Recombinant expression and purification of functional vascular endothelial growth factor-121 in the baculovirus expression system

Objective: To express human Vascular endothelial growth factor121(VEGF121) in insect cells. Methods: A gene construct containing VEGF was cloned in the p Fast Bac-HTA vector, followed by transformation in DH10 BAC. The recombinant bacmid was then extracted, and transfected into Sf9 insect cells. The transfected cells were harvested, and then VEGF expression was confirmed by Western blotting using specific antibodies. The tube formation assay was used for functional assessment of VEGF. Results: Our results showed that VEGF could be successfully expressed in the baculovirus system. Purified VEGF was able to stimulate in vitro tube formation of human endothelial cells. Conclusions: Results from this study demonstrated that the recombinantly-produced VEGF can be considered as a promising candidate for therapeutic purposes.

Stable Surface Expression of a Gene for Helicobacter pylori Toxic Porin Protein with pBAD Expression System

Helicobacterpylori （1f. pylori） infection causes peptic and duodenal diseases in humans. Among a 32-protein family of outer membrane proteins, a porin-like protein, HopE, has been a subject of note, mainly for its conservative nature among 11. pylori, and for its potential as a vaccine candidate. To achieve stable surface expression of this host cell-toxic protein, hopE gene was introduced into pBAD expression system. After induction with arabinose, all 15 randomly-chosen E. coli LMG 194 colonies from 3 successive passages could express Hope protein, while only 1 from 5 E. coli colonies that contained lac operon-regulated plasmid encoding hopE gene could express HopE. Indirect immunofluorescence confirmed the expression of HopE on E. coli cell surface

Establishment of a tetracycline-off and heat shock-on gene expression system in tobacco

The tetracycline （Tet）-off gene expression regulation system based on the TetR-VP16/Top10 construct has not been widely utilized in plants, for its highly expressed TetR-VP16 activator is toxic to some plants and repeatedly replenishing tetracycline to turn off the constitutively active system is a tedious process. To solve these problems, a Tet-off and heat shock （HS）-on gene expression regulation system was constructed in this study. This system is composed of a chimeric transactivator gene TetR-HSF that is derived from a Tet repressor （TetR） and a HS transcription factor （HSF） controlled by a HS promoter HSP70m, and a Tet operator containing hybrid promoter, Om35S, that drives expression of the β-glucuronidase （GUS） gene. The resultant system yields a GUS expression pattern similar to that of the HSP70m promoter under inducing temperatures and at 35 and 40℃ drives GUS expression to a similar level as the Cauliflower mosaic virus （CaMV） 35S promoter. Further examination revealed that the TetR-HSF and GUS genes were induced by HS, reaching peak expression after 1 and 6 h treatment, respectively, and the HS induction of the expression system could be inhibited by Tet. This system will provide a useful tool for transgenic studies of plants in the laboratory and in the field, including transgene function analysis, agronomic trait improvement, biopharmaceutical protein production and others.

Construction of Multi-ribozyme Expression System and Its Characterization of Cleavage on the MDR1/MRP1 Double Target Substrate in vitro

To improve catalytic activity of ribozyme on its substrate, the multi-ribozyme expression system was designed and constructed from 20 cis-acting hammerhead ribozymes undergoing self-cleavage with 10 trans-acting hammerhead ribozymes inserted alternatively regularly and the plasmid of pGEM-MDR1/MRP1 used to transcribe the MDR1/MRPl（196/210） substrate containing double target sites was also constructed by DNA recombination. Endonuclease digestion analysis and DNA sequencing indicate all the recombinant plasmids were correct. The clea- vage activities were evaluated for the multi-ribozyme expression system on the MDR1/MRP1 substrate in the cell free system. The results demonstrate that the cis-acting hammerhead ribozymes in the multi-ribozyme expression system were able to cleave themselves and the 72 nt of 196Rz and the 71 nt of 210Rz trans-acting hammerhead ribozymes were liberated effectively, and the trans-acting hammerhead ribozymes released were able to act on the MDR1/MRP1 double target RNA substrate and cleave the target RNA at specific sites effectively. The multi- ribozyme expression system of the [Coat＇A196Rz/Coat＇B210Rz]5 is more significantly superior to that of the [Coat＇A 196Rz/Coat＇B210Rz] 1 in cleavage of RNA substrate. The fractions cleaved by [Coat＇A 196Rz/Coat＇B210Rz]5 on the MDR1/MRP1 substrate for 8 h at observed temperatures showed no marked difference. The studies of Mg^2＋ on cleavage efficiency indicate that cleavage reaction is dependent on Mg^2＋ ions concentration. The plot of lg（kobs） vs. lgc（Mg^2＋） displays a linear relationship between 2.5 mmol/L and 20 mmol/L Mg^2＋. It suggests that Mg^2＋ ions play a crucial role in multi-ribozyme cleavage on the substrate.

