A new p65 isoform that bind the glucocorticoid hormone and is expressed in inflammation liver diseases and COVID-19

Inflammation is a physiological process whose deregulation causes some diseases including cancer. Nuclear Factor kB (NF-kB) is a family of ubiquitous and inducible transcription factors, in which the p65/p50 heterodimer is the most abundant complex, that play critical roles mainly in inflammation. Glucocorticoid Receptor (GR) is a ligand-activated transcription factor and acts as an anti-inflammatory agent and immunosuppressant. Thus, NF-kB and GR are physiological antagonists in the inflammation process. Here we show that in mice and humans there is a spliced variant of p65, named p65 iso5, which binds the corticosteroid hormone dexamethasone amplifying the effect of the glucocorticoid receptor and is expressed in the liver of patients with hepatic cirrhosis and hepatocellular carcinoma (HCC). Furthermore, we have quantified the gene expression level of p65 and p65 iso5 in the PBMC of patients affected by SARS-CoV-2 disease. The results showed that in these patients the p65 and p65 iso5 mRNA levels are higher than in healthy subjects. The ability of p65 iso5 to bind dexamethasone and the regulation of the glucocorticoid (GC) response in the opposite way of the wild type improves our knowledge and understanding of the anti-inflammatory response and identifies it as a new therapeutic target to control inflammation and related diseases.

BIOL-09. HNRNPA1 SPLICED VARIANT: KEY RESISTANT GENE SIGNATURE IN GLIOMAS

Glioblastoma is inevitably a recurrent cancer. Despite of recent advancement, temozolomide remain the prescribed lifeline drug, after the surgery. Inadvertently, MGMT (O6-methylguanine-DNA-methyltransferase) expression mechanistically linked with Temozolomide (alkylating drug) glioma resistant development. To understand the resistant against Temozolomide sought to deciphered, by making invitro drug resistant glioma cell lines. RNA seq analysis over a illumina platform; drug resistant glioma cell lines showed various critical key factor such as splice factor hnRNPA1 and deubiquitinating enzymes were showed to highly upregulated in resistant cell lines. Commonly, from our previous study, the stability of hnRNPA1 in presence of USP5 were showed to promote cell survival, whereas knocking down of USP5 significantly lower down the telomerase activity and NAD/NADH ratio enlarge. Furthermore, expression of MGMT was showed significantly downregulated in hnRNPA1 knock down T98G glioma cells, as well as in U87 Temozolomide resistant cells. Extrinsic apoptosis pathway was showed more prevalent in hnRNPA1 knock down glioma cells in presence of Trail ligand. Interestingly, we found one more spliced variants of hnRNPA1 exclusively expressing in drug resistant cells is new finding. Selectively knocking down of hnRNPA1 splice variant promotes apoptosis. RNA seq analysis followed the comparison between two hnRNPA1 spliced variant knock down, drug resistant glioma cell lines showed differentially expressed transcript support our finding to be distinctly regulated by hnRNPA1 spliced variants. Spliced variant of hnRNPA1 showed a potential therapeutic candidate signature.

HGG-27. HNRNPA1 SPLICED VARIANT SENSITIZATION EFFECT DISCLOSED IN GLIOMA CELLS

Glioblastoma is aggressive brain tumor. Glioma heterogeneity builds in hypoxic condition due to its intrinsic high apoptosis rate cause to develop a high selection clonal pressure. HnRNPA1 plays a key role in developing glycolytic tumor, shows its high expression exclusively in hypoxic glioma cells. Recently we observed one more spliced variant of hnRNPA1, encoding higher isoform, exclusively abundant in resistant glioma cell lines. Widely around the scientific community HnRNPA1 splice factor family protein was found distinctly regulating resistant glioma phenotype. To support our hypothesis, methodology we perform includes various apoptosis assays to critically understand hnRNPA1 spliced variant dependent pathway in Temozolomide resistant U87 glioma cells. Proteomic based apoptotic array and angiogenic array enable us to visualize selective knock down of hnRNPA1 has dominant role in promoting apoptotic cascade. Additionally, flow cytometry base annexin V-PI staining technique to understand early and late apoptosis was measured in selective hnRNPA1 spliced variant knockdown cells in presence or absence of PI3 kinase inhibitor wortmannin (5 micro molar). Results showed hnRNPA1 higher isoform knock down promotes more apoptosis compare to lower isoform. Interestingly, overexpression of HnRNPA1 higher isoform or lower isoform alone doesn’t promote apoptosis, however is prominently higher apoptosis in Bortezomib treated U87 glioma cells. These both isoforms are presently majorly in gliomas, but somehow for long was not recognized. Conclusion is to explore more related novel finding or therapeutic strategy to target higher isoform of hnRNPA1, using invivo mouse xenograft model.

Endoplasmic Reticulum Stress (ER Stress) and Unfolded Protein Response (UPR) Occur in a Rat Varicocele Testis Model

Using a surgically induced varicocele rat model, we show here strong evidence that the misfolded/unfolded protein response that is part of the stress response of the endoplasmic reticulum (ER) is activated in the varicocele testis (VCL), leading to the induction of apoptosis. To support this hypothesis, it is observed that the spliced variant of the X-box protein 1 (XBP1s), resulting from the activation of the inositol-requiring enzyme 1 (IRE1) membrane sensor, is significantly more represented in VCL testicular extracts. The activation of the IRE1/XBP1s pathway is also supported by the observation that the VCL testes show an increase phosphorylation of the c-Jun-kinase (JNK) known to be one intermediate of this pathway and an increased level of caspase-3, the terminal apoptotic effector, partly explaining the apoptotic status of the VCL testis.

CD44 Alternative Splice Variants v6 and v10

CD44 glycoprotein is ubiqutously present and situated at the extracellular surface of cell. Its role as an adhesion recptor was modified depending upon the state of post addition of glycomoeties under varied microenvironment. Many alternative spliced variants of CD44 were expressed by human cells depending upon the attainment during mutifunctional signal disposition. The function of splice factors, and hnRNPA family (Heterogenous ribonucleoproteins A) were dicussed in context to CD44 alternative variants. Glioma cell cline U87 MG were used in this study to search more about the affect of hnRNPA1 and hnRNPA2/B1 knockdown in CD44 V6 and CD44V10 expression. We found in preliminary study that splice factor knock down reduces the alternative splice variants of CD44 such as V6 & V10. Therefore, potential of this study to elaborate further the mechanism in vivo model, to validate the alternative spliced variant in context to interaction with extracellular microenvironment.