Biology subjects

Cao, J.

Publications and source records attributed to Cao, J..

At least 181 records · Page 10Linked to original sources

bioRxiv2020

A DNAmRep epigenetic fingerprint for determining cellular replication age

ii.Aging is known to elicit dramatic changes to DNA methylation (DNAm), although the causes and consequences of such alterations are not clear and require further exploration. Our ability to experimentally uncover mechanisms of epigenetic aging will be greatly enhanced by our ability to study and manipulate these changes using in vitro models. However, it remains unclear whether the changes elicited by cells in culture can serve as a model of what is observed in aging tissues in vivo. To test this, we serially passaged mouse embryonic fibroblasts (MEFs) and assessed changes in DNAm at each time-point via RRBS. By developing a measure that tracked cellular aging in vitro, we tested whether it tracked physiological aging in various mouse tissues and whether anti-aging interventions modulate this measure. Our measure, termed DNAmCULTURE, was shown to strongly increase with age when examined in multiple tissues (liver, lung, kidney, blood, and adipose). As a control, we confirmed that the measure was not a marker of cellular senescence, suggesting that it reflects a distinct yet progressive cellular aging phenomena that can be induced in vitro. Furthermore, we demonstrated slower epigenetic aging in animals undergoing caloric restriction and a resetting of our measure in lung and kidney fibroblasts when re-programmed to iPSCs. Enrichment and clustering analysis implicated SUZ12, EED and Polycomb group (PcG) factors as potentially important chromatin regulators in translational culture aging phenotypes. Overall, this study supports the concept that physiologically relevant aging changes can be induced in vitro and moving forward, used to uncover mechanistic insights into epigenetic aging.

pathology↗

bioRxiv2020

Nanopore sequencing reveals full-length Tropomyosin 1 isoforms and their regulation by RNA binding proteins during rat heart development

Alternative splicing (AS) contributes to the diversity of the proteome by producing multiple isoforms from a single gene. Although short-read RNA sequencing methods have been the gold standard for determining AS patterns of genes, they have a difficulty in defining full length mRNA isoforms assembled using different exon combinations. Tropomyosin 1 (TPM1) is an actin binding protein required for cytoskeletal functions in non-muscle cells and for contraction in muscle cells. Tpm1 undergoes AS regulation to generate muscle versus non-muscle TPM1 protein isoforms with distinct physiological functions. It is unclear which full length Tpm1 isoforms are produced via AS and how they are regulated during heart development. To address these, we utilized nanopore long-read cDNA sequencing without gene-specific PCR amplification. In rat hearts, we identified full length Tpm1 isoforms composed of distinct exons with specific exon linkages. We showed that Tpm1 undergoes AS transitions during embryonic heart development such that muscle-specific exons are connected together generating predominantly muscle specific Tpm1 isoforms in adult hearts. We found that the RNA binding protein RBFOX2 controls AS of rat Tpm1 exon 6a, which is important for cooperative actin binding. Furthermore, RBFOX2 regulates Tpm1 AS of exon 6a antagonistically to the RNA binding protein PTBP1. In sum, we defined full length Tpm1 isoforms with different exon combinations that are tightly regulated during cardiac development and provided insights into regulation of Tpm1 AS by RNA binding proteins. Our results demonstrate that nanopore sequencing is an excellent tool to determine fulllength AS variants of muscle enriched genes.

molecular biology↗

bioRxiv2020

Screening of Botanical Drugs Against Lassa Virus Entry

Lassa virus (LASV) belongs to the Old World Mammarenavirus genus (family Arenaviridae) and is classified as a category A biological threat agent. At present, there are no approved drugs or vaccines specific for LASV. In this study, high-throughput screening of a botanical drug library was performed against LASV entry using a pseudotype virus bearing the LASV envelope glycoprotein (GPC). Two hit compounds, bergamottin and casticin, were identified as LASV entry inhibitors in the micromolar range. A mechanistic study revealed that casticin inhibited LASV entry by blocking low pH-induced membrane fusion. Adaptive mutant analyses demonstrated that the F446L mutation, located in the transmembrane domain of GP2, conferred resistance to casticin. Furthermore, casticin extended its antiviral spectrum to the New World (NW) pathogenic mammarenaviruses, and mutation of the conserved F446 conferred NW resistance to casticin. Unlike casticin, bergamottin has little effect on LASV GPC-mediated membrane fusion, while it inhibited LASV entry by blocking endocytic trafficking. Our study shows that both bergamottin and casticin are candidates for LASV therapy, indicating that the conserved F446 plays important roles in drug resistance in mammarenaviruses. IMPORTANCECurrently, there is no approved therapy to treat Lassa fever (LASF); we aimed to find candidates for LASF therapy. Herein, we screened a botanical drug library and identified two compounds, bergamottin and casticin, that inhibited LASV entry via different mechanisms.