A Transient Expression System for the Functional Assessment of Early Response Genes on the Powdery Mildew Infected Barley or Wheat Leaves

The principle and the basic steps of the transient assay system for the functional assessment of early response genes on the powdery mildew infected barley or wheat leaves were summarized in brief. The development of this technology and its extensive application were reviewed. Future studies on this approach were recommended in this paper.

Construction and Cleavage Characterization of Single Targeted Multi-ribozyme Expression Systems in vitro

In order to clarify cleavage efficiency of a multi-ribozyme expression system on their substrates,plasmids containing 20 cis-acting hammerhead ribozymes for self-cleavage and 10 trans-acting hammerhead ribozymes targeted on MDR1 and MRP1 substrates,the target plasmids pGEM-MDR1 and pGEM-MRP1 were constructed by employing PCR and DNA recombination.The endonuclease＇s digestion and DNA sequencing confirmed exactness of the multi-ribozyme expression systems and the target plasmids.The repeated self-cleavage tests revealed that the cisacting hammerhead ribozymes in the multi-ribozyme expression system were able to cleave themselves and trans-acting hammerhead ribozymes 196 Rz and 210 Rz were released.The cleavage efficiencies of liberated ribozymes on their targets were evaluated in vitro and the results indicate that 196 Rz and 210 Rz were able to cleave the MDR1 and MRP1 RNA substrates.The profile of cleavage reaction shows that the cleavage efficiencies were correlated with the numbers of transacting hammerhead ribozymes contained in the multi-ribozyme expression systems,as well as the multi-ribozyme system is much better than the single ribozyme.Test of various concentrations of Mg^2＋ reveals that ribozyme cleavage reactions were dependent on Mg^2＋ concentration.The plot of lg（kobs） vs.lg[Mg^2＋] displays a linear relationship in the concentration range observed（2.5―20 mmol/L）.

Strategies for production of active eukaryotic proteins in bacterial expression system

Bacteria have long been the favorite expression system for recombinant protein production.However,the flaw of the system is that insoluble and inactive proteins are co-produced due to codon bias,protein folding,phosphorylation,glycosylation,mRNA stability and promoter strength.Factors are cited and the methods to convert to soluble and active proteins are described,for example a tiglit control of Escherichia coli milieu,refolding from inclusion body and through fusion technology.

An improved protein expression system for T3SS genes regulation analysis in Xanthomonas oryzae pv. oryzae

Xanthomonas oryzea pv.oryzae(Xoo)is the causal agent of bacterial blight of rice,which is a significant threat to many of rice-growing regions.The type Ⅲ secretion system(T3SS)is an essential virulence factor in Xoo.Expression of the T3SS is often induced in the host environment or in hrp-inducing medium but is repressed in nutrient-rich medium.The elucidation of molecular mechanism underlying induction of T3SS genes expression is a very important step to lift the veil on global virulence regulation network in Xoo.Thus,an efficient and reliable genetic tool system is required for detection of the T3SS proteins.In this study,we constructed a protein expression vector pH3-flag based on the backbone of pHM1,a most widely used vector in Xoo strains,especially a model strain PXO99A.This vector contains a synthesized MCS-FLAG cassette that consists of a multiple cloning site(MCS),containing a modified pUC18 polylinker,and Flag as a C-terminal tag.The cassette is flanked by transcriptional terminators to eliminate interference of external transcription enabling detection of accurate protein expression.We evaluated the potential of this expression vector as T3SS proteins detection system and demonstrated it is applicable in the study of T3SS genes expression regulation in Xoo.This improved expression system could be very effectively used as a molecular tool in understanding some virulence genes expression and regulation in Xoo and other Xanthomonas spp.

