Gene RNA Research Articles

Abstract 3665: Stratifying prostate cancer patients through circulating genes related to prostate cell subtypes, drug targets, and therapeutic resistance

Abstract Stratification remains an obstacle for the optimal treatment of prostate cancer (PCa) patients throughout the treatment trajectory. Targeting the androgen receptor (AR) kills the majority of luminal-like cells, which are AR-positive and express Prostate Specific Antigen (PSA). However, a subset of surviving AR-positive cells develop resistance to treatments while AR-negative cells of the neuroendocrine (NE) or stem-like phenotypes emerge through selection. We reported on the clinical relevance of cell-subtype genes in whole blood RNA of advanced PCa patients. Here, we studied an expanded panel of genes to include drug targets and therapeutic resistance as predictive biomarkers to stratify patients at diagnosis and at the advanced stage of disease.Genes were chosen based on literature review, results of clinical trials in PCa, and PCa transcriptomic data. Technical validation of TaqMan RT-qPCR assays was carried out for each gene, including rigorous testing of reproducibility. Whole blood RNA was extracted from 26 healthy controls with no prostatic disease, 16 patients prior to prostatectomy, and 43 blood samples from 28 metastatic cases. Gene overexpression was defined as the 99.5% confidence interval of expression in controls. Clinical data were retrieved from patients’ charts.A panel of 64 genes was built, showing overexpression in advanced PCa but low or no expression in normal blood (including whole blood, white and red blood cell populations and platelets). Testing in control blood showed no correlation with age. The proportions of patients’ white blood cells did not correlate with gene expression in their blood. Overall, up to 44/64 genes were overexpressed in at least one patient sample. Patients with prostatic intraductal carcinoma at prostatectomy showed more circulating genes, including more NE and stemness genes, and more PCa cell subtypes represented. Intermediate and high-risk patients showed more circulating NE genes. In metastatic patients, signatures of luminal, NE, stemness, and resistance to AR inhibitors or taxanes were associated with progression. PCa-specific luminal genes were associated with shorter overall survival. Treatment resistance genes correlated with lines of treatment and current taxanes. Two targetable NE genes were overexpressed in distinct categories of patients, suggesting that they may benefit from more specific treatments.In conclusion, phenotypic and functional differences in circulating gene patterns of PCa patients correlate with pathological features, treatments, progression. They may be clinically meaningful to stratify patients and predict therapeutic response. Circulating genes encoding drug targets may justify clinical trials to offer personalized treatments and impact on lethal PCa. Citation Format: Seta Derderian, Edouard Jarry, Arynne Santos, Mohanachary Amaravadi, Quentin Vesval, Lucie Hamel, Raphael Sanchez-Salas, Alexis Rompré-Brodeur, Wassim Kassouf, Raghu Rajan, Marie Duclos, Fadi Brimo, Armen Aprikian, Simone Chevalier. Stratifying prostate cancer patients through circulating genes related to prostate cell subtypes, drug targets, and therapeutic resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3665.

An ex vitro hairy root system from petioles of detached soybeanleaves for in planta screening of target genes and CRISPR strategies associated with nematode bioassays.

The ex vitro hairy root system from petioles of detached soybeanleaves allows the functional validation of genes using classical transgenesis and CRISPR strategies (e.g., sgRNA validation, gene activation) associated with nematode bioassays. Agrobacterium rhizogenes-mediated root transformation has been widely used in soybean for the functional validation of target genes in classical transgenesis and single-guide RNA (sgRNA) in CRISPR-based technologies. Initial data showed that in vitro hairy root induction from soybean cotyledons and hypocotyls were not the most suitable strategies for simultaneous performing genetic studies and nematode bioassays. Therefore, an ex vitro hairy root system was developed for in planta screening of target molecules during soybean parasitism by root-knot nematodes (RKNs). Applying this method, hairy roots were successfully induced by A. rhizogenes from petioles of detached soybeanleaves. The soybean GmPR10 and GmGST genes were then constitutively overexpressed in both soybean hairy roots and tobacco plants, showing a reduction in the number of Meloidogyne incognita-induced galls of up to 41% and 39%, respectively. In addition, this system was evaluated for upregulation of the endogenous GmExpA and GmExpLB genes by CRISPR/dCas9, showing high levels of gene activation and reductions in gall number of up to 58.7% and 67.4%, respectively. Furthermore, morphological and histological analyses of the gallsweresuccessfully performed. These collective data validate the ex vitro hairy root system for screening target genes, using classical overexpression and CRISPR approaches, directly in soybean in a simple manner and associated with nematode bioassays. This system can also be used in other root pathosystems for analyses of gene function and studies of parasite interactions with plants, as well as for other purposes such as studies of root biology and promoter characterization.

