Gene editing for corneal disease management

Gene editing has recently emerged as a promising technology to engineer genetic modifications precisely in the genome to achieve long-term relief from corneal disorders.Recent advances in the molecular biology leading to the development of clustered regularly interspaced short palindromic repeats(CRISPRs) and CRISPR-associated systems,zinc finger nucleases and transcription activator like effector nucleases have ushered in a new era for high throughput in vitro and in vivo genome engineering.Genome editing can be successfully used to decipher complex molecular mechanisms underlying disease pathophysiology,develop innovative next generation gene therapy,stem cell-based regenerative therapy,and personalized medicine for corneal and other ocular diseases.In this review we describe latest developments in the field of genome editing,current challenges,and future prospects for the development of personalized genebased medicine for corneal diseases.The gene editing approach is expected to revolutionize current diagnostic and treatment practices for curing blindness.

Inhibitory Effect of Isoflavones on Prostate Cancer Cells and PTEN Gene

Objective To explore the mechanisms by which genistein and daidzein inhibit the growth of prostate cancer cells. Methods LNCaP and PC-3 cells were exposed to genistein and daldzein and cell viability was determined by MTF assay and cytotoxicity of the drugs by LDH test. Flow cytometry （FCM） was used to assess the cell cycle in LNCaP and PC-3 cells. Reverse transcription-polymerase chain reaction （RT-PCR） was applied to examine the expression of PTEN gene （a tumor suppressor gene）, estrogen receptor alpha gene （ERα）, estrogen receptor beta gene （ERβ）, androgen receptor gene （AR） and vascular endothelial growth factor gene （VEGF）. Results The viability of PC-3 and LNCaP cells decreased with increasing concentrations and exposure time of genistein and daidzein. Genistein increased G2/M phase cells in PC 3 cells while decreased S phase cells in LNCaP cells in a dose-dependent manner. Daidzein exerted no influence on the cell cycle of LNCaP and PC-3 cells, but the apoptosis percentage of LNCaP cells was elevated significantly by daidzein, Genistein induced the expression of PTEN gene in PC-3 and LNCaP cells. Daidzein induced the expression of PTEN gene in LNCaP but not in PC-3 cells. The expression of VEGF, ERα and ERβ genes decreased and AR gene was not expressed after incubation with genistein and daldzein in PC-3 cells. In LNCaP cells, the expression of VEGF and AR gene decreased but there was no change in the expression of ERα and ERβ gene after incubation with genistein and daidzein. Conclusion Genistein and daidzein exert a timeand dose-dependent inhibitory effect on PC-3 and LNCaP cells. The down-regulation of ER gene by daidzein influences the growth of PC-3 ceils directly, The inhibition of PC-3 cells by genistein and that of LNCaP cells by genistein and daidzein may be via Alct pathway that is repressed by PTEN gene, which subsequently down-regulates the expression of AR and VEGF genes. Our results suggest that the expression of PTEN gene plays a key role and several pathways may be involved in the suppression of prostate cancer cells by genistein and daidzein.

Bioinformatic identification of key candidate genes and pathways in axon regeneration after spinal cord injury in zebrafish

Zebrafish and human genomes are highly homologous;however,despite this genomic similarity,adult zebrafish can achieve neuronal proliferation,regeneration and functional restoration within 6–8 weeks after spinal cord injury,whereas humans cannot.To analyze differentially expressed zebrafish genes between axon-regenerated neurons and axon-non-regenerated neurons after spinal cord injury,and to explore the key genes and pathways of axonal regeneration after spinal cord injury,microarray GSE56842 was analyzed using the online tool,GEO2R,in the Gene Expression Omnibus database.Gene ontology and protein-protein interaction networks were used to analyze the identified differentially expressed genes.Finally,we screened for genes and pathways that may play a role in spinal cord injury repair in zebrafish and mammals.A total of 636 differentially expressed genes were obtained,including 255 up-regulated and 381 down-regulated differentially expressed genes in axon-regenerated neurons.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment results were also obtained.A protein-protein interaction network contained 480 node genes and 1976 node connections.We also obtained the 10 hub genes with the highest correlation and the two modules with the highest score.The results showed that spectrin may promote axonal regeneration after spinal cord injury in zebrafish.Transforming growth factor beta signaling may inhibit repair after spinal cord injury in zebrafish.Focal adhesion or tight junctions may play an important role in the migration and proliferation of some cells,such as Schwann cells or neural progenitor cells,after spinal cord injury in zebrafish.Bioinformatic analysis identified key candidate genes and pathways in axonal regeneration after spinal cord injury in zebrafish,providing targets for treatment of spinal cord injury in mammals.