Cloning of Porcine Lactoferrin Gene and Construction of Expression System in Recombinant Lactobacillus

Lactobacillus was selected as a bacterial carrier for expression of N-lobe of porcine lactoferrin （PLFN）. A pair of primers was designed with Oligo6.0 and used to amplify PLFN gene. It was in accordance with the characters of translational fusions from gene and expression vector plasmid. A 1 077 bp fragment of the gene from PLF was cloned from mammary gland tissue of the lactating sow on the third day by RT-PCR; the gene was connected with the vector plasmid pPG612.1 and transformed into the host strain JM109. The recombinant expression vector plasmid pPG612-PLFN was created and identified by using plasmid extraction, PCR, restriction enzyme digestion and sequence analysis. The recombinant plasmid was transformed into Lactobacillus casei ATCC393, Lactobacillus plantarum KLDS 1.0344, Lactobacillus paracasei KLDS 1.0652 and Lactobacillus pentosus KLDS 1.0413 by electroporation, and produced the recombinant strains of pPG612-PLFN/L, casei, pPG612-PLFN/L, plantarum, pPG612-PLFN/ L. paracasei and pPG612-PLFN/L, pentosus, respectively. The results indicated that PLFN gene had inserted into the expression vectors and achieved multiple Laetobacillus expression systems. It electes the base for the expression and production of recombinant porcine lactoferrin in Lactobaeillus

Genome-wide identification and expression profiling of photosystem II(PsbX)gene family in upland cotton(Gossypium hirsutum L.)

Background Photosystem II(PSII)constitutes an intricate assembly of protein pigments,featuring extrinsic and intrinsic polypeptides within the photosynthetic membrane.The low-molecular-weight transmembrane protein PsbX has been identified in PSII,which is associated with the oxygen-evolving complex.The expression of PsbX gene protein is regulated by light.PsbX’s central role involves the regulation of PSII,facilitating the binding of quinone molecules to the Qb(PsbA)site,and it additionally plays a crucial role in optimizing the efficiency of photosynthesis.Despite these insights,a comprehensive understanding of the PsbX gene’s functions has remained elusive.Results In this study,we identified ten PsbX genes in Gossypium hirsutum L.The phylogenetic analysis results showed that 40 genes from nine species were classified into one clade.The resulting sequence logos exhibited substantial conservation across the N and C terminals at multiple sites among all Gossypium species.Furthermore,the ortholo-gous/paralogous,Ka/Ks ratio revealed that cotton PsbX genes subjected to positive as well as purifying selection pressure might lead to limited divergence,which resulted in the whole genome and segmental duplication.The expression patterns of GhPsbX genes exhibited variations across specific tissues,as indicated by the analysis.Moreover,the expression of GhPsbX genes could potentially be regulated in response to salt,intense light,and drought stresses.Therefore,GhPsbX genes may play a significant role in the modulation of photosynthesis under adverse abiotic conditions.Conclusion We examined the structure and function of PsbX gene family very first by using comparative genom-ics and systems biology approaches in cotton.It seems that PsbX gene family plays a vital role during the growth and development of cotton under stress conditions.Collectively,the results of this study provide basic information to unveil the molecular and physiological function of PsbX genes of cotton plants.

Development of an efficient expression system with large cargo capacity for interrogation of gene function in bamboo based on bamboo mosaic virus

Bamboo is one of the fastest growing plants among monocotyledonous species and is grown extensively in subtropical regions.Although bamboo has high economic value and produces much biomass quickly,gene functional research is hindered by the low efficiency of genetic transformation in this species.We therefore explored the potential of a bamboo mosaic virus(BaMV)-mediated expression system to investigate genotype-phenotype associations.We determined that the sites between the triple gene block proteins(TGBps)and the coat protein(CP)of BaMV are the most efficient insertion sites for the expression of exogenous genes in both monopodial and sympodial bamboo species.Moreover,we validated this system by individually overexpressing the two endogenous genes ACE1 and DEC1,which resulted in the promotion and suppression of intemode elongation,respectively.In particular,this system was able to drive the expression of three 2A-linked betalain biosynthesis genes(more than 4 kb in length)to produce betalain,indicating that it has high cargo capacity and may provide the prerequisite basis for the development of a DNA-free bamboo genome editing platform in the future.Since BaMV can infect multiple bamboo species,we anticipate that the system described in this study will greatly contribute to gene function research and further promote the molecular breeding of bamboo.