microbiology↗

bioRxiv2020

Virus-free and live-cell visualizing SARS-CoV-2 cell entry for studies of neutralizing antibodies and compound inhibitors

The ongoing COVID-19 pandemic, caused by SARS-CoV-2 infection, has resulted in hundreds of thousands of deaths. Cellular entry of SARS-CoV-2, which is mediated by the viral spike protein and host ACE2 receptor, is an essential target for the development of vaccines, therapeutic antibodies, and drugs. Using a mammalian cell expression system, we generated a recombinant fluorescent protein (Gamillus)-fused SARS-CoV-2 spike trimer (STG) to probe the viral entry process. In ACE2-expressing cells, we found that the STG probe has excellent performance in the live-cell visualization of receptor binding, cellular uptake, and intracellular trafficking of SARS-CoV-2 under virus-free conditions. The new system allows quantitative analyses of the inhibition potentials and detailed influence of COVID-19-convalescent human plasmas, neutralizing antibodies and compounds, providing a versatile tool for high-throughput screening and phenotypic characterization of SARS-CoV-2 entry inhibitors. This approach may also be adapted to develop a viral entry visualization system for other viruses.

microbiology↗

bioRxiv2020

FER-mediated tyrosine phosphorylation and PIK3R2/p85β recruitment on IRS4 promotes the PI3K-AKT signaling pathway and tumorigenesis in ovarian cancer

Tyrosine phosphorylation, orchestrated by tyrosine kinases and phosphatases, modulates a multi-layered signaling network in a time and space dependent manner. Dysregulation of this post-translational modification is inevitably associated with pathological diseases. Our previous work has demonstrated that non-receptor tyrosine kinase FER is upregulated in ovarian cancer. Knockdown of the kinase attenuates metastatic phenotypes in tumor cells. Here we employed mass spectrometry and biochemical approaches to identify IRS4 as a novel substrate of FER. Using a proximity-based tagging system, we determined that FER-mediated phosphorylation of Tyr779 enables IRS4 to recruit PIK3R2/p85{beta}, the regulatory subunit of PI-3K, and activate the PI3K-AKT pathway. Rescuing IRS4-null ovarian tumor cells with phosphorylation-defective mutant, but not WT IRS4, delayed tumor cell proliferation both in vitro and in vivo. Overall, we revealed a kinase-substrate regulatory mode between FER and IRS4, and the pharmacological inhibition of FER kinase may be beneficial for ovarian cancer patients with PI3K-AKT hyperactivation.

molecular biology↗

bioRxiv2020

A tropane-based ibogaine analog rescues folding-deficient SERT and DAT

Missense mutations that give rise to protein misfolding are rare, but collectively, defective protein folding diseases are consequential. Folding deficiencies are amenable to pharmacological correction (pharmacochaperoning), but the underlying mechanisms remain enigmatic. Ibogaine and its active metabolite noribogaine correct folding defects in the dopamine transporter (DAT), but they rescue only a very limited number of folding-deficient DAT mutants, which give rise to infantile Parkinsonism and dystonia. Herein, a series of analogs was generated by reconfiguring the complex ibogaine ring system and exploring the structural requirements for binding to wild type transporters, and for rescuing two equivalent synthetic folding-deficient mutants, SERT-PG601,602AA and DAT-PG584,585AA. The most active tropane-based analog (9b) was also an effective pharmacochaperone in vivo, in Drosophila harboring DAT-PG584,585AA and rescued six out of 13 disease-associated human DAT mutants in vitro. Hence, a novel lead pharmacochaperone has been identified that demonstrates medication development potential for patients harboring DAT mutants.

