Hsp Inhibitor is Affective Against Adenocarcinomic Human Alveolar Basal Epithelial Cells Through Modulating ERK/MAPK Signaling Pathway.

The extracellular signal-regulated kinase (ERK) - mitogen-activated protein kinase (MAPK) pathway regulates cell proliferation, differentiation, and apoptosis. Heat Shock Protein 90 (HSP90) is required to activate proto-oncogenic protein kinases and promotes tumor growth through anti-apoptotic effects on A549-non-small cell lung cancer (NSCLC). Therefore, deregulation of the ERK-MAPK pathway and abnormal expression of HSP90 are reasonably frequent events in NSCLC. In this study, novel perimidine-pyrazole compounds employed to block ERK-MAPK deregulation through inhibiting HSP dependent cancer cell survival mechanisms. A set of perimidine-pyrazole derivatives effects was monitored on NSCLC cell line. Array experiments performed to understand the effect of the compounds on signaling pathways and results were analyzed by gene enrichment analysis. Further, senescence and apoptosis experiments were performed to support the enrichment results along with in silico methods to determine perimidine-pyrazole/HSP interactions. Treatment of NSCLC cells with perimidine-pyrazole derivatives displayed cancer-inhibitory, pro-senescent and pro-apoptotic effects on NSCLC cells through ERK/MAPK pathway and these compounds are promising templates for designing anticancer drugs.

Silver(I) Complexes Based on Oxadiazole-Functionalized α-Aminophosphonate: Synthesis, Structural Study, and Biological Activities.

Two silver(I) complexes, bis{diethyl[(5-phenyl-1,3,4-oxadiazol-2-yl-κN3:κN4-amino) (4-trifluoromethylphenyl)methyl]phosphonate-(tetrafluoroborato-κF)}-di-silver(I) and tetrakis-{diethyl[(5-phenyl-1,3,4-oxadiazol-2-yl-κN3-amino)(4-trifluoromethylphenyl)methyl]phosphonate} silver(I) tetrafluoroborate, were prepared starting from the diethyl[(5-phenyl-1,3,4-oxadiazol-2-yl-amino)(4-trifluoromethylphenyl)methyl]phosphonate (1) ligand and AgBF4 salt in Ag/ligand ratios of 1/1 and 1/4, respectively. The structure, stoichiometry, and geometry of the silver complexes were fully characterized by elemental analyses, infrared, single-crystal X-ray diffraction studies, multinuclear NMR, and mass spectroscopies. The binuclear complex ([Ag2(1)2(BF4)2]; 2) crystallizes in the monoclinic asymmetric space group P21/c and contains two silver atoms adopting a {AgN2F} planar trigonal geometry, which are simultaneously bridged by two oxadiazole rings of two ligands, while the mononuclear complex ([Ag(1)4]BF4; 3) crystallizes in the non-usual cubic space group Fd-3c in which the silver atom binds to four distinct electronically enriched nitrogen atoms of the oxadiazole ring, in a slightly distorted {AgN4} tetrahedral geometry. The α-aminophosphonate and the monomeric silver complex were evaluated in vitro against MCF-7 and PANC-1 cell lines. The silver complex is promising as a drug candidate for breast cancer and the pancreatic duct with half-maximal inhibitory concentration (IC50) values of 8.3 ± 1.0 and 14.4 ± 0.6 µM, respectively. Additionally, the interactions of the ligand and the mononuclear complex with Vascular Endothelial Growth Factor Receptor-2 and DNA were evaluated by molecular docking methods.

ATPase inhibition by omeprazole reveals role of heat shock proteins on testicular torsion.

Testicular torsion leads ischaemic injury and generates reactive oxygen species. Reactive oxygen species triggers lipid peroxidation, protein degradation and DNA damage. These biochemical processes trigger tissue damage. Heat shock proteins (HSPs) are important in spermatogenesis, and this work elucidates role of HSPs at the testicular torsion-detorsion process. A proton-pump inhibitor, omeprazole, tested to reveal the drug's curative effect since HSP functions through ATP hydrolysis. Thirty-two male Wistar Albino rats were divided into four groups: sham, control, omeprazole and serum physiologic groups. Right testis was torsed, while left ones remained untorsed. Protein peroxidation, DNA damage and lipid hydroperoxide levels as well as HSP expression were measured. Further, the effects were visualised with histopathologic imaging. HSP expression increases at the torsed right testis compared to the contralateral testis. Although HSP70 and HSP90 help antioxidant enzymes to keep their native structure, their anti-apoptotic properties accelerate the tissue damage. Omeprazole a proton-pump inhibitor employed to impair electron transfer chain and to inhibit HSP ATPase function. Omeprazole effectively inhibits HSPs and alleviates lipid peroxidation and DNA damage levels both at molecular and at tissue level, and the drug has profound curative effect on testicular torsion recovery.