Astrocytic endothelin-1 overexpression impairs learning and memory ability in ischemic stroke via altered hippocampal neurogenesis and lipid metabolism

Vascular etiology is the second most prevalent cause of cognitive impairment globally.Endothelin-1,which is produced and secreted by endothelial cells and astrocytes,is implicated in the pathogenesis of stroke.However,the way in which changes in astrocytic endothelin-1 lead to poststroke cognitive deficits following transient middle cerebral artery occlusion is not well understood.Here,using mice in which astrocytic endothelin-1 was overexpressed,we found that the selective overexpression of endothelin-1 by astrocytic cells led to ischemic stroke-related dementia(1 hour of ischemia;7 days,28 days,or 3 months of reperfusion).We also revealed that astrocytic endothelin-1 overexpression contributed to the role of neural stem cell proliferation but impaired neurogenesis in the dentate gyrus of the hippocampus after middle cerebral artery occlusion.Comprehensive proteome profiles and western blot analysis confirmed that levels of glial fibrillary acidic protein and peroxiredoxin 6,which were differentially expressed in the brain,were significantly increased in mice with astrocytic endothelin-1 overexpression in comparison with wild-type mice 28 days after ischemic stroke.Moreover,the levels of the enriched differentially expressed proteins were closely related to lipid metabolism,as indicated by Kyoto Encyclopedia of Genes and Genomes pathway analysis.Liquid chromatography-mass spectrometry nontargeted metabolite profiling of brain tissues showed that astrocytic endothelin-1 overexpression altered lipid metabolism products such as glycerol phosphatidylcholine,sphingomyelin,and phosphatidic acid.Overall,this study demonstrates that astrocytic endothelin-1 overexpression can impair hippocampal neurogenesis and that it is correlated with lipid metabolism in poststroke cognitive dysfunction.

Human-Computer Interaction Using Deep Fusion Model-Based Facial Expression Recognition System

A deep fusion model is proposed for facial expression-based human-computer Interaction system.Initially,image preprocessing,i.e.,the extraction of the facial region from the input image is utilized.Thereafter,the extraction of more discriminative and distinctive deep learning features is achieved using extracted facial regions.To prevent overfitting,in-depth features of facial images are extracted and assigned to the proposed convolutional neural network(CNN)models.Various CNN models are then trained.Finally,the performance of each CNN model is fused to obtain the final decision for the seven basic classes of facial expressions,i.e.,fear,disgust,anger,surprise,sadness,happiness,neutral.For experimental purposes,three benchmark datasets,i.e.,SFEW,CK+,and KDEF are utilized.The performance of the proposed systemis compared with some state-of-the-artmethods concerning each dataset.Extensive performance analysis reveals that the proposed system outperforms the competitive methods in terms of various performance metrics.Finally,the proposed deep fusion model is being utilized to control a music player using the recognized emotions of the users.

Effects of Manganese on the Antioxidant System and Related Gene Expression Levels in the “Hong Yang” Kiwifruit Seedlings

To explore how manganese affects the antioxidant system and the expression levels of related genes of“Hong yang”seedlings,the leaves of its tissue cultured seedlings were taken as test materials,and single factor treatment was performed by changing the manganese chloride(MnCl_(2)·4H_(2)O)solution concentration when spraying the leaves.The expression levels of Mn-SOD,POD64 and POD27 genes in leaves were quantitatively analyzed by real-time quantitative PCR(qRT-PCR)at different determination times.Meanwhile,the contents of malondial-dehyde(MDA),hydrogen peroxide(H_(2)O_(2)),the activities of antioxidant enzymes,including catalase(CAT),peroxidase(POD),and superoxide dismutase(SOD).The results showed that the SOD,CAT,POD,ascorbate peroxidase(APX),and reduced glutathione(GSH)activities in leaves were the highest at 12 h post-treatment with 50μM MnCl_(2)·4H_(2)O.Furthermore,the contents of MDA and H_(2)O_(2) in leaves also peaked when the concentration of H_(2)O_(2) is 50μM,which is the minimum value.Additionally at 50μM Mn^(2+),the Mn-SOD and POD27 expression was up-regulated as compared to the control,which promoted the expression of their respective enzyme activities.However,POD64 expression increased with the increasing Mn^(2+) concentration.Therefore,50μM is the optimal concentration of Mn when exogenously applied on“Hong yang”,which improve the antioxidant enzyme activity and regulate the plant’s physiological and biochemical functions.