pharmacology and toxicology↗

bioRxiv2020

fruitless tunes functional flexibility of courtship circuitry during development

Drosophila male courtship is controlled by the male-specific products of the fruitless (fruM gene and its expressing neuronal circuitry. fruM is considered a master gene that controls all aspects of male courtship. By temporally and spatially manipulating fruM expression, we found that fruM is required during a critical developmental period for innate courtship towards females, and its function during adulthood is relatively trivial. By altering or eliminating fruM expression, we generated males that are innately heterosexual, homosexual, bisexual, or without innate courtship but could acquire such behavior in an experience-dependent manner. These findings show that fruM is not absolutely necessary for courtship but is critical during development to build a sex circuitry with reduced flexibility and enhanced efficiency and provide a new view about how fruM tunes functional flexibility of a sex circuitry.

neuroscience↗

bioRxiv2020

Electron microscopic observation of infected CCB and whole genome analysis of Koi herpesvirus isolate GY01

BackgroundCyprinid herpesvirus- 3 (CyHV-3), commonly known as Koi herpesvirus (KHV) can induce infectious and acute viremia in common/koi carp (Cyprinus carpio). In an earlier study in this laboratory a KHV isolate GY1506 (KHV GY01 in GeneBank) was isolated from diseased common carp, replicated on CCB cells and identified by PCR targeted on and phylogenetic analysis of thymidine kinase (TK) gene. Electron microscopy examination of GY01 infected CCB cell line and whole genome analysis was studied for further characteristics and epidemiological features of this strain. ResultsElectron microscopy examination of CCB infected with KHV GY01 strain revealed destruct of infected cells including incomplete nuclear membranes, deformed nucleus, marginalized nuclear chromosome, and the virus of different development stages and morphologies in the cytoplasm and nucleus, which resembles the herpesvirus. MEGA X and phyML were used for multiple alignment and phylogenetic analysis of whole genomes of GY01 and other 21 KHV strains available in GeneBank. analysis showed that GY01 was more close to E and KHV-I and was predicted it originated from the same ancestor as the E and KHV-I. Pairwise alignment of strain GY01 and strain E by Geneious software and YASS online version revealed that two strains had high identity(99.1%) at the nucleotide sequence level although variations and disagreement existed. The number and structure arrangement of open reading frames (orfs) or protein-encoding genes of GY01 is very similar to KHV E, and also to KHV-U but different from KHV-I. The characteristics and function of each orf need further study in the future. ConclusionsPathogenic changes of infected CCB cells and morphologies of KHV GY01 resembles the herpesvirus. Pairwise, multiple alignment and phylogenetic analysis of whole genomes of GY01 and other 21 KHV strains available in GeneBank demonstrated that the GY01 is closely related to strain KHV E and KHV-I and suggested it originated from the same ancestor as the E and KHV-I.

microbiology↗

bioRxiv2020

Microbubble-Mediated Cavitation Promotes Apoptosis and Suppresses Invasion in AsPC-1 Cells

This study aimed to identify the potential and mechanisms of microbubble-mediated cavitation in promoting apoptosis and suppressing invasion in cancer cells. AsPC-1 cells were used and divided into four groups: control group, microbubble-only (MB) group, ultrasound-only (US) group, and ultrasound combined with microbubbles (US+MB) group. Pulse ultrasound was used with a frequency of 360 kHz and an intensity of 0.7 W/cm2 for 1 min (duty rate=50%). Then cells in four groups were cultured for 24 h. Cell counting kit-8 illustrated that US+MB could decrease cell viability. Western blot confirmed that US+MB increased cleaved Caspase-3, Bcl-2-associated X protein (BAX), and decreased the B-cell lymphoma-2 (Bcl-2) levels. Besides, US+MB increased intracellular calcium ions and down-regulated cleaved Caspase-8. For proliferation ability, cells in US+MB group had a lower expression of Ki67 and the weakened colony formation ability. The transwell invasion assay showed that US+MB could decrease invasion ability in AsPC-1 cells. Further evidence showed that cells conducted with US+MB had the lower level of hypoxia-inducible factor-1 (HIF-1) and Vimentin and the higher expression of E-cadherin than the other three groups. Finally, cells conducted with US+MB had less invadopodia formation. In conclusion, these results suggested that microbubble-mediated cavitation promoted apoptosis and suppressed invasion in AsPC-1 cells. SignificanceCancer cells have a high rate of cell division and a high rate of cell division can speed up the development of hypoxia which lead to promote cancer invasion and metastasis. Here we used microbubble-mediated cavitation to generate a mechanical shock wave which could effectively kill cancer cells through physical damage and intrinsic signaling pathway. Furthermore, effective killing of cancer cells could restrain the development of hypoxia and prevent invasion ability of cancer cells.