Bioinformatics analyses of differentially expressed genes associated with spinal cord injury:a microarray-based analysis in a mouse model

Gene spectrum analysis has shown that gene expression and signaling pathways change dramatically after spinal cord injury,which may affect the microenvironment of the damaged site.Microarray analysis provides a new opportunity for investigating diagnosis,treatment,and prognosis of spinal cord injury.However,differentially expressed genes are not consistent among studies,and many key genes and signaling pathways have not yet been accurately studied.GSE5296 was retrieved from the Gene Expression Omnibus DataSet.Differentially expressed genes were obtained using R/Bioconductor software(expression changed at least two-fold;P < 0.05).Database for Annotation,Visualization and Integrated Discovery was used for functional annotation of differentially expressed genes and Animal Transcription Factor Database for predicting potential transcription factors.The resulting transcription regulatory protein interaction network was mapped to screen representative genes and investigate their diagnostic and therapeutic value for disease.In total,this study identified 109 genes that were upregulated and 30 that were downregulated at 0.5,4,and 24 hours,and 3,7,and 28 days after spinal cord injury.The number of downregulated genes was smaller than the number of upregulated genes at each time point.Database for Annotation,Visualization and Integrated Discovery analysis found that many inflammation-related pathways were upregulated in injured spinal cord.Additionally,expression levels of these inflammation-related genes were maintained for at least 28 days.Moreover,399 regulation modes and 77 nodes were shown in the protein-protein interaction network of upregulated differentially expressed genes.Among the 10 upregulated differentially expressed genes with the highest degrees of distribution,six genes were transcription factors.Among these transcription factors,ATF3 showed the greatest change.ATF3 was upregulated within 30 minutes,and its expression levels remained high at28 days after spinal cord injury.These key genes screened by bioinformatics tools can be used as biological markers to diagnose diseases and provide a reference for identifying therapeutic targets.

Axon regeneration induced by environmental enrichment-epigenetic mechanisms

Environmental enrichment is known to be beneficial for cognitive improvement.In many animal models of neurological disorders and brain injury,EE has also demonstrated neuroprotective benefits in neurodegenerative diseases and in improving recovery after stroke or traumatic brain injury.The exact underlying mechanism for these phenomena has been unclear.Recent findings have now indicated that neuronal activity elicited by environmental enrichment induces Ca2+influx in dorsal root ganglion neurons results in lasting enhancement of CREB-binding protein-mediated histone acetylation.This,in turn,increases the expression of pro-regeneration genes and promotes axonal regeneration.This mechanism associated with neuronal activity elicited by environmental enrichment-mediated pathway is one of several epigenetic mechanisms which modulate axon regeneration upon injury that has recently come to light.The other prominent mechanisms,albeit not yet directly associated with environmental enrichment,include DNA methylation/demethylation and N6-methyladenosine modification of transcripts.In this brief review,I highlight recent work that has shed light on the epigenetic basis of environmental enrichment-based axon regeneration,and discuss the mechanism and pathways involved.I further speculate on the implications of the findings,in conjunction with the other epigenetic mechanisms,that could be harness to promote axon regeneration upon injury.

Molecular Cloning and Bioinformatics Analysis of sucC Gene of Vibrio alginolyticus Strain HY9901

[Objectives]To clone the sucC gene of Vibrio alginolyticus strain HY9901 and conduct the bioinformatics analysis.[Methods]Based on the sucC gene of V.alginolyticus strain HY9901,specific primers were designed to amplify the full length sequence by PCR and make further analysis.[Results]The theoretical molecular weight of SucC protein was about 41528.45 Da,and the full length was 1167 bp,encoding 388 amino acids.It has no signal peptide and transmembrane region,and has a variety of functional sites.It is predicted that it is mainly located in the cytoplasm,and the ubiquitin and lactate modification sites overlap,and it has high gene homology with Vibrio parahaemolyticus.Theα-helix,random coil and extended strand are the main secondary structures.The similarity between the constructed three-level structure model and the template is high.[Conclusions]This study reveals the structural characteristics and functional potential of SucC protein,and provides a theoretical basis for the study of drug resistance mechanism and prevention strategies.