Effects of pyraclostrobin on growth,oxidative stress,and gene expression in relation to stress and ATP-binding cassette transporters in Tetrahymena thermophila

Pyraclostrobin(PYR),a widely used fungicide,has negative effects on fish and algae,but its toxicity in protozoa remains unclear.In this study,the effects of PYR on the growth,oxidative stress,and gene expression related to stress and ATP-binding cassette(ABC)transporters in Tetrahymena thermophila were investigated.The result showed that the 96-h IC_(50)of PYR against T.thermophila was 17.2 mg/L.Moreover,PYR inhibited the growth of T.thermophila in concentration-or time-dependent manner.A morphological study revealed that the shape and size of T.thermophila changed,and damage of cell membrane surface was observed by scanning electron microscopy after 96 h of PYR exposure.The activities of superoxide dismutase(SOD)and catalase(CAT)increased throughout the experiment.In contrast,the glutathione(GSH)content was increased at 24 h and 48 h of exposure and decreased at 96 h.Moreover,a significant increase in malondialdehyde(MDA)level was observed in T.thermophila after96 h of exposure.Furthermore,PYR upregulated the HSP703,HSP705,GPx2,and ABAC15 gene expression in the 0.1–5-mg/L groups and downregulated the HSP704,HSP90,TGR,and ABCC52 mRNA levels at 96 h of exposure.These results suggest that PYR may exert adverse effects on T.thermophila by inducing oxidative stress and changing the gene expression related to ABC transporters and stress,which may enrich the understanding of the toxicity mechanism of PYR in aquatic organisms and provide reference data for aquatic ecological risk assessments.

Expression of cyclin-dependent kinase 9 is positively correlated with the autophagy level in colon cancer

BACKGROUND Cyclin-dependent kinase 9(CDK9)expression and autophagy in colorectal cancer(CRC)tissues has not been widely studied.CDK9,a key regulator of transcription,may influence the occurrence and progression of CRC.The expression of auto-phagy-related genes BECN1 and drug resistance factor ABCG2 may also play a role in CRC.Under normal physiological conditions,autophagy can inhibit tumorigenesis,but once a tumor forms,autophagy may promote tumor growth.Therefore,understanding the relationship between autophagy and cancer,partic-ularly how autophagy promotes tumor growth after its formation,is a key motivation for this research.AIM To investigate the relationship between CDK9 expression and autophagy in CRC,assess differences in autophagy between left and right colon cancer,and analyze the associations of autophagy-related genes with clinical features and prognosis.METHODS We collected tumor tissues and paracarcinoma tissues from colon cancer patients with liver metastasis to observe the level of autophagy in tissues with high levels of CDK9 and low levels of CDK9.We also collected primary tissue from left and right colon cancer patients with liver metastasis to compare the autophagy levels and the expression of BECN1 and ABCG2 in the tumor and paracarcinoma tissues.RESULTS The incidence of autophagy and the expression of BECN1 and ABCG2 were different in left and right colon cancer,and autophagy might be involved in the occurrence of chemotherapy resistance.Further analysis of the rela-tionship between the expression of autophagy-related genes CDK9,ABCG2,and BECN1 and the clinical features and prognosis of colorectal cancer showed that the high expression of CDK9 indicated a poor prognosis in colorectal cancer.CONCLUSION This study laid the foundation for further research on the combination of CDK9 inhibitors and autophagy inhibitors in the treatment of patients with CRC.

High patatin like phospholipase domain containing 8 expression as a biomarker for poor prognosis of colorectal cancer

BACKGROUND Patatin like phospholipase domain containing 8(PNPLA8)has been shown to play a significant role in various cancer entities.Previous studies have focused on its roles as an antioxidant and in lipid peroxidation.However,the role of PNPLA8 in colorectal cancer(CRC)progression is unclear.AIM To explore the prognostic effects of PNPLA8 expression in CRC.METHODS A retrospective cohort containing 751 consecutive CRC patients was enrolled.PNPLA8 expression in tumor samples was evaluated by immunohistochemistry staining and semi-quantitated with immunoreactive scores.CRC patients were divided into high and low PNPLA8 expression groups based on the cut-off va-lues,which were calculated by X-tile software.The prognostic value of PNPLA8 was identified using univariate and multivariate Cox regression analysis.The over-all survival(OS)rates of CRC patients in the study cohort were compared with Kaplan-Meier analysis and Log-rank test.RESULTS PNPLA8 expression was significantly associated with distant metastases in our cohort(P=0.048).CRC patients with high PNPLA8 expression indicated poor OS(median OS=35.3,P=0.005).CRC patients with a higher PNPLA8 expression at either stage I and II or stage III and IV had statistically significant shorter OS.For patients with left-sided colon and rectal cancer,the survival curves of two PN-PLA8-expression groups showed statistically significant differences.Multivariate analysis also confirmed that high PNPLA8 expression was an independent prog-nostic factor for overall survival(hazard ratio HR=1.328,95%CI:1.016-1.734,P=0.038).

