In this study, the AGO protein assisted gene silencing mechanism with miR-181c-5p target genes in CTE was performed via bioinformatics approach. The mRNA-miRNA-AGO associated gene silencing mechanism is a known fact at the molecular level and well studies in mammalian steam cells. The gene silencing by RNA-induced silencing complex (RISC) is well-studied cytoplasmic post transcriptional mechanism [15]. Through this concept, we performed the analysis of gene 9 (GNAQ, SAMHD1, CALM1, MAP2K1, ATM, MYH10, TRPC6, DERL, and LAMA) with miRNA181c-5p based on their MFE score in miRTarbase.
Target locations in the experimental validation have been minimized and the process of predicting target genes has been simplified by computational methods. A novel and cutting-edge method for identifying target RNAs that bind to the miRISC complex is the CLIP-seq method [49]. In this investigation, we validated the miR-181c-5p prediction using four distinct target prediction techniques. Each gene expression database has a different validation process, using techniques including Western blot, qPCR, microarrays, and next-generation sequencing. [50]. In our search, we found 86 target genes in total from three different databases; similarly, research on Alzheimer's disease found 37 target genes in total for several miRs, including miR-107, miR-125b, miR-146, and miR-181c. [51].
The pathway analysis showed that the nine genes were associated with multiple pathways in the following areas: signaling pathways regulating stem cell pluripotency, vascular smooth muscle contraction, long-term potentiation, GnRH signaling pathway, melanogenesis, estrogen signaling pathway, Apelin signaling pathway, cGMP-PKG signaling pathway and human immunodeficiency virus 1 infection. GNAQ gene encodes the Guanine nucleotide-binding protein G (q) subunit alpha protein. In a study employing, a mouse model exposed to repeated blasts under excessive pressure, this protein decreased. This study discovered a number of pathways, including those connected to muscular dystrophy and WHIM syndrome. [52]. During proteomic profiling of mTBI mice brains, the gene was also evaluated for the dopaminergic synapse pathway [53]. The microglia-driven chronic cortical inflammation and neuronal dysfunction are mediated by the CALM1 gene, which is involved in calcium homeostasis and signalling [54]. Shen et al, confirmed the hypothesis that decreased ATM signaling in AD causes neuronal death. The downregulated indicators include nuclear translocation and histone trimethylation. [55].
The nine genes were used to perform the Clustal Omega sequence similarity structure. Although the structure of CALM, and MAP2K1 were found to be comparable, there is no proof that a miRNA-mRNA complex has formed based on the data. Likewise, the research has also tracked the prediction of the miRNA-mRNA complex using the MFE score obtained from the miRTarbase. The ATM − 10.20 kcal/mol MFE score. In align to with our scoring, a study on the miRNAs associated with solid cancer tumours also found that the PTEN gene has a good MFE score of -14.60 (kcal/mol).[56]. We also looked at the h-bond and hydrophobic interactions that the docked AGO protein had with the miRNA-mRNA complex. Among these are the complex's cysteine group and ARG amino acid, which are separated by 3.1Å. However, research has shown that strongly hydrophobic aliphatic amino acids provide stability during molecular interactions during protein-ligand docking, corroborating the findings. More equilibrium is found in the binding stability when amino acids with hydrophobic aromatic side chains are used [17, 57].
The current investigation found that the miRNA-mRNA complex is strongly bound by the miRNA-MAPT predicted secondary structure. For RNA secondary structure, there are primarily three prediction techniques: AveRNA [58] RNAfold[34], and Mfold[59] Each of the three employs a distinct methodology, including dynamic programming, pre-directed MFE structures based on thermodynamic parameters, and the method of combining secondary structures. RNAfold not only predicts the miRNA-mRNA as a single sequence but also provides the structure of the dot bracket. Three character alphabets are string formed in the dot bracket notation [60]. The nucleotide's structural context is not included in the depiction [61]. This structural exhaustion method is addressed by Sankoff's dynamic programming algorithm; however, the bioinformatics approach makes it quite difficult [62].
The stability of the complex is protected by the binding affinity of the complex, and the AGO protein helped miRNA silence the miRNA-mRNA. The target gene MAPT comes first, then ATM and TTBK1. Targets for osteoblast-like cells in virtual model-based research have been verified using miRNA-214 in conjunction with the TEAF3 and ATF4 genes. This work also raises concerns about how single miRNA silencing may alter downstream target genes[17]. A study on hepatocellular carcinoma (HCC) revealed that the ATM's 3 UTR structure forms the RISC complex when it binds to miR-181c. The same study found that miR suppressed ATM expression, which in turn affected downstream targets like Akt and Chk2 [63]. The aforementioned study supports our idea to target the miR-181c-5p and its mRNA complex as a potential treatment for CTE.