喉鳞状细胞癌中Skp2、PTEN及p27蛋白的表达及其意义

目的探讨Skp2、PTEN及p27蛋白表达与喉鳞状细胞癌(LSCC)发生、发展的关系。方法采用免疫组化SABC法检测65例LSCC组织中Skp2、PTEN及p27蛋白的表达情况。结果Skp2、PTEN及p27蛋白在LSCC组织中总的阳性表达率分别为60.0%,49.2%和41.5%,Skp2过表达与LSCC的临床分期及淋巴结转移正相关(P均<0.05),低分化、伴颈部淋巴结转移及Ⅲ～Ⅳ期LSCC组织中PTEN阳性率明显降低(P均<0.05),p27在颈部淋巴结转移组中阳性率显著降低(P<0.05)。结论联合检测Skp2、PTEN及p27的表达有助于综合评估LSCC的生物学行为。

抑癌基因PTEN、一氧化氮合酶与胃癌的关系及临床意义

目的检测抑癌基因PTEN编码蛋白、iNOS在正常胃粘膜及胃癌组织中的表达情况,探讨PTEN、iNOS与胃癌发生发展的关系及作用机理。方法采用免疫组织化学S-P法进行检测。结果10例正常胃粘膜均可见PTEN蛋白的强表达,40例胃癌组织中28例PTEN蛋白表达较正常胃粘膜明显下降或缺失,差异有显著性(P<0.01),与胃癌的分化程度、浸润深度、TNM分期、淋巴结转移显著相关(P<0.05)。iNOS在胃癌组织中的表达显著高于正常胃粘膜组,(72.5%vs10%,P<0.01),与胃癌的浸润深度、淋巴结转移、TNM分期显著相关(P<0.05)。在PTEN蛋白阴性的胃癌组中,iNOS阳性率为93.8%,显著高于PTEN阳性组(P<0.05)。结论抑癌基因PTEN编码蛋白在胃癌及胃癌发展过程中表达逐渐下降或缺失,与iNOS之间可能存在相互作用,协同参与了胃癌的发展、转移及浸润。PTEN蛋白表达可作为判定胃癌生物学行为的临床参考指标。

PTEN和VEGF在非小细胞肺癌中的表达及相关性研究

目的探讨PTEN和VEGF基因在非小细胞肺癌中的表达及两者的相互关系。方法应用免疫组织化学法检测65例非小细胞肺癌组织及18例肺良性组织中PTEN和VEGF基因的表达。结果PTEN和VEGF基因在非小细胞肺癌组织中表达显著高于肺良性组织,PTEN基因表达与肺癌患者的细胞分化程度、PTNM分期、淋巴结转移密切相关(P<0.05)。VEGF基因表达与肺癌患者的肿瘤大小、细胞分化程度、PTNM分期、淋巴结转移密切相关(P<0.05)。PTEN与VEGF基因呈明显负相关(P<0.05)。结论PTEN和VEGF与非小细胞肺癌的临床特征和生物学行为密切相关,二者的检测将有助于评估肺癌患者的恶性程度和预后。

乳腺癌发生过程中p53、BRCA1、BRCA2、PTEN、Rb蛋白异常表达

目的探讨p53、BRCA1、BRCA2、PTEN、Rb基因蛋白异常表达在乳腺癌发生中的作用。方法选取同时存在浸润癌、导管内癌、不典型增生和单纯性增生的乳腺癌档案蜡块,应用免疫组化SP法检测p53、BRCA1、BRCA2、PTEN、Rb基因蛋白在各例的异常表达。结果(1)在24.3%(17/70)的乳腺癌及其癌旁不典型增生中检测到突变型p53蛋白表达,分别在2.9%(2/69)、6.3%(4/63)、5.1%(3/59)、5.4%(3/56)的乳腺癌及其癌旁不典型增生中分别检测到BRCA1、BRCA2、PTEN、Rb表达缺失。(2)在44.6%的病例同时检测到2种基因蛋白异常表达,在5.4%的病例同时检测到3种基因蛋白异常表达,在3.6%的病例同时检测到4种基因蛋白异常表达。结论(1)p53、BRCA1、BRCA2、PTEN、Rb蛋白异常表达可出现于乳腺癌发生的早期阶段,可能在乳腺癌发生过程中起作用。(2)多种抑癌基因的失活共同参与了乳腺癌的发生。