Differential Expression of Subsets of Genes Related to HDL Metabolism and Atherogenesis in the Peripheral Blood in Coronary Artery Disease.

Differential expression of genes (DEGs) in coronary artery disease (CAD) and the association between transcript level and high-density lipoprotein cholesterol (HDL-C) were studied with 76 male patients with CAD and 63 control patients. The transcript level of genes related to HDL metabolism (24 genes) and atherosclerosis-prone (41 genes) in RNA isolated from peripheral blood mononuclear cells was measured by real-time RT-PCR. Twenty-eight DEGs were identified. The expression of cholesterol transporters, ALB, APOA1, and LCAT was down-regulated, while the expression of AMN, APOE, LDLR, LPL, PLTP, PRKACA, and CETP was up-regulated. The systemic inflammation in CAD is evidenced by the up-regulation of IL1B, TLR8, CXCL5, and TNFRSF1A. For the controls, TLR8 and SOAT1 were negative predictors of the HDL-C level. For CAD patients, PRKACG, PRKCQ, and SREBF1 were positive predictors, while PRKACB, LCAT, and S100A8 were negative predictors. For CAD patients, the efficiency of reverse cholesterol transport is 73-79%, and intracellular free cholesterol seems to accumulate at hyperalphalipoproteinemia. Both atheroprotective (via S100A8) and proatherogenic (via SREBF1, LCAT, PRKACG, PRKACB, and PRKCQ) associations of gene expression with HDL-C determine HDL functionality in CAD patients. The selected key genes and involved pathways may represent HDL-specific targets for the diagnosis and treatment of CAD and atherosclerosis.

Analysis of BRCA1 and BRCA2 alternative splicing in predisposition to ovarian cancer

BackgroundThe mRNA splicing is regulated on multiple levels, resulting in the proper distribution of genes' transcripts in each cell and maintaining cell homeostasis. At the same time, the expression of alternative transcripts can change in response to underlying genetic variants, often missed during routine diagnostics. AimThe main aim of this study was to define the frequency of aberrant splicing in BRCA1 and BRCA2 genes in blood RNA extracted from ovarian cancer patients who were previously found negative for the presence of pathogenic alterations in the 25 most commonly analysed ovarian cancer genes, including BRCA1 and BRCA2. Material and methodsFrequency and spectrum of splicing alterations in BRCA1 and BRCA2 genes were analysed in blood RNA from 101 ovarian cancer patients and healthy controls (80 healthy women) using PCR followed by gel electrophoresis and Sanger sequencing. The expression of splicing events was examined using RT-qPCR. ResultsWe did not identify any novel, potentially pathogenic splicing alterations. Nevertheless, we detected six naturally occurring transcripts, named BRCA1ΔE9-10, BRCA1ΔE11, BRCA1ΔE11q, and BRCA2ΔE3, BRCA2ΔE12 and BRCA2ΔE17-18 of which three (BRCA1ΔE11q, BRCA1ΔE11 and BRCA2ΔE3) were significantly higher expressed in the ovarian cancer cohort than in healthy controls (p ≤ 0.0001). ConclusionsThis observation indicates that the upregulation of selected naturally occurring transcripts can be stimulated by non-genetic mechanisms and be a potential systemic response to disease progression and/or treatment. However, this hypothesis requires further examination.

Multisite validation of a host response signature for predicting likelihood of bacterial and viral infections in patients with suspected influenza.