The Role of Cysteine Cathepsins in Cancer Progression and Drug Resistance.

Cysteine cathepsins are lysosomal enzymes belonging to the papain family. Their expression is misregulated in a wide variety of tumors, and ample data prove their involvement in cancer progression, angiogenesis, metastasis, and in the occurrence of drug resistance. However, while their overexpression is usually associated with highly aggressive tumor phenotypes, their mechanistic role in cancer progression is still to be determined to develop new therapeutic strategies. In this review, we highlight the literature related to the role of the cysteine cathepsins in cancer biology, with particular emphasis on their input into tumor biology.

Isolation and characterization of Heat Shock Protein 100-Batu1 from Toxoplasma gondii RH strain.

Toxoplasma gondii is an intracellular parasitic protozoon which infects human and most warm-blooded animals. Almost one-third of the world's population is affected by life-threatening infection of T. gondii tachyzoites form. Slow growing, transmissible and encysted bradyzoites forms are composed after tachyzoites stage. Cellular and environmental stresses induce conversion of tachyzoites from bradyzoites and this condition is associated with Heat Shock Protein (Hsps) family. Hsp100 is a member of this protein family, and coordinates to disassemble protein aggregates with Hsp70 and Hsp40 in an ATP dependent manner. Several proteins are involved during this stage differentiation and Hsp100 may help them to be in their native soluble form to perform their function as observed in other organisms. For this purpose, Hsp100-Batu1 was isolated from T. gondii RH strain to characterize its biochemical properties in this current study. Hsp100 proteins play a role in survival and virulence of pathogens as shown in the literature. Therefore, manipulation of protein-protein interaction may perturb T. gondii infection and impair conversion to tachyzoites by inhibiting Hsp100 function. Therefore, results of this work present a potential route for vaccination or immunotherapy.

Therapeutic Targeting of microRNAs in Cancer: Future Perspectives.

Preclinical Research The discovery of microRNAs (miRNAs) and their link with cancer has opened a new era in cancer therapeutics. Approximately, 18 - 24 nucleotides long, miRNAs can up-regulate or down-regulate gene expression in many cancer types and are respectively categorized as oncogenes (oncomirs) or tumor suppressors. Expression profiles of miRNAs with biomarker potential can be used for the classification, diagnosis, therapeutic treatment, and prognosis of different cancer types. miRNA mimics and miRNA antagonists are the two main approaches to miRNA-based cancer therapies that respectively inhibit oncomirs or restore the expression of tumor suppressive miRNAs. This review serves to provide some general insight into miRNA biogenesis, cancer related miRNAs, and miRNA therapeutics.

Dynamic fluctuations provide the basis of a conformational switch mechanism in apo cyclic AMP receptor protein.

Escherichia coli cyclic AMP Receptor Protein (CRP) undergoes conformational changes with cAMP binding and allosterically promotes CRP to bind specifically to the DNA. In that, the structural and dynamic properties of apo CRP prior to cAMP binding are of interest for the comprehension of the activation mechanism. Here, the dynamics of apo CRP monomer/dimer and holo CRP dimer were studied by Molecular Dynamics (MD) simulations and Gaussian Network Model (GNM). The interplay of the inter-domain hinge with the cAMP and DNA binding domains are pre-disposed in the apo state as a conformational switch in the CRP's allosteric communication mechanism. The hinge at L134-D138 displaying intra- and inter-subunit coupled fluctuations with the cAMP and DNA binding domains leads to the emergence of stronger coupled fluctuations between the two domains and describes an on state. The flexible regions at K52-E58, P154/D155 and I175 maintain the dynamic coupling of the two domains. With a shift in the inter-domain hinge position towards the N terminus, nevertheless, the latter correlations between the domains loosen and become disordered; L134-D138 dynamically interacts only with the cAMP and DNA binding domains of its own subunit, and an off state is assumed. We present a mechanistic view on how the structural dynamic units are hierarchically built for the allosteric functional mechanism; from apo CRP monomer to apo-to-holo CRP dimers.