bioengineering↗

bioRxiv2020

RBFOX2 Is Critical For Maintaining Alternative Polyadenylation And Mitochondrial Health In Myoblasts

RBFOX2, which has a well-established role in alternative splicing, is linked to heart diseases. However, it is unclear whether RBFOX2 has other roles in RNA processing that can influence gene expression/function in muscle cells, contributing to disease pathology. Here, we employed both 3-end and nanopore cDNA sequencing to reveal a previously unrecognized role for RBFOX2 in maintaining alternative polyadenylation (APA) signatures in myoblasts. We found that RBFOX2-mediated APA modulates both mRNA levels and isoform expression of a collection of genes including contractile and mitochondrial genes. We identified the key muscle-specific contractile gene, Tropomyosin 1 and essential mitochondrial gene, Slc25a4 as APA targets of RBFOX2. Unexpectedly, depletion of RBFOX2 adversely affected mitochondrial health in myoblasts that is in part mediated by disrupted APA of mitochondrial gene Slc25a4. Mechanistically, we found that RBFOX2 regulation of Slc25a4 APA is mediated through consensus RBFOX2 binding motifs near the distal polyadenylation site enforcing the use of the proximal polyadenylation site. In sum, our results unveiled a new role for RBFOX2 in fine tuning expression levels of mitochondrial and contractile genes via APA in myoblasts relevant to heart diseases.

molecular biology↗

bioRxiv2020

The mechanics of Temporal Interference stimulation

We utilize single neuron models to understand mechanisms behind Temporal Interference (TI) stimulation (also called "Interferential Stimulation"). We say that a neuron exhibits TI stimulation if it does not fire for a high-frequency sinusoidal input, but fires when the input is a low-frequency modulation of the high-frequency sinusoid (specifically that generated by addition of two high frequency sinusoids with a small difference in their frequencies), while the maximum amplitude is kept the same in both cases. Our key observation - that holds for both FitzHugh-Nagumo and Hodgkin-Huxley neuron models - is that for neuron models that do exhibit TI stimulation, a high frequency pure sinusoidal input results in a current balance between inward and outward currents. This current balance leads to a subthreshold periodic orbit that keeps the membrane potential from spiking for sinusoidal inputs. However, the balance is disturbed when the envelope of the sinusoids is modulated with a high slope: the fast-changing envelope activates fast depolarizing currents without giving slow outward currents time to respond. This imbalance causes the membrane potential to build up, causing the neuron to fire. This mechanistic understanding can help design current waveforms for neurons that exhibit TI stimulation, and also help classify which neuron-types may or may not exhibit TI stimulation.

neuroscience↗

bioRxiv2020

Horseshoe crab genomes reveal the evolutionary fates of genes and microRNAs after three rounds (3R) of whole genome duplication

Whole genome duplication (WGD) has occurred in relatively few sexually reproducing invertebrates. Consequently, the WGD that occurred in the common ancestor of horseshoe crabs ~135 million years ago provides a rare opportunity to decipher the evolutionary consequences of a duplicated invertebrate genome. Here, we present a high-quality genome assembly for the mangrove horseshoe crab Carcinoscorpius rotundicauda (1.7Gb, N50 = 90.2Mb, with 89.8% sequences anchored to 16 pseudomolecules, 2n = 32), and a resequenced genome of the tri-spine horseshoe crab Tachypleus tridentatus (1.7Gb, N50 = 109.7Mb). Analyses of gene families, microRNAs, and synteny show that horseshoe crabs have undergone three rounds (3R) of WGD, and that these WGD events are shared with spiders. Comparison of the genomes of C. rotundicauda and T. tridentatus populations from several geographic locations further elucidates the diverse fates of both coding and noncoding genes. Together, the present study represents a cornerstone for a better understanding of the consequences of invertebrate WGD events on evolutionary fates of genes and microRNAs at individual and population levels, and highlights the genetic diversity with practical values for breeding programs and conservation of horseshoe crabs.