Indiscriminate use of antimicrobials and antimicrobial resistance is a public health threat. IMX-BVN-1, a 29-host mRNA classifier, provides two separate scores that predict likelihoods of bacterial and viral infections in patients with suspected acute infections. We validated the performance of IMX-BVN-1 in adults attending acute health care settings with suspected influenza. We amplified 29-host response genes in RNA extracted from blood by NanoString nCounter. IMX-BVN-1 calculated two scores to predict probabilities of bacterial and viral infections. Results were compared against the infection status (no infection; highly probable/possible infection; confirmed infection) determined by clinical adjudication. Amongst 602 adult patients (74.9% ED, 16.9% ICU, 8.1% outpatients), 7.6% showed in-hospital mortality and 15.5% immunosuppression. Median IMX-BVN-1 bacterial and viral scores were higher in patients with confirmed bacterial (0.27) and viral (0.62) infections than in those without bacterial (0.08) or viral (0.21) infection, respectively. The AUROC distinguishing bacterial from nonbacterial illness was 0.81 and 0.87 when distinguishing viral from nonviral illness. The bacterial top quartile's positive likelihood ratio (LR) was 4.38 with a rule-in specificity of 88%; the bacterial bottom quartile's negative LR was 0.13 with a rule-out sensitivity of 96%. Similarly, the viral top quartile showed an infinite LR with rule-in specificity of 100%; the viral bottom quartile had a LR of 0.22 and a rule-out sensitivity of 85%. IMX-BVN-1 showed high accuracy for differentiating bacterial and viral infections from noninfectious illness in patients with suspected influenza. Clinical utility of IMX-BVN will be validated following integration into a point of care system.

LncRNA ENST869 Targeting Nestin Transcriptional Region to Affect the Pharmacological Effects of Chidamide in Breast Cancer Cells.

Breast cancer is one of the leading threats to the health of women. It has the highest incidence and mortality in women worldwide. Although progress has been made in the development and application of anti-breast cancer drugs such as Chidamide and others, the occurrence of drug resistance limits the effective application of chemotherapies. The purpose of this study is to explore the role of LncRNA in the pharmacological effect of Chidamide in breast cancer therapy. The human breast cancer MCF-7 or MDA-MB-231 cells were used as the research cell models. The RNA library screening and high-throughput sequencing comparative analysis was conducted. The binding of LncRNA and its downstream target genes in RNA and protein levels was tested. The results showed that the expression of LncRNA ENST869 in cells treated with Chidamide increased significantly, as demonstrated by real-time PCR and cell viability assay. RNAplex analysis showed that LncRNA ENST869 and Nestin mRNA may interact. RNA interference and Western blot analysis indicated that LncRNA ENST869 could target and regulate the expression of Nestin. Luciferase assay and RNA-protein pulldown showed that LncRNA ENST869 affected Nestin transcription. There might be a highly active binding region of LncRNA ENST869 in regulating Nestin transcriptional activity within the site of 250 bp upstream of the transcription starting point of Nestin. In addition, LncRNA ENST869 did not directly interact with Nestin protein to affect its activity. In conclusion, our results demonstrated that LncRNA ENST869 could affect the function of Nestin in breast cancer cells treated with Chidamide. Nestin is a key player in influencing the pharmacological activity of Chidamide and an essential factor in drug resistance of breast cancer cells.

Effect of lung pericyte-like cell ablation on the bleomycin model of injury and repair.

We previously showed that pericyte-like cells derived from the FoxD1-lineage contribute to myofibroblasts following bleomycin-induced lung injury. However, their functional significance in lung fibrosis remains unknown. In this study, we used a model of lung pericyte-like cell ablation to test the hypothesis that pericyte-like cell ablation attenuates lung fibrosis in bleomycin-induced lung injury. Lung fibrosis was induced by intratracheal instillation of bleomycin. To ablate pericyte-like cells in the lung, diphtheria toxin (DT) was administered to Foxd1-Cre;Rosa26-iDTR mice at two different phases of bleomycin-induced lung injury. For early ablation, we coadministered bleomycin with DT and harvested mice at days 7 and 21. To test the effect of ablation after acute injury, we delivered DT 7 days after bleomycin administration. We assessed fibrosis by lung hydroxyproline content and semiquantitative analysis of picrosirius red staining. We performed bronchoalveolar lavage to determine cell count and differential. We also interrogated mRNA expression of fibrosis-related genes in whole lung RNA. Compared with DT-insensitive littermates where pericyte-like cells were not ablated, DT-sensitive animals exhibited no difference in fibrosis at day 21 both in the early and late pericyte ablation models. However, early ablation of pericytes reduced acute lung inflammation, as indicated by decreased inflammatory cells. Our data confirm a role for pericytes in regulating pulmonary inflammation in early lung injury.

Identification of lncRNAs based on different patterns of immune infiltration in gastric cancer.