Metabologenomics and network pharmacology to understand the molecular mechanism of cancer research.

In this editorial I comment on the article "Network pharmacological and molecular docking study of the effect of Liu-Wei-Bu-Qi capsule on lung cancer" published in the recent issue of the World Journal of Clinical Cases 2023 November 6; 11 (31): 7593-7609. Almost all living forms are able to manufacture particular chemicals-metabolites that enable them to differentiate themselves from one another and to overcome the unique obstacles they encounter in their natural habitats. Numerous methods for chemical warfare, communication, nutrition acquisition, and stress prevention are made possible by these specialized metabolites. Metabolomics is a popular technique for collecting direct measurements of metabolic activity from many biological systems. However, confusing metabolite identification is a typical issue, and biochemical interpretation is frequently constrained by imprecise and erroneous genome-based estimates of enzyme activity. Metabolite annotation and gene integration uses a biochemical reaction network to obtain a metabolite-gene association so called metabologenomics. This network uses an approach that emphasizes metabolite-gene consensus via biochemical processes. Combining metabolomics and genomics data is beneficial. Furthermore, computer networking proposes that using metabolomics data may improve annotations in sequenced species and provide testable hypotheses for specific biochemical processes.

Synthesis of novel carbazole hydrazine-carbothioamide scaffold as potent antioxidant, anticancer and antimicrobial agents.

BACKGROUND: Carbazole-based molecules containing thiosemicarbazide functional groups are recognized for their diverse biological activities, particularly in enhancing therapeutic anticancer effects through inhibiting crucial pathways. These derivatives also exhibit noteworthy antioxidant properties. OBJECTIVES: This study aims to synthesize, characterize, and evaluate the antioxidant and anticancer activities of 18 novel carbazole derivatives. METHODS: The radical scavenging capabilities of the compounds were assessed using the 2,2-diphenyl-1-picrylhydrazyl assay. Antiproliferative activities were evaluated on MCF-7 cancer cell lines through viability assays. Additionally, the modulation of the PI3K/Akt/mTOR pathway, apoptosis/necrosis induction, and cell cycle analysis were conducted for the most promising anticancer agents. RESULTS: nine compounds showed potent antioxidant activities with IC50 values lower than the positive control acarbose, with compounds 4 h and 4y exhibiting the highest potency (IC50 values of 0.73 and 0.38 µM, respectively). Furthermore, compounds 4o and 4r displayed significant anticancer effects, with IC50 values of 2.02 and 4.99 µM, respectively. Compound 4o, in particular, exhibited promising activity by targeting the PI3K/Akt/mTOR signaling pathway, inhibiting tumor survival, inducing apoptosis, and causing cell cycle arrest in MCF-7 cell lines. Furthermore, compound 4o was showed significant antimicrobial activities against S. aureus and E. coli, and antifungal effect against C. albicans. Its potential to overcome drug resistance through this pathway inhibition highlights its promise as an anticancer agent. Molecular docking simulations supported these findings, revealing favorable binding profiles and interactions within the active sites of the enzymes PI3K, AKT1, and mTOR. Moreover, assessing the druggability of the newly synthesized thiosemicarbazide derivatives demonstrated optimal physicochemical properties, further endorsing their potential as drug candidates.

Synthesis and anticancer activity of acyl thioureas bearing pyrazole moiety.

In this work novel organic based compounds, acyl thiourea derivatives were synthesized and their anticancer activities were investigated. A new series of acyl thiourea derivatives containing pyrazole ring were prepared in good yield through one pot reaction of 4-benzoyl-1, 5-diphenyl-1H-pyrazole-3-carbonyl chloride with ammonium thiocyanate and various amines. The structures of the newly synthesized compounds were confirmed by IR, (1)H NMR, (13)C NMR and elemental analysis. Anticancer activities of synthesized compounds were evaluated on human colon, liver and leukemia cancer cell lines. Cell culture studies have demonstrated significant toxicity of the compounds on the cell lines, and the levels of toxicity have altered in the presence of various side groups. These results confirm that novel pyrazolyl acyl thioureas derived compounds may be utilized for cancer treatment. Furthermore, these compounds have a great potential and significance for further investigations.

Pralatrexate for Peripheral T-Cell Lymphoma (PTCL): Chance Only Supports The Prepared Mind.