evolutionary biology↗

bioRxiv2020

A Large-scale Drug Repositioning Survey for SARS-CoV-2 Antivirals

The emergence of novel SARS coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19). To date, more than 2.1 million confirmed cases and 139,500 deaths have been reported worldwide, and there are currently no medical countermeasures available to prevent or treat the disease. As the development of a vaccine could require at least 12-18 months, and the typical timeline from hit finding to drug registration of an antiviral is >10 years, repositioning of known drugs can significantly accelerate the development and deployment of therapies for COVID-19. To identify therapeutics that can be repurposed as SARS-CoV-2 antivirals, we profiled a library of known drugs encompassing approximately 12,000 clinical-stage or FDA-approved small molecules. Here, we report the identification of 30 known drugs that inhibit viral replication. Of these, six were characterized for cellular dose-activity relationships, and showed effective concentrations likely to be commensurate with therapeutic doses in patients. These include the PIKfyve kinase inhibitor Apilimod, cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825, and ONO 5334, and the CCR1 antagonist MLN-3897. Since many of these molecules have advanced into the clinic, the known pharmacological and human safety profiles of these compounds will accelerate their preclinical and clinical evaluation for COVID-19 treatment.

microbiology↗

bioRxiv2020

Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells

The global pandemic of Coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable in vitro neutralization assay is very important for the development of neutralizing antibodies, vaccines and other inhibitors. In this study, G protein-deficient vesicular stomatitis virus (VSVdG) bearing full-length and truncated spike (S) protein of SARS-CoV-2 were evaluated. The virus packaging efficiency of VSV-SARS-CoV-2-Sdel18 (S with C-terminal 18 amino acid truncation) is much higher than VSV-SARS-CoV-2-S. A neutralization assay for antibody screening and serum neutralizing titer quantification was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and human angiotensin-converting enzyme 2 (ACE2) overexpressed BHK21 cell (BHK21-hACE2). The experimental results can be obtained by automatically counting EGFP positive cell number at 12 hours after infection, making the assay convenient and high-throughput. The serum neutralizing titer of COVID-19 convalescent patients measured by VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with live SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting receptor binding domain (RBD) of SARS-CoV-2-S were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines.

microbiology↗

bioRxiv2020

C Terminus of DJ-1 Determines Its Homodimerization, Deglycation Activity and Suppression of Ferroptosis

DJ-1 is a multi-functional protein related to cancer and autosomal early-onset Parkinson disease (PD). Besides the well-documented antioxidative stress activity, recent studies suggest that DJ-1 has the deglycation enzymatic activity and the anti-ferroptosis function. Although it has been demonstrated that DJ-1 forms the homodimerization, which dictates its antioxidative stress activity, the relationship between the dimeric structure and newly reported activities remains largely elusive. In this study, we find that the deletion mutation of the last 3 amino acids at C terminus of DJ-1 disrupts its homodimerization in both transfected and purified DJ-1 protein. Further study shows that hydrophobic L187 residue is of great importance for DJ-1 homodimerization. In addition, the ability in methylglyoxal detoxification is almost abolished in the mutation of deleting last 3 residues at C terminus ({Delta}C3) and point mutant L187E compared with wild type DJ-1 (DJ-1 WT). We also find that the suppression of ferroptosis is fully inhibited by {Delta}C3 and L187E while partially suppressed by V51C. Thus, our findings show that C terminus of DJ-1 is crucial for its homodimerization, deglycation activity and suppression of ferroptosis.

molecular biology↗

bioRxiv2020

High-Throughput 5' UTR Engineering for Enhanced Protein Production in Non-Viral Gene Therapies