Expression of vascular endothelial growth factor and its role in oncogenesis of human gastric carcinoma

AIM: To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly. METHODS: The expression of VEGF and its receptor kinase-domain insert containing receptor (KDR) in human gastric cancer tissue were observed by immunohistochemical staining. VEGF levels were manipulated in human gastric cancer cell using eukaryotic expression constructs designed to express the complete VEGF(165) complimentary DNA in either the sense or antisense orientation. The biological changes of the cells were observed in which VEGF was up-regulated or down-regulated. RESULTS: VEGF-positive rate was 50%, and VEGF was mainly localized in the cytoplasm and membrane of the tumor cells, while KDR was mainly located in the membrane of vascular endothelial cells in gastric cancer tissues and peri-cancerous tissue. In 2 cases of 50 specimens, the gastric cancer cells expressed KDR, localized in both the cytoplasm and membrane. Introduction of VEGF(165) antisense into human gastric cancer cells (SGC-7901, immunofluorescence intensity, 31.6%)) resulted in a significant reduction in VEGF-specific messenger RNA and total and cell surface VEGF protein (immunofluorescence intensity, 8.9%) (P【0.05). Conversely, stable integration of VEGF(165) in the sense orientation resulted in an increase in cellular and cell surface VEGF (immunofluorescence intensity, 75.4%) (P【0.05). Lowered VEGF levels were associated with a marked decrease in the growth of nude mouse xenografted tumor (at 33 days postimplantation, tumor volume: 345.40 +/- 136.31 mm3)(P【0.05 vs control SGC-7901 group: 1534.40 +/- 362.88 mm3), whereas up-regulation of VEGF resulted in increased xenografted tumor size (at 33 days postimplantation, tumor volume: 2350.50 +/- 637.70 mm3) (P【0.05 vs control SGC-7901 group). CONCLUSION: This study provides direct evidence that VEGF plays an important role in the oncogenesis of human gastric cancer.

Adeno-associated virus mediated endostatin gene therapy in combination with topoisomerase inhibitor effectively controls liver tumor in mouse model

AIM:rAAV mediated endostatin gene therapy has been examined as a new method for treating cancer.However, a sustained and high protein delivery is required to achieve the desired therapeutic effects.We evaluated the impact of topoisomerase inhibitors in rAAV delivered endostatin gene therapy in a liver tumor model. METHODS:rAAV containing endostatin expression cassettes were transduced into hepatoma cell lines.To test whether the topoisomerase inhibitor pretreatment increased the expression of endostatin,Western blotting and ELISA were performed.The biologic activity of endostatin was confirmed by endothelial cell proliferation and tube formation assays. The anti-tumor effects of the rAAV-endostatin vector combined with a topoisomerase inhibitor,etoposide,were evaluated in a mouse liver tumor model. RESULTS:Topoisomerase inhibitors,including camptothecin and etoposide,were found to increase the endostatin exPression level in vitro.The over-expressed endostatin, as a result of pretreatment with a topoisomerase inhibitor, was also biologically active.In animal experiments,the combined therapy of topoisomerase inhibitor,etoposide with the rAAV-endostatin vector had the best tumor- suppressive effect and tumor foci were barely observed in livers of the treated mice.Pretreatment with an etoposide increased the level of endostatin in the liver and serum of rAAV-endostatin treated mice.Finally,the mice treated With rAAV-endostatin in combination with etoposide showed the longest survival among the experimental models. CONCLUSION:rAAV delivered endostatin gene therapy in combination with a topoisomerase inhibitor pretreatment is an effective modality for anticancer gene therapy.