Gastric cancer is one of the most common malignant tumors in the world, which brings great challenges to people's life and health. The purpose of this study was to investigate immune related-lncRNAs and identify new biomarkers for the prognosis of gastric cancer (GC). We downloaded data from The Cancer Genome Atlas (TCGA) and used R software to determine the ESTIMATEScore, ImmuneScore, and StromalScore of each tumor sample. We performed prognostic analysis and identified the differentially expressed lnRNAs, which were then used to construct a prognostic model. Among the 44 hub genes in the competitive endogenous RNA (ceRNA) network, 3 differentially expressed genes were verified by qPCR. Based on the degree of immune infiltration, cluster A had a higher ESTIMATEScore, ImmuneScore, and StromalScore and higher expression levels of PD-L1 (CD274) and CTLA4 than cluster B. Univariate Cox analysis was conducted for these differential lncRNAs, and 57 lncRNAs were found to have prognostic value (P<0.05). gene cluster A had a worse prognosis than gene cluster B (P=0.021). Then, a prognostic model was constructed. The low-risk group had a significantly higher survival rate. Finally, the qPCR results showed that the expression levels of BMPER, PRUNE2, and RBPMS2 were low in GC cell lines. We identified a risk score of 19 lncRNAs as a prognostic marker of GC. There was a relationship between these 19 prognostic-related lncRNAs and the subtypes of infiltrating immune cells. An approach for predicting the prognosis of GC was therefore provided in this study.

Prunus necrotic ringspot virus in Turkey: an immigrant population

An outbreak in northwestern Turkey of prunus necrotic ringspot virus (PNRSV, genus Ilarvirus, family Bromoviridae) was sampled in 2016-2018. Gene sequences from these isolates, together with all of the gene sequence data for this virus in the GenBank database (>300 non-recombinant coat protein (CP) genes and 20 complete genomic sequences) were analysed to determine the relationship of the Turkish PNRSV isolates to those from other parts of the world. Phylogenetic and population genetic methods independently showed that the most recent common ancestor of the world PNRSV population was probably American, not Eurasian. PNRSV has spread to Turkey on several occasions, as its CP sequences are among the terminal branches of three of the most sampled CP phylogroups. The complete PNRSV genome consists of three segments (RNA1, RNA2, and RNA3), with the larger two encoding replicases and the smallest encoding the movement protein and the CP. One quarter of the RNA1 and RNA2 genes were recombinants. The phylogenies of the CP and MP genes (i.e., different regions of RNA3) were closely correlated but did not correlate with those of RNA1 and RNA2, indicating that some of the isolates were reassortants. However, the non-reassortant ancestor could not be identified, probably because none of the complete genome sequences were from isolates from the basal CP phylogroups. Our results emphasize the importance of strict quarantine, both international and local, for the world's stone fruit crops.

Vitamin D supplementation in systemic lupus erythematosus: relationship to disease activity, fatigue and the interferon signature gene expression

BackgroundIn addition to the well-known role of vitamin D in calcium homeostasis and bone metabolism, vitamin D is important in the modulation of the immune system and inflammatory processes. Vitamin D deficiency is common in patients with systemic lupus erythematosus (SLE), possibly as a result of sun avoidance. The aim of this prospective open-label study was to assess the effect of the treatment of vitamin D deficiency and insufficiency in SLE patients, particularly with regards to disease activity, fatigue and interferon signature gene expression.Methods31 SLE patients, 13 with vitamin D deficiency and 18 with vitamin D insufficiency were treated with vitamin D3. They were supplemented with vitamin D3 8000 IU daily for 8 weeks if they were vitamin D deficient, or 8000 IU daily for 4 weeks if they were insufficient. This was followed by 2000 IU daily maintenance. They were assessed at baseline, after 6 and 12 months by means of an interview, filling in questionnaires and blood tests. The expression of 12 interferon signature genes in RNA extracted from whole blood was measured by using QuantiGene Plex technology.ResultsAn improvement in disease activity measured by systemic lupus erythematosus disease activity index-2K (SLEDAI-2K; p = 0.028) and fatigue measured by fatigue severity scale (FSS; p = 0.071) at 12 months were noted. A significant decrease in anti-double stranded deoxyribonucleic acid (dsDNA) titre (p = 0.045) was also noted. The mean interferon signature gene expression score decreased from baseline to 6 months, however statistical significance was not achieved (p = 0.165).ConclusionsImproved disease activity and fatigue have been noted when Vitamin D has been supplemented in vitamin D deficient/insufficient SLE patients. One possible mechanism could be the suppression of the interferon signature gene expression.Trial registration: The study was registered with the ISRCTN registry on 12/04/2021 (Trial ID: ISRCTN59058825).