BACKGROUND: Due to their primary effects on DNA synthesis, antimetabolites are most effective against actively dividing cells and are significantly specific to the cell cycle phase. Pralatrexate (PDX), an antifolate metabolite designed to accumulate in cancer cells, was the first new agent approved by the US Food and Drug Administration for the treatment of resistant/recurrent peripheral T-cell lymphomas. PDX was a drug that is frequently used not only for PTCL, but also for cutaneous T-cell lymphoma (CTCL), extranodal natural killer (NK) / T-cell lymphoma. OBJECTIVE: This article reviews Pralatrexate's history, pharmacokinetics, clinical phase studies including phases I, II and III, types of cancers it is effective on, drug side effects, inhibition mechanism and even its use in the treatment of other cancers with innovative methods, including its antiviral effect against SARS-CoV-2 infection. METHODS: A comprehensive internet-based research was planned, covering all published and unpublished studies on the subject. We conducted this review in accordance with Preferred Reporting Items for systematic reviews and metaanalysis (PRISMA-P), and Cochrane Collaboration reporting items for systematic reviews and meta-analysis. The results of the studies in the articles were recorded to include all phase studies. RESULTS: Pralatrexate was structurally designed to have enhanced cellular transport via RFC (reduced folate carrier type) and be subject to more polyglutamation compared to methotrexate. The enhanced polyglutamylation ability of pralatrexate is associated with increased tumor cell death and ultimately improved anticancer activity. Pralatrexate is considered a promising drug for patients with recurrent and treatment-resistant PTCL with a good survival advantage. At the same time, it is an antifolate agent with a significant advantage over methotrexate as it does not cause myelosuppression. CONCLUSION: While there are manageable side effects such as thrombocytopenia, neutropenia, and mucositis, it is critical to explore new approaches, targeted agents, novel cellular therapies, and immunotherapies to determine optimal pretreatment in the rare but heterogeneous disease PTCL, and future studies and experienced haematologists are needed.

A Novel 6,8,9-Trisubstituted Purine Analogue Drives Breast Cancer Luminal A Subtype MCF-7 to Apoptosis and Senescence through Hsp70 Inhibition.

BACKGROUND: Cancer cells restrain apoptotic and senescence pathways through intracellular heat shock protein 70 (Hsp 70). These cells aid stimulus-independent growth, and their higher metabolism rate requires Hsps. Hsps compensate abnormally increased substrate protein folding rate of cancer cells. OBJECTIVE: Misfolding of substrate proteins especially signaling substrate proteins, may not function properly. Therefore, Hsp70 folds these substrate proteins into their native-fully functional states, and this mode of action helps cancer cell survival. METHODS: Targeting Hsps is promising cancer therapy, and in this study, 6,8,9-trisubstituted purine derivatives were designed and synthesized to inhibit Hsp70 and drive cancer cells to apoptosis. Further, oncogenic stimuli through inhibitors can induce an irreversible senescent state and senescence is a barrier to transformation. RESULTS: Hsp70 helps cancer cells to bypass the cellular senescence program, however, binding of N6-(4- isopropylaniline) analogue (7) depletes Hsp70 function as evidenced by aggregation assay and Hsp70 depletion induces senescence pathway. CONCLUSION: The purine-based inhibitor-compound 7 effectively inhibits MCF-7 cell line. Moreover, the therapeutic potential with regard to the senescence-associated secretory phenotype has complementary action. Dual action of the inhibitor not only drives the cells to apoptosis but also force the cells to be in the senescence state and provides promising results specially for luminal A type breast cancer therapy.

Triad pyrazole-thiazole-coumarin heterocyclic core effectively inhibit HSP and drive cancer cells to apoptosis.

Intensive studies on hepatocellular carcinoma (HCC), which is spreading rapidly around the world and has a high mortality rate, is due to the lack of adequate preventive or curative treatment methods. Treating patients with HCC has become very challenging because of the heterogeneity in the patient population lead activation of different signaling pathways, and pathway crosstalk for patients. Therefore, understanding these molecular mechanisms and combining drugs with molecular therapies to overcome these drawbacks has become an area of utmost importance. In this study, the biological activities of the designed and characterized triad Pyrazole-Thiazol-Coumarin (PTC) compounds were determined by performing cell viability, qPCR array, apoptosis and cell cycle assays. One of the compounds (PTC10) implicitly suppresses multiple pathways (RAS/MAP kinase and PI3K-AKT) simultaneously. This action is provided by (i) arresting cancer cells at G2 phase, (ii) driving cancer cells to apoptosis and (iii) inhibiting HSP network. Remarkably, HSP is an apoptotic factor and help cancer cell to survive. HSP90 also coordinates with Cdk4/Cdc37, therefore inhibiting HSP both drives cells to arrest and apoptosis. ATP hydrolysis and aggregation assay further displayed specific HSP inhibition. Therefore, PTC provides a unique drug template for HCC treatment.