Screening biomarkers for spinal cord injury using weighted gene co-expression network analysis and machine learning

Immune changes and inflammatory responses have been identified as central events in the pathological process of spinal co rd injury.They can greatly affect nerve regeneration and functional recovery.However,there is still limited understanding of the peripheral immune inflammato ry response in spinal cord inju ry.In this study.we obtained microRNA expression profiles from the peripheral blood of patients with spinal co rd injury using high-throughput sequencing.We also obtained the mRNA expression profile of spinal cord injury patients from the Gene Expression Omnibus(GEO)database(GSE151371).We identified 54 differentially expressed microRNAs and 1656 diffe rentially expressed genes using bioinformatics approaches.Functional enrichment analysis revealed that various common immune and inflammation-related signaling pathways,such as neutrophil extracellular trap formation pathway,T cell receptor signaling pathway,and nuclear factor-κB signal pathway,we re abnormally activated or inhibited in spinal cord inju ry patient samples.We applied an integrated strategy that combines weighted gene co-expression network analysis,LASSO logistic regression,and SVM-RFE algorithm and identified three biomarke rs associated with spinal cord injury:ANO10,BST1,and ZFP36L2.We verified the expression levels and diagnostic perfo rmance of these three genes in the original training dataset and clinical samples through the receiver operating characteristic curve.Quantitative polymerase chain reaction results showed that ANO20 and BST1 mRNA levels were increased and ZFP36L2 mRNA was decreased in the peripheral blood of spinal cord injury patients.We also constructed a small RNA-mRNA interaction network using Cytoscape.Additionally,we evaluated the proportion of 22 types of immune cells in the peripheral blood of spinal co rd injury patients using the CIBERSORT tool.The proportions of naive B cells,plasma cells,monocytes,and neutrophils were increased while the proportions of memory B cells,CD8^(+)T cells,resting natural killer cells,resting dendritic cells,and eosinophils were markedly decreased in spinal cord injury patients increased compared with healthy subjects,and ANO10,BST1 and ZFP26L2we re closely related to the proportion of certain immune cell types.The findings from this study provide new directions for the development of treatment strategies related to immune inflammation in spinal co rd inju ry and suggest that ANO10,BST2,and ZFP36L2 are potential biomarkers for spinal cord injury.The study was registe red in the Chinese Clinical Trial Registry(registration No.ChiCTR2200066985,December 12,2022).

Reversal of Multidrug Resistance and Inhibition of Phosphorylation of AKT in Human Ovarian Cancer Cell Line by Wild-type PTEN Gene

The reversing effect of wild-type PTEN gene on resistance of C 13K cells to cisplatin and its inhibitory effect on the phosphorylation of protein kinase B （AKT） were studied. The expression of PTEN mRNA and protein in OV2008 cells and C13K cells were semi-quantitatively detected by using RT-PCR and Western blotting. Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine2000. The expression of PTEN mRNA was monitored by RT-PCR and the expression of PTEN, Akt, p-Akt protein were ana- lyzed by Western blotting in PTEN-transfected and non-transfected C13K cells. Proliferation and chemosensitivity of cells to DDP were measured by MTT, and cell apoptosis was detected by flow cytometry after treatment with cisplatin. The expression of PTEN mRNA and protein in OV2008 cells were significantly higher than those in C13K cells. After transfection with PTEN gene for 48 h, the expression of PTEN mRNA and protein in C 13K cells were 2.04 ± 0.10, 0.94± 0.04 respectively and the expression of p-Akt protein （ 0.94± 0.07） was lower than those in control groups （1.68 ±0.14, 1.66± 0.10） （P〈 0.05）. The IC50 of DDP to C 13 K cells transfected with PTEN （7.2± 0.3 la mol/L） was obviously lower than those of empty-vector transfected cells and non-transfected cells （12.7±0.4 lamol/1, 13.0±0.3 lamol/L） （P〈0.05）. The apopototis ratio of wild-type PTEN-transfected, empty vector transfected and non-transfected C13K cells were （41.65___0.87）%, （18.61 ±0.70）% and （15.28±0.80）% respectively, and the difference was statistically significant （P〈0.05）. PTEN gene plays an important role in ovarian cancer multidrug resistance. Transfection of PTEN could increase the expression of PTEN and restore drug sensitivity to cisplatin in human ovarian cancer cell line C 13K with multidrug-resistance by decreasing the expression of p-Akt.