Phylogenetics of tobacco rattle virus isolates from potato (Solanum tuberosum L.) in the USA: a multi-gene approach to evolutionary lineage.

Tobacco rattle virus (TRV) is an important soil-borne virus of potato that is transmitted by stubby-root nematodes. TRV causes corky ringspot, a tuber disease of economic importance to potato production. Utilizing protein-coding regions of the whole genome and a range of computational tools, the genetic diversity, and population structure of TRV isolates from several potato-growing regions (Colorado, Idaho, Indiana, Minnesota, Nebraska, North Dakota, and Washington State) in the USA were determined. Phylogenetic analyses based on RNA2 nucleotide sequences, the coat protein (CP) and nematode transmission (2b) genes, showed geographical clustering of USA isolates with previously known American isolates, while European isolates grouped in a distinct cluster. This was corroborated by the observed genetic differentiation and infrequent gene flow between American and European isolates. Low genetic diversity was revealed among American isolates compared to European isolates. Phylogenetic clustering based on RNA1 genes (RdRp, RdRp-RT, and 1a) were all largely incongruent to that of 1b gene (virus suppressor of RNA silencing). This genetic incongruence suggested the influence of recombination. Furthermore, the RdRp, RdRp-RT, and 1a genes were predicted to be more conserved and under negative selection, while the 1b gene was less constrained. Different evolutionary lineages between TRV RNA1 and RNA2 genomic segments were revealed.

Hypermethylation of Genes in New Long Noncoding RNA in Ovarian Tumors and Metastases: A Dual Effect.

The role of methylation in the regulation of genes of long noncoding RNA (lncRNA) is still poorly understood. We revealed new hypermethylated lncRNA genes in ovarian tumors and their effect on metastasis of ovarian cancer. A multiple and significant (p<0.001) increase in methylation of a group of lncRNA genes (MEG3, SEMA3B-AS1, ZNF667-AS1, and TINCR) was shown by quantitative methylation-specific PCR using the non-parametric Mann-Whitney test. Moreover, methylation of SEMA3B-AS1, ZNF667-AS1, and TINCR genes in ovarian cancer tumors was detected for the first time. Comparative analysis of 19 samples of peritoneal metastases and paired primary tumors showed a significant decrease in the methylation level of the same 4 genes: MEG3 (p=0.004), SEMA3B-AS1 (p=0.002), TINCR (p=0.002), and ZNF667-AS1 (p<0.001). Reduced methylation of suppressor lncRNA genes in peritoneal metastases is probably associated with the involvement of these lncRNA in the regulation of plastic reversion of the epithelial-mesenchymal transition to the mesenchymal-epithelial transition. Thus, the effect of lncRNA and their methylation on the development of tumors and metastases of ovarian cancer was demonstrated, which is important for understanding of the pathogenesis and mechanisms of metastasis of ovarian cancer. New properties of lncRNA can find application in the development of new approaches in the therapy of ovarian cancer.

Expression of glucose and magnesium transport-associated genes in whole blood RNA of lactating ewes supplemented with magnesium

Lactation is characterized by an increase in energy demand and glucose consumption, which may also be linked to the concurrent compromise in immune function during early lactation. Magnesium (Mg) could be involved in this link based on its role for more than 600 metabolic reactions associated with energy metabolism, in addition to its essential role in glucose metabolism. Therefore, the current study aimed to investigate the influence of Mg supplementation on the expression of glucose and Mg transport-associated genes in blood cells (predominantly leukocytes) of lactating ewes and to identify possible correlations between the expression of these genes and leukocyte subpopulations. Pregnant ewes were divided into a control group (Control, n = 8) and a Mg group (Mg, n = 8), the latter supplemented with magnesium oxide at a daily Mg intake of approximately 0.30% and 0.38% of dry matter during the last four weeks of gestation and the first four weeks after lambing, respectively. At the end of the supplementation period, blood samples were collected in PAXgene tubes in order to determine the mRNA expression of glucose transporters (GLUT1, GLUT3 and GLUT4) and genes linked to Mg transport (SLC41A1, CNNM2, TRPM6, TRPM7 and MagT1) in blood cells. Mg supplementation tended to increase the expression of GLUT1 in blood cells (P = 0.087). Expression values of GLUT3, SLC41A1, CNNM2, TRPM6, TRPM7, and MagT1 were not significantly changed (P > 0.1). Interestingly, GLUT4 was not detected in either group. Counts of circulating neutrophils, lymphocytes and monocytes showed no significant differences between the two groups. Furthermore, no correlations were observed between the expression of the investigated genes and specific leukocyte subpopulation percentages/frequenciies. In conclusion, the expression of a variety of glucose and Mg transport-associated genes did not respond to dietary Mg supplementation. Nonetheless, a tendency towards higher expression of GLUT1 in the Mg group might support an involvement of Mg in the regulation of glucose metabolism of ovine blood cells.