Involvement of Metabolites and Non-coding RNAs in Diseases.

Non-coding RNAs have a role in gene regulation and cellular metabolism control. Metabolism produces metabolites which are small molecules formed during the metabolic process. So far, a direct relationship between metabolites and genes is not fully established; however, pseudogenes and their progenitor genes regulate health and disease states. Other non-coding RNAs also contribute to this regulation at different cellular processes. Accumulation and depletion of metabolites accompany the dynamic equilibrium of health and disease state. In this study, metabolites, their roles in the cell, and the link between metabolites and non-coding RNAs are discussed.

Neglected role of cAMP receptor protein monomer.

Lac operon transcription activation through CRP dimer depends on cAMP second messenger. The formation of CRP homodimers is mediated by protein-protein interactions between the monomers. Cyclic AMP ligand binding brings CRP dimer to an active state via conformational changes. Molecular modeling studies in our lab showed the importance of monomer in transcription activation through its pre-existing conformational state. Until now CRP experiments were carried out at protein concentrations higher than that of CRP dimer dissociation value making all CRP monomers dimer and ignore the importance of CRP monomer in allosteric activation. Labeling CRP monomers with fluorophores exterminate using excess protein concentration and allow monitoring CRP monomer behavior. CRP monomer exchange accelerates in the presence of non specific DNA whereas the exchange is inhibited in the presence of specific DNA and cAMP ligand. Degree of subunit exchange depends on the stability of CRP dimer. Cyclic AMP forms a single molecule from two monomers and addition of specific DNA further stabilizes CRP dimer and decreases monomer exchange. On the other hand, addition of non specific DNA increases CRP monomer exchange and may explain the mechanism of CRP monomer removal and dissociation of CRP dimer:cAMP:DNA complex. The exchange behavior of CRP in the presence of different factors implies importance of monomer in transcription complex association and dissociation.

Pseudogenes.

Pseudogenes are ubiquitous and abundant in genomes. Pseudogenes were once called "genomic fossils" and treated as "junk DNA" several years. Nevertheless, it has been recognized that some pseudogenes play essential roles in gene regulation of their parent genes, and many pseudogenes are transcribed into RNA. Pseudogene transcripts may also form small interfering RNA or decrease cellular miRNA concentration. Thus, pseudogenes regulate tumor suppressors and oncogenes. Their essential functions draw the attention of our research group in my current work on heat shock protein 90: a chaperone of oncogenes. The paper reviews our current knowledge on pseudogenes and evaluates preliminary results of the chaperone data. Current efforts to understand pseudogenes interactions help to understand the functions of a genome.

Computational Analysis of Drug Resistance Network in Lung Adenocarcinoma.

BACKGROUND: Lung cancer is a significant health problem and accounts for one-third of the deaths worldwide. A great majority of these deaths are caused by Non-Small Cell Lung Cancer (NSCLC). Chemotherapy is the leading treatment method for NSCLC, but resistance to chemotherapeutics is an important limiting factor that reduces the treatment success of patients with NSCLC. OBJECTIVE: In this study, the relationship between differentially expressed genes affecting the survival of the patients, according to the bioinformatics analyses, and the mechanism of drug resistance is investigated for nonsmall cell lung adenocarcinoma patients. METHODS: Five hundred thirteen patient samples were compared with fifty-nine control samples. The employed dataset was downloaded from The Cancer Genome Atlas (TCGA) database. The information on how the drug activity altered against the expressional diversification of the genes was extracted from the NCI-60 database. Four hundred thirty-three drugs with known Mechanism of Action (MoA) were analyzed. Diversifications of the activity of these drugs related to genes were considered based on nine lung cancer cell lines virtually. The analyses were performed using R programming language, GDCRNATools, rcellminer, and Cytoscape. RESULTS: This work analyzed the common signaling pathways and expressional alterations of the proteins in these pathways associated with survival and drug resistance in lung adenocarcinoma. Deduced computational data demonstrated that proteins of EGFR, JNK/MAPK, NF-κB, PI3K /AKT/mTOR, JAK/STAT, and Wnt signaling pathways were associated with the molecular mechanism of resistance to anticancer drugs in NSCLC cells. CONCLUSION: To understand the relationships between resistance to anticancer drugs and EGFR, JNK/MAPK, NF-κB, PI3K /AKT/mTOR, JAK/STAT, and Wnt signaling pathways is an important approach to design effective therapeutics for individuals with NSCLC adenocarcinoma.