Characteristics and advantages of adeno-associated virus vector-mediated gene therapy for neurodegenerative diseases

Common neurodegenerative diseases of the central nervous system are characterized by progressive damage to the function of neurons, even leading to the permanent loss of function. Gene therapy via gene replacement or gene correction provides the potential for transformative therapies to delay or possibly stop further progression of the neurodegenerative disease in affected patients. Adeno-associated virus has been the vector of choice in recent clinical trials of therapies for neurodegenerative diseases due to its safety and efficiency in mediating gene transfer to the central nervous system. This review aims to discuss and summarize the progress and clinical applications of adeno-associated virus in neurodegenerative disease in central nervous system. Results from some clinical trials and successful cases of central neurodegenerative diseases deserve further study and exploration.

Analysis of the autophagy gene expression profile of pancreatic cancer based on autophagy-related protein microtubule-associated protein 1A/1B-light chain 3

BACKGROUND Pancreatic cancer is a highly invasive malignant tumor. Expression levels of the autophagy-related protein microtubule-associated protein 1 A/1 B-light chain 3(LC3) and perineural invasion(PNI) are closely related to its occurrence and development. Our previous results showed that the high expression of LC3 was positively correlated with PNI in the patients with pancreatic cancer. In this study, we further searched for differential genes involved in autophagy of pancreatic cancer by gene expression profiling and analyzed their biological functions in pancreatic cancer, which provides a theoretical basis for elucidating the pathophysiological mechanism of autophagy in pancreatic cancer and PNI.AIM To identify differentially expressed genes involved in pancreatic cancer autophagy and explore the pathogenesis at the molecular level.METHODS Two sets of gene expression profiles of pancreatic cancer/normal tissue(GSE16515 and GSE15471) were collected from the Gene Expression Omnibus.Significance analysis of microarrays algorithm was used to screen differentially expressed genes related to pancreatic cancer. Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis were used to analyze the functional enrichment of the differentially expressed genes. Protein interaction data containing only differentially expressed genes was downloaded from String database and screened. Module mining was carried out by Cytoscape software and ClusterOne plug-in. The interaction relationship between the modules was analyzed and the pivot nodes between the functional modules were determined according to the information of the functional modules and the data of reliable protein interaction network.RESULTS Based on the above two data sets of pancreatic tissue total gene expression, 6098 and 12928 differentially expressed genes were obtained by analysis of genes with higher phenotypic correlation. After extracting the intersection of the two differential gene sets, 4870 genes were determined. GO analysis showed that 14 significant functional items including negative regulation of protein ubiquitination were closely related to autophagy. A total of 986 differentially expressed genes were enriched in these functional items. After eliminating the autophagy related genes of human cancer cells which had been defined, 347 differentially expressed genes were obtained. KEGG pathway analysis showed that the pathways hsa04144 and hsa04020 were related to autophagy. In addition,65 clustering modules were screened after the protein interaction network was constructed based on String database, and module 32 contains the LC3 gene,which interacts with multiple autophagy-related genes. Moreover, ubiquitin C acts as a pivot node in functional modules to connect multiple modules related to pancreatic cancer and autophagy.CONCLUSION Three hundred and forty-seven genes associated with autophagy in human pancreatic cancer were concentrated, and a key gene ubiquitin C which is closely related to the occurrence of PNI was determined, suggesting that LC3 may influence the PNI and prognosis of pancreatic cancer through ubiquitin C.