A Novel Deep Learning-Based Model for Classification of Wheat Gene Expression

Deep learning(DL)plays a critical role in processing and converting data into knowledge and decisions.DL technologies have been applied in a variety of applications,including image,video,and genome sequence analysis.In deep learning the most widely utilized architecture is Convolutional Neural Networks(CNN)are taught discriminatory traits in a supervised environment.In comparison to other classic neural networks,CNN makes use of a limited number of artificial neurons,therefore it is ideal for the recognition and processing of wheat gene sequences.Wheat is an essential crop of cereals for people around the world.Wheat Genotypes identification has an impact on the possible development of many countries in the agricultural sector.In quantitative genetics prediction of genetic values is a central issue.Wheat is an allohexaploid(AABBDD)with three distinct genomes.The sizes of the wheat genome are quite large compared to many other kinds and the availability of a diversity of genetic knowledge and normal structure at breeding lines of wheat,Therefore,genome sequence approaches based on techniques of Artificial Intelligence(AI)are necessary.This paper focuses on using the Wheat genome sequence will assist wheat producers in making better use of their genetic resources and managing genetic variation in their breeding program,as well as propose a novel model based on deep learning for offering a fundamental overview of genomic prediction theory and current constraints.In this paper,the hyperparameters of the network are optimized in the CNN to decrease the requirement for manual search and enhance network performance using a new proposed model built on an optimization algorithm and Convolutional Neural Networks(CNN).

Effect of Chuanzhifang component (ZGC) on macrophage inflammatory injury based on whole gene expression profile

Objective: The effect of Chuanzhi Fang (ZGC) on the whole genome expression profile of RAW264.7 cells activated by lipopolysaccharide (LPS) was analyzed, and to explore the possible mechanism of action and core target of this formula on macrophage inflammatory injury at the overall level. Methods: A model of LPS-induced inflammation in RAW264.7 cells was constructed, and the effect of ZGC intervention on the genome-wide expression of inflammatory macrophages 3was examined by gene microarray technology, GO/KEGG enrichment analysis was performed for significantly differentially expressed genes among each group. Results: The results of genome-wide expression profiling microarray analysis showed that the ZGC intervention group upregulated the expression of 5 genes including C4bp and inhibited the expression of 22 genes including Mgat3, Psma6, and Siglecg relative to the LPS model group. KEGG signaling pathway analysis results showed that ZGC mainly acted through cytokine receptor interaction and the C-type lectin receptor signaling pathway. Conclusion: ZGC can interfere with the abnormal expression of 27 genes in inflammatory macrophages, and the related genes may exert corresponding anti-inflammatory effects by affecting cytokine receptor interactions, C-type lectin receptor signaling pathway, and TLR4/ NF-κB signaling pathway.

Decoding Retinoblastoma: Differential Gene Expression

Background: Retinoblastoma, the most common intraocular pediatric cancer, presents complexities in its genetic landscape that necessitate a deeper understanding for improved therapeutic interventions. This study leverages computational tools to dissect the differential gene expression profiles in retinoblastoma. Methods: Employing an in silico approach, we analyzed gene expression data from public repositories by applying rigorous statistical models, including limma and de seq 2, for identifying differentially expressed genes DEGs. Our findings were validated through cross-referencing with independent datasets and existing literature. We further employed functional annotation and pathway analysis to elucidate the biological significance of these DEGs. Results: Our computational analysis confirmed the dysregulation of key retinoblastoma-associated genes. In comparison to normal retinal tissue, RB1 exhibited a 2.5-fold increase in expression (adjusted p Conclusions: Our analysis reinforces the critical genetic alterations known in retinoblastoma and unveils new avenues for research into the disease’s molecular basis. The discovery of chemoresistance markers and immune-related genes opens potential pathways for personalized treatment strategies. The study’s outcomes emphasize the power of in silico analyses in unraveling complex cancer genomics.