Impact of hormonal biomarkers on response to hormonal therapy in advanced and recurrent endometrial cancer

Approximately 20% of women with endometrial cancer have advanced-stage disease or suffer from a recurrence. For these women, prognosis is poor, and palliative treatment options include hormonal therapy and chemotherapy. Lack of predictive biomarkers and suboptimal use of existing markers for response to hormonal therapy have resulted in overall limited efficacy. This study aimed to improve the efficacy of hormonal therapy by relating immunohistochemical expression of estrogen and progesterone receptors and estrogen receptor pathway activity scores to response to hormonal therapy. Patients with advanced or recurrent endometrial cancer and available biopsies taken before the start of hormonal therapy were identified in 16 centers within the European Network for Individualized Treatment in Endometrial Cancer and the Dutch Gynecologic Oncology Group. Tumor tissue was analyzed for estrogen and progesterone receptor expressions and estrogen receptor pathway activity using a quantitative polymerase chain reaction-based messenger RNA model to measure the activity of estrogen receptor-related target genes in tumor RNA. The primary endpoint was response rate defined as complete and partial response using the Response Evaluation Criteria in Solid Tumors. The secondary endpoints were clinical benefit rate and progression-free survival. Pretreatment biopsies with sufficient endometrial cancer tissue and complete response evaluation were available in 81 of 105 eligible cases. Here, 22 of 81 patients (27.2%) with a response had estrogen and progesterone receptor expressions of >50%, resulting in a response rate of 32.3% (95% confidence interval, 20.9-43.7) for an estrogen receptor expression of >50% and 50.0% (95% confidence interval, 35.2-64.8) for a progesterone receptor expression of >50%. Clinical benefit rate was 56.9% for an estrogen receptor expression of >50% (95% confidence interval, 44.9-68.9) and 75.0% (95% confidence interval, 62.2-87.8) for a progesterone receptor expression of >50%. The application of the estrogen receptor pathway test to cases with a progesterone receptor expression of >50% resulted in a response rate of 57.6% (95% confidence interval, 42.1-73.1). After 2 years of follow-up, 34.3% of cases (95% confidence interval, 20-48) with a progesterone receptor expression of >50% and 35.8% of cases (95% confidence interval, 20-52) with an estrogen receptor pathway activity score of >15 had not progressed. The prediction of response to hormonal treatment in endometrial cancer improves substantially with a 50% cutoff level for progesterone receptor immunohistochemical expression and by applying a sequential test algorithm using progesterone receptor immunohistochemical expression and estrogen receptor pathway activity scores. However, results need to be validated in the prospective Prediction of Response to Hormonal Therapy in Advanced and Recurrent Endometrial Cancer (PROMOTE) study.

METTL3 regulates viral m6A RNA modification and host cell innate immune responses during SARS-CoV-2 infection.

It is urgent and important to understand the relationship of the widespread severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) with host immune response and study the underlining molecular mechanism. N6-methylation of adenosine (m6A) in RNA regulates many physiological and disease processes. Here, we investigate m6A modification of the SARS-CoV-2 gene in regulating the host cell innate immune response. Our data show that the SARS-CoV-2 virus has m6A modifications that are enriched in the 3′ end of the viral genome. We find that depletion of the host cell m6A methyltransferase METTL3 decreases m6A levels in SARS-CoV-2 and host genes, and m6A reduction in viral RNA increases RIG-I binding and subsequently enhances the downstream innate immune signaling pathway and inflammatory gene expression. METTL3 expression is reduced and inflammatory genes are induced in patients with severe coronavirus disease 2019 (COVID-19). These findings will aid in the understanding of COVID-19 pathogenesis and the design of future studies regulating innate immunity for COVID-19 treatment.

