE and BMP4 on hRMEC viability was detected by thiazole blue colorimetric method. The effects of 4-HNE and BMP4 on cell migration was determined by cell scratch test. The relative expression of BMP4 mRNA in the cells of the control group and 4-HNE treatment group and the mRNA expression of the control group, the fibronectin (FN) of BMP4 group, laminin (Laminin), α-smooth muscle contractile protein (α-SMA), and collagen type Ⅰ (Collagen Ⅰ), vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) were detected by real-time quantitative polymerase chain reaction (qRT-PCR). Western blot was used to detect the relative expression of BMP4 protein in the control group and 4-HNE group. The control group and 4-HNE group were compared by t test. Results:Compared with the control group, cell viability ( t=12.73, 16.26, P=0.000 2, <0.000 1), cell migration rate ( t=28.17, 37.48, P<0.000 1, <0.000 1) in 4-HNE group and BMP4 group were significantly increased, and the difference was statistically significant; the relative expression of BMP4 mRNA and protein in the 4-HNE group was significantly increased, and the difference was statistically significant ( t=16.36, 69.35, P=0.000 1, <0.000 1). The qRT-PCR test results showed that compared with the control group, the relative expression of VEGF, FN, Laminin, α-SMA, Collagen Ⅰ, and CTGF mRNA in the cells of the BMP4 group was significantly increased, and the difference was statistically significant ( t=10.61, 17.00, 14.85, 7.78, 12.02, 10.61, P=0.0004, <0.000 1, 0.000 1, 0.001 5, 0.000 1, 0.000 4). Conclusion:BMP4 can induce the proliferation and migration of hRMEC; it can also regulate the expression of angiogenesis factors and fibrosis-related factors in hRMEC.

Cholesterol is an important precursor of many endogenous molecules. Disruption of cholesterol homeostasis can cause many pathological changes, leading to liver and cardiovascular diseases. CYP1A is widely involved in cholesterol metabolic network, but its exact function has not been fully elucidated. Here, we aim to explore how CYP1A regulates cholesterol homeostasis. Our data showed that CYP1A1/2 knockout (KO) rats presented cholesterol deposition in blood and liver. The serum levels of low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and total cholesterol were significantly increased in KO rats. Further studies found that the lipogenesis pathway (LXRα-SREBP1-SCD1) of KO rats was activated, and the key protein of cholesterol ester hydrolysis (CES1) was inhibited. Importantly, lansoprazole can significantly alleviate rat hepatic lipid deposition in hypercholesterolemia models by inducing CYP1A. Our findings reveal the role of CYP1A as a potential regulator of cholesterol homeostasis and provide a new perspective for the treatment of hypercholesterolemia.