Intron retention phenomenon in phospholipase C gamma 1 isoform: how the Harpagophytum procumbens extract works? A cellular and bioionformatic approach.
Harpagophytum procumbens is an African plant, commonly known as devil's claw, used since the times of traditional medicine as a natural remedy for Osteoarthritis (OA) treatment, the most common joint disease. Nowadays, there is no cure for OA disease; all the therapies are aimed to reduce pain and inflammation and improve the patient quality of life. Given the widespread of pathology and its early onset in patients, the use of a natural remedy for the treatment of symptoms in the early stages of the disease allows to avoid the prolonged administration of analgesics and anti-inflammatory drugs responsible of the onset of several side effects, and at the same time achieving good therapeutic results. In our experimental conditions, the analgesic and anti-inflammatory effect of a H. procumbens extract (HPE) was tested in vitro on human primary synoviocytes isolated from OA patients that underwent total knee replacement surgery. In last years, we demonstrated an anti-nociceptive role of the HPE on endocannabinoid type 2 receptors (CB2) and an antinflammatory effect on MAPK pathway, and we investigated in depth its effects on several signaling pathways. In particular, performing PCR analyses on both HPE-treated and un-treated synoviocytes, we observed a very interesting result on the phospholipase C ?1 enzyme gene expression. Both samples showed two PCR products, a lower band at 300 bp which was the expected product, and a higher band at 500 bp which was an unexpected product. The surprising result of this experiment concerned the HPE ability to modulate the expression of these two PCR products. While in the untreated sample the product at 300 bp was the most expressed, the HPE treatment induced a decrease in this product expression, increasing that of the product with more base pairs. By investigating the composition of this product through a Sanger sequencing analysis, it has been observed that HPE interferes with the PLC ?1 gene splicing process by stimulating the intron retention during the mRNA maturation. We verified that the introns that were retained in our experimental conditions were also described by the Human and Vertebrate Analysis and Annotation (HAVANA) group, however nobody in literature has attributed this effect to HPE by explaining its molecular mechanisms. Moreover, we demonstrate that samples from different patients had diverse treatment responses regarding the phenomenon of intron retention. Indeed, it has been observed that only in approximately 37% of the HPE treated samples the intron retention phenomenon occurred, while in the remaining part of the samples no modulation was observed. However, as well as the mechanism by which the extract inhibited the splicing process was not clear enough, similarly we have not been able to understand what mentioned inter-individual variability depends on. For these reasons, in the coming months, we plan to conduct and analyze RNAseq experiments in order to understand how HPE works in the intron retention and what factors influence the treatment response among patients. In recent years, much progress has been made in the analysis and identification of retained introns considered, but still many challenges remain open. It has been shown that this phenomenon is involved in many pathologies, such as rheumatoid arthritis, Alzheimer's disease and carcinogenic processes, with potential biological consequences for gene expression and cell survival. For this reason, understanding how a substance is able to modulate the intron retention phenomenon is the main innovative aspect of this study and opens up new perspectives in several disease therapeutic treatment. Overall design: Comparative gene expression profiling analysis of RNA-seq data among HPE-treated synoviocytes and untreated cells and also among patients with different mRNA expression profile (Intron retention or normal splicing).