MicroRNA Targeting.

MicroRNAs (miRNAs) are small noncoding elements that play essential roles in the posttranscriptional regulation of biochemical processes. miRNAs recognize and target multiple mRNAs; therefore, investigating miRNA dysregulation is an indispensable strategy to understand pathological conditions and to design innovative drugs. Targeting miRNAs in diseases improve outcomes of several therapeutic strategies thus, this present study highlights miRNA targeting methods through experimental assays and bioinformatics tools. The first part of this review focuses on experimental miRNA targeting approaches for elucidating key biochemical pathways. A growing body of evidence about the miRNA world reveals the fact that it is not possible to uncover these molecules' structural and functional characteristics related to the biological processes with a deterministic approach. Instead, a systemic point of view is needed to truly understand the facts behind the natural complexity of interactions and regulations that miRNA regulations present. This task heavily depends both on computational and experimental capabilities. Fortunately, several miRNA bioinformatics tools catering to nonexperts are available as complementary wet-lab approaches. For this purpose, this work provides recent research and information about computational tools for miRNA targeting research.

MicroRNAs and Heat Shock Proteins in Breast Cancer Biology.

Breast cancer has five major immune types; luminal A, luminal B, HER2, Basal-like, and normal-like. Cells produce a family of protein called heat shock proteins (Hsps) in response to exposure to thermal and other proteotoxic stresses play essential roles in cancer metabolism and this large family shows a diverse set of Hsp involvement in different breast cancer immune types. Recently, Hsp members categorized according to their immune type roles. Hsp family consists of several subtypes formed by molecular weight; Hsp70, Hsp90, Hsp100, Hsp40, Hsp60, and small molecule Hsps. Cancer cells employ Hsps as survival factors since most of these proteins prevent apoptosis. Several studies monitored Hsp roles in breast cancer cells and reported Hsp27 involvement in drug resistance, Hsp70 in tumor cell transformation-progression, and interaction with p53. Furthermore, the association of Hsp90 with steroid receptors and signaling proteins in patients with breast cancer directed research to focus on Hsp-based treatments. miRNAs are known to play key roles in all types of cancer that are upregulated or downregulated in cancer which respectively referred to as oncogenes (oncomirs) or tumor suppressors. Expression profiles of miRNAs may be used to classify, diagnose, and predict different cancer types. It is clear that miRNAs play regulatory roles in gene expression and this work reveals miRNA correlation to Hsp depending on specific breast cancer immune types. Deregulation of specific Hsp genes in breast cancer subtypes allows for identification of new targets for drug design and cancer treatment. Here, we performed miRNA network analysis by recruiting Hsp genes detected in breast cancer subtypes and reviewed some of the miRNAs related to aforementioned Hsp genes.

44 Current Challenges in miRNomics.

Mature microRNAs (miRNAs) are short RNA sequences about 18-24 nucleotide long, which provide the recognition key within RISC for the posttranscriptional regulation of target RNAs. Considering the canonical pathway, mature miRNAs are produced via a multistep process. Their transcription (pri-miRNAs) and first processing step via the microprocessor complex (pre-miRNAs) occur in the nucleus. Then they are exported into the cytosol, processed again by Dicer (dsRNA) and finally a single strand (mature miRNA) is incorporated into RISC (miRISC). The sequence of the incorporated miRNA provides the function of RNA target recognition via hybridization. Following binding of the target, the mRNA is either degraded or translation is inhibited, which ultimately leads to less protein production. Conversely, it has been shown that binding within the 5' UTR of the mRNA can lead to an increase in protein product. Regulation of homeostasis is very important for a cell; therefore, all steps in the miRNA-based regulation pathway, from transcription to the incorporation of the mature miRNA into RISC, are under tight control. While much research effort has been exerted in this area, the knowledgebase is not sufficient for accurately modelling miRNA regulation computationally. The computational prediction of miRNAs is, however, necessary because it is not feasible to investigate all possible pairs of a miRNA and its target, let alone miRNAs and their targets. We here point out open challenges important for computational modelling or for our general understanding of miRNA-based regulation and show how their investigation is beneficial. It is our hope that this collection of challenges will lead to their resolution in the near future.