Despite significant clinical progress in cell and gene therapies, maximizing protein expression in order to enhance potency remains a major challenge. One approach to increase protein expression is by optimizing translation through the engineering of 5 untranslated regions (5 UTRs). Here, we developed a high-throughput strategy to design, screen, and optimize novel 5UTRs that enhance protein expression from a strong human cytomegalovirus (CMV) promoter. We first identified naturally occurring 5 UTRs with high translation efficiencies and used this information with in silico genetic algorithms to generate synthetic 5 UTRs. A total of [~]12,000 5 UTRs were then screened using a recombinase-mediated integration strategy that greatly enhances the sensitivity of high-throughput screens by eliminating copy number and position effects that limit lentiviral approaches. Using this approach, we identified three synthetic 5 UTRs that outperformed commonly used non-viral gene therapy plasmids in expressing protein payloads. Furthermore, combinatorial assembly of these 5 UTRs enabled even higher protein expression than obtained with each individual 5 UTR. In summary, we demonstrate that high-throughput screening of 5 UTR libraries with recombinase-mediated integration can identify genetic elements that enhance protein expression, which should have numerous applications for engineered cell and gene therapies.

synthetic biology↗

bioRxiv2020

Potentiating the anti-tumor response of tumor infiltrated T cells by NAD+ supplementation

Tumor immunotherapies have provided clinical benefits, yet great potential remains for optimizing therapeutic effects. Here, we show that low NAD+ levels restrict the function of tumor infiltrating T lymphocytes (TILs). TILs harvested from human ovarian tumor tissues showed decreased NAD+ levels compared with T cells from paired peripheral blood samples. The combination of whole-genome CRISPR and large-scale metabolic inhibitor screens implicated the NAD+ biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT) is required for T cell activation. Further isotopic labeling and LC-MS studies confirmed that NAD+ depletion suppressed mitochondrial energy biosynthesis in T cells. Excitingly, NAD+ supplementation significantly enhanced the tumor cell-killing efficacy of CAR-T cells ex vivo, and extended animal survive in both adoptive CAR-T model and immune checkpoint blockade treatment models in vivo. This study demonstrates an over-the-counter nutrient supplement NAD+ could robustly boost the efficacy of T cell-based immunotherapy and provides insights into the cellular basis of T cell metabolic reprogramming in treating cancers. One Sentence SummaryNAD+ supplementation during cancer immunotherapies significantly enhances T cell activation and tumor killing capacity.

immunology↗

bioRxiv2020

Comprehensive analysis of prognostic immune-related genes in the tumor microenvironment of lung adenocarcinoma

BackgroundLung adenocarcinoma (LUAD) is the most common primary lung cancer, and increasing evidence indicates the clinical importance of the microenvironment in LUAD. Tumor-infiltrating immune cells play an important role in promoting or inhibiting tumor growth. This study aimed to identify immune-related prognostic genes that were associated with the LUAD microenvironment. MethodsWe used the "estimate" R package to calculate the immune/stromal scores of each sample of GSE72094 based on the ESTIMATE algorithm. Then we looked up relationships between patients characteristics and immune/stromal scores. After that, we divided the samples into two groups: high and low scores, identified the common differentially expressed genes (DEGs), and performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) on the common DEGs. After conducting the overall survival analysis of the common DEGs, prognostic genes were harvested. Then we constructed the protein-protein interaction network and performed the enrichment of GO and KEGG for the prognostic genes. Crucial prognostic genes were obtained after validating in two independent data sources (GSE68465 and TIMER). Finally, we investigated the immune correlates of the crucial prognostic genes based on the TIMER. ResultsImmune scores did not vary with gender, age, smoking history, tumor stage, and EGFR status, but vary with the status of KRAS, STK11, and TP53. For the stromal scores, only the status of STK11 and TP53 mattered. Reduced immune score predicted poor prognosis of LUAD. 357 common DEGs were found, of which 108 were identified as prognostic genes after overall survival analysis. GO and KEGG analysis found that common DEGs and prognostic genes were both mainly involved in immune-related items. After validation in two independent data sources, 12 genes were validated to be crucial prognostic genes linked to prognosis. After investigated the TIMER, all 12 genes were correlated with the main immune cell types. Conclusion12 immune-related prognostic genes were discovered relating to the microenvironment in LUAD. These findings suggest that the composition of the tumor microenvironment affects the clinical outcomes of LUAD, and it may provide a basis for the development of novel prognostic biomarkers and immunotherapy for LUAD.

cancer biology↗