Short communication: Molecular markers for epithelial cells across gastrointestinal tissues and fecal RNA in preweaning dairy calves

The objective of this study was to compare the transcription of gene markers for gastrointestinal (GI) epithelial cells, including fatty acid binding protein 2 (FABP2) and cytokeratin 8 (KRT8), and tight junction complex genes (TJP1, CLDN1, CLDN4) in fecal RNA against several GI tract tissue sections in dairy calves. Eight healthy Jersey calves were euthanized at 5 wk of age, and postmortem samples were collected from rumen, duodenum, jejunum, ileum, large intestine, cecum, and feces for total RNA isolation. Tissues and fecal samples were immediately frozen in liquid nitrogen until RNA isolation. A real-time quantitative PCR analysis was performed using a single standard curve composited of equal amounts of all samples, including cDNA from fecal and GI tract tissues. The mRNA expression of the tight junctions TJP1, CLDN1, and CLDN4 was greater in fecal RNA compared with lower GI tract tissues (i.e., duodenum, jejunum, ileum, large intestine, and cecum). Similar to fecal RNA, rumen tissue had greater expression of tight junctions CLDN1 and CLDN4 than lower GI tract tissues. Similarly, rumen tissue had greater expression of TPJ1 than all lower GI tract tissues except duodenum. The expression of TJP1 and CLDN4 was greater in fecal RNA than in rumen tissue; in contrast, CLDN1 mRNA expression was greater in rumen tissue than in the fecal RNA. The expression of FABP2 was greater in duodenum in comparison to all tissue except ileum. The mRNA expression of FABP2 in fecal samples was similar to jejunum and ileum. The expression of KRT8 in fecal samples was similar to duodenum, large intestine, and cecum. The fecal RNA had a greater expression of KRT8 in comparison to jejunum and ileum. The rumen tissue had the lowest mRNA expression of KRT8. The expression levels of FABP2, KRT8, and tight junction genes observed in fecal transcripts suggest that a considerable amount of RNA derived from GI tract epithelial cells can be detected in fecal RNA, which is in agreement with previous data in neonatal dairy calves and other biological models including humans, rodents, and primates. The greater expression of tight junctions in fecal RNA in comparison to sections of the low GI remains to be understood, and due to the importance of tight junctions in GI physiology, further clarification of this effect is warranted. The similarities in mRNA expression of FABP2 and KRT8 between fecal RNA and intestinal sections add up to the accumulating evidence that fecal RNA can be used to investigate molecular alterations in the GI tract of neonatal dairy calves. Further research in this area should include high-throughput transcriptomic analysis via RNA-seq to uncover novel molecular markers for specific sections of the GI tract of neonates.

1971P Neuroendocrine (NE) expression profiling in non-castrate tumours is associated with poor therapy benefit and adverse clinical outcome in metastatic castration-resistant prostate cancer (mCRPC) patients

NE dedifferentiation is associated to clinical aggressiveness and resistance to androgen receptor (AR) inhibition in prostate cancer. We analyzed the correlation of a NE expression signature in non-castrate tumors with the clinical outcome of mCRPC patients treated with taxanes or AR signaling inhibitors (ARSI; abiraterone or enzalutamide). Patients with mCRPC were retrospectively tested for a customized signature of 45 NE-related genes in total RNA from formalin-fixed paraffin-embedded non-castrate tumor samples by the nCounter platform (Nanostring Technologies). Patients were grouped as high and low NE profile according to the unsupervised clustering of gene expression data and correlated with treatment response and clinical outcome. Ninety-six patients were included, with a median follow-up of 20 (1.17-81.3) months. Median age was 69.2 (55.8-87.1) years. All patients received taxanes (68 docetaxel, 10 cabazitaxel and 18 both), 29 received ARSI as first line in CRPC and 53 received ARSI after taxanes. Patients classified as high NE expression profile (59 vs. 37 patients) were associated with a shorter time of CRPC development (median 12.6 vs. 30.8 mo, HR 3.3, 95%CI 2-5.5, P<0.001) and shorter OS from CRPC diagnosis (median 24.3 vs. 42.2 mo, HR 2.2, 95%CI 1.4-3.4, P=0.001). Regarding taxanes outcome, high NE profile patients correlated with lower PSA-PFS (HR 1.7, 95%CI 1.1-2.6, P=0.011), radiologic (RX)-PFS (HR 1.8, 95%CI 1.1-2.8, P=0.012) and overall survival (OS) (HR 1.7, 95%CI 1.1-2.6, P=0.009), and they were independently associated with shorter PSA-PFS (HR 1.8, 95%CI 1.1-3, P=0.023). In first-line ARSI-treated patients, high NE profile patients (15 vs. 14 patients) correlated with shorter PSA-PFS (HR 3, 95%CI 1.3-7, P=0.011), RX-PFS (HR 2.5, 95%CI 1-5.9, P=0.042) and OS (HR 5.8, 95%CI 2.3-14.9, P<0.001). NE-related gene expression in non-castrate tumor samples is associated with adverse clinical outcome and both poor taxanes and ARSI benefit in mCRPC patients. Thus, molecular characterization of primary tumors may be useful to guide treatment strategies in mCRPC.