The concept of gene therapy for glaucoma:the dream that has not come true yet

Gene therapies,despite of being a relatively new therapeutic approach,have a potential to become an important alternative to current treatment strategies in glaucoma.Since glaucoma is not considered a single gene disease,the identified goals of gene therapy would be rather to provide neuroprotection of retinal ganglion cells,especially,in intraocular-pressure-independent manner.The most commonly reported type of vector for gene delivery in glaucoma studies is adeno-associated virus serotype 2 that has a high tro pism to retinal ganglion cells,res ulting in long-term expression and low immunogenic profile.The gene thera py studies recruit inducible and genetic animal models of optic neuropathy,like DBA/2J mice model of high-tension glaucoma and the optic nerve crush-model.Reported gene therapy-based neuroprotection of retinal ganglion cells is targeting specific genes translating to growth factors(i.e.,brain derived neurotrophic factor,and its receptor TrkB),regulation of apoptosis and neurodegeneration(i.e.,Bcl-xl,Xiap,FAS system,nicotinamide mononucleotide adenylyl transferase 2,Digit3 and Sarm1),immunomodulation(i.e.,Crry,C3 complement),modulation of neuroinflammation(i.e.,e rythropoietin),reduction of excitotoxicity(i.e.,Com KIlα)and transcription regulation(i.e.,Max,Nrf2).On the other hand,some of gene therapy studies focus on lowering intra ocular pressure,by impacting genes involved in both,decreasing aqueous humor production(i.e.,aquaporin 1),and increasing outflow facility(i.e.,COX2,prostaglandin F2a receptor,RhoA/RhoA kinase signaling pathway,MMP1,Myocilin).The goal of this review is to summarize the current stateof-art and the direction of development of gene therapy strategies for glaucomatous neuropathy.

Expression of E-Cadherin and the PTEN Gene in Relation to Invasion and Metastasis of Gastric Carcinomas

OBJECTIVE To observe the expression of PTEN and E-Cadherin in gastric carcinomas (GCs), and to investigate the relationship between their expression and the pathology and prognosis of patients with GC.METHODS The proposed markers were detected inmmunohistochemically by using the SABC method in 100 post-operated specimens of GC. The results were statically analyzed by the chi-square and log rank tests.RESULTS Both E-Cadherin and PTEN proteins were expressed in noncancerous mucosa. They were reduced or lost in GCs. The abnormal rate of expression of E-Cadherin was 42.0%. The decreased rate of expression in the diffuse-type GC (48.6%) was significantly higher than in the intestinaltype GC (26.7%, P<0.05). The abnormal expression of E-Cadherin closely correlated to the depth of invasion (P<0.05). The degree of loss of the PTEN protein was 59.0% in GCs. In the diffuse-type GC, the rate of loss of PTEN was (65.7%) which was significantly higher than that in the intestinal-type GC (43.3%, P<0.05). The rate of loss of PTEN (64.5%) in GCs with lymph node metastasis was significantly higher than that in GCs without metastasis(41.7%, P<0.05). The prognosis of patients with a loss of PTEN protein was worse than the patients with positive expression of PTEN (P=0.0066). E-Cadherin was normally expressed in 65.9% of GCs with positive expression of PTEN.CONCLUSION The loss of E-Cadherin and PTEN markers correlated with infusion and metastasis of GC. The expression of PTEN showed a close relationship to the prognosis of patients. Detection of the 2 markers together aided in the correct prediction of the prognosis of the GC patients and provided information for clinical treatment.

Exogenous PTEN Gene Induces Apoptosis in Breast Carcinoma Cell Line MDA468

The effects and mechanisms of exogenous phosphatase and tensin homolog deleted from chromosome ten （PTEN） gene on phosphatase activity-dependent apoptosis of breast cancer cell line MDA468 were investigated. PTEN gene packaged with lipofectin was transferred into breast cancer cell line MDA468 and parental MDA468 cells served as controls. RT-PCR and Western blot were done to detect the expression of target genes. The expression of phosphospecific protein kinase B （PKB/Akt） and focal adhesion kinase （FAK） protein stimulated by epidermal growth factor （EGF） was also detected. Apoptosis was determined by flow cytometry with a double-staining method using FITC-conjugated annexin V and PI. MDA468 cells transfected with PTEN gene could express PTEN mRNA and protein. PTEN decreased the phosphorylation level of AKT protein and down-regulated FAK protein expression in MDA468 stimulated by EGE The apoptosis rate was 21.68%. PTEN induced breast cancer apoptosis phosphatase activity-dependently, The mechanism is possibly related with phosphoinositide 3-kinase （PI3K）/protein kinase B （PKB）/AKT signaling pathway. Those results may provide new clues on the gene therapy in breast cancer.