Gene Signatures of Early Response to Anti-TNF Drugs in Pediatric Inflammatory Bowel Disease.

Around a 20–30% of inflammatory bowel disease (IBD) patients are diagnosed before they are 18 years old. Anti-TNF drugs can induce and maintain remission in IBD, however, up to 30% of patients do not respond. The aim of the work was to identify markers that would predict an early response to anti-TNF drugs in pediatric patients with IBD. The study population included 43 patients aged <18 years with IBD who started treatment with infliximab or adalimumab. Patients were classified into primary responders (n = 27) and non-responders to anti-TNF therapy (n = 6). Response to treatment could not be analyzed in 10 patients. Response was defined as a decrease in over 15 points in the disease activity indexes from week 0 to week 10 of infliximab treatment or from week 0 to week 26 of adalimumab treatment. The expression profiles of nine genes in total RNA isolated from the whole-blood of pediatric IBD patients taken before biologic administration and after 2 weeks were analyzed using qPCR and the 2−∆∆Ct method. Before initiation and after 2 weeks of treatment the expression of SMAD7 was decreased in patients who were considered as non-responders (p value < 0.05). Changes in expression were also observed for TLR2 at T0 and T2, although that did not reach the level of statistical significance. In addition, the expression of DEFA5 decreased 1.75-fold during the first 2 weeks of anti-TNF treatment in responders, whereas no changes were observed in non-responders. Expression of the SMAD7 gene is a pharmacogenomic biomarker of early response to anti-TNF agents in pediatric IBD. TLR2 and DEFA5 need to be validated in larger studies.

Inhibition of hepatitis C virus genotype 4 replication using siRNA targeted to the viral core region and the CD81 cellular receptor.

Hepatitis C virus (HCV) is one of the most important causative agents of hepatitis worldwide. The current study aimed to evaluate the silencing effect of the small interference RNA (siRNA) molecules designed against the core region of HCV genotype 4 (HCV-4) and the CD81 gene, which is the cellular receptor for HCV in the human hepatocytes. RT-PCR was used to measure the changes in both the viral HCV core and the cellular CD81 genes induced by the specific siRNA molecules. Additionally, the fluctuations in either the viral or the cellular proteins of the target regions were tested by flow cytometry and immunofluorescence. The results showed the effectiveness of the used siRNA molecules against the target genes in either RNA or protein levels. The effect of 100nM of siCD81 and 40nM of siCore was more evident at 24 and 48h post-transfection. The combination of the two siRNA molecules resulted in an extra inhibitory effect of the HCV core at both the RNA (85.6%) and protein (98.5%) levels. The current study suggested that targeting of the CD81 cellular receptor and/or the viral HCV core region by the small interference molecules might be a suitable choice in the suppression of HCV-4 replication. This might assist the development of new antiviral medications and provides a new alternative strategy for the targeting and treatment of HCV genotype 4.

Prevalence and genetic diversity of grapevine fabavirus isolates from different grapevine cultivars and regions in China

A total of 288 grapevine samples of 61 different grapevine cultivars, collected from 22 provinces and regions, were analyzed by reverse transcription-PCR (RT-PCR) for the presence of grapevine fabavirus (GFabV). PCR detection results showed the incidences of GFabV were 12.8% (30/235) and 48.1% (25/52) in the asymptomatic and symptomatic vines, respectively. The genetic diversity of GFabV isolates was analyzed based on partial nucleotide and encoded amino acid sequences of the RNA1 and RNA2 polyprotein genes. Phylogenetic analyses of the RNA1 and RNA2 gene sequences divided the GFabV isolates into five well-defined groups. Groups 1, 2, and 4 comprised only Chinese isolates. This article represents the first report for the prevalence and genetic diversity of GFabV in grapevines grown in China.