Serum miR-215-5p, miR-192-5p and miR-378a-5p as novel diagnostic biomarkers for periampullary adenocarcinoma.

OBJECTIVE: MicroRNAs (miRNAs) are present in human serum in a stable form. Circulating miRNAs are increasingly recognized as promising biomarkers for early cancer detection. The aim of this study was to identify serum miRNAs as biomarkers for periampullary adenocarcinoma (PAC). PATIENTS AND METHODS: 68 patients with PAC and 50 healthy controls (HCs) subjects were recruited in this study. The expression levels of 11 selected miRNAs were determined in serum samples using the SYBR-green quantitative reverse transcription polymerase chain reaction (qRT-PCR) method. Receiver operating characteristic (ROC) analysis was used to evaluate the diagnostic potential of serum miRNAs. RESULTS: The expression levels of three miRNAs (miR-215-5p, miR-192-5p, and miR-378a-5p) were significantly upregulated in the serum samples derived from the PAC patients compared with those from the HC (p < 0.001). The ROC analysis showed that all three significantly altered miRNAs (miR-215-5p, miR-192-5p, and miR-378a-5p) could potentially discriminate patients with PAC from HC with AUC value of 0.771 (95% CI: 0.684-0.843), 0.877 (95% CI: 0.799-0.927) and 0.768 (95% CI: 0.674-0.853) respectively. Further comparisons showed that these three serum miRNAs (miR-215-5p, miR-192-5p, and miR-378a-5p) can strongly discriminate early-stage PAC patients from HC with an AUC value of 0.802 (95% CI: 0.719-0.886), 0.870 (95% CI: 0.793-0.974) and 0.793 (95% CI: 0.706-0.880) respectively, may aid in early detection of PAC. CONCLUSIONS: Taken together, our findings demonstrated that these three serum miRNAs (miR-215-5p, miR-192-5p, and miR-378a-5p) may serve as noninvasive biomarkers for the early detection of PAC.

Long non-coding RNAs in cancer: multifaceted roles and potential targets for immunotherapy.

Cancer remains a major global health concern with high mortality rates mainly due to late diagnosis and poor prognosis. Long non-coding RNAs (lncRNAs) are emerging as key regulators of gene expression in human cancer, functioning through various mechanisms including as competing endogenous RNAs (ceRNAs) and indirectly regulating miRNA expression. LncRNAs have been found to have both oncogenic and tumor-suppressive roles in cancer, with the former promoting cancer cell proliferation, migration, invasion, and poor prognosis. Recent research has shown that lncRNAs are expressed in various immune cells and are involved in cancer cell immune escape and the modulation of the tumor microenvironment, thus highlighting their potential as targets for cancer immunotherapy. Targeting lncRNAs in cancer or immune cells could enhance the anti-tumor immune response and improve cancer immunotherapy outcomes. However, further research is required to fully understand the functional roles of lncRNAs in cancer and the immune system and their potential as targets for cancer immunotherapy. This review offers a comprehensive examination of the multifaceted roles of lncRNAs in human cancers, with a focus on their potential as targets for cancer immunotherapy. By exploring the intricate mechanisms underlying lncRNA-mediated regulation of cancer cell proliferation, invasion, and immune evasion, we provide insights into the diverse therapeutic applications of these molecules.

Development of an efficient single-cell cloning and expansion strategy for genome edited induced pluripotent stem cells.

BACKGROUND: Disease-specific human induced pluripotent stem cells (hiPSCs) can be generated directly from individuals with known disease characteristics or alternatively be modified using genome editing approaches to introduce disease causing genetic mutations to study the biological response of those mutations. The genome editing procedure in hiPSCs is still inefficient, particularly when it comes to homology directed repair (HDR) of genetic mutations or targeted transgene insertion in the genome and single cell cloning of edited cells. In addition, genome editing processes also involve additional cellular stresses such as poor cell viability and genetic stability of hiPSCs. Therefore, efficient workflows are desired to increase genome editing application to hiPSC disease models and therapeutic applications. METHODS AND RESULTS: To this end, we demonstrate an efficient workflow for feeder-free single cell clone generation and expansion in both CRISPR-mediated knock-out (KO) and knock-in (KI) hiPSC lines. Using StemFlex medium and CloneR supplement in conjunction with Matrigel cell culture matrix, we show that cell viability and expansion during single-cell cloning in edited and unedited cells is significantly enhanced. Keeping all factors into account, we have successfully achieved hiPSC single-cell survival and cloning in both edited and unedited cells with rates as maximum as 70% in less than 2 weeks. CONCLUSION: This simplified and efficient workflow will allow for a new level of sophistication in generating hiPSC-based disease models to promote rapid advancement in basic research and also the development of novel cellular therapeutics.

Antigene and Antiproliferative Effects of Triplex-Forming Oligonucleotide (TFO) Targeted on hmgb1 Gene in Human Hepatoma Cells.

BACKGROUND: The high mobility group box 1 (hmgb1) is one of the frequently over-expressed genes whose aberrant expression is reported in a number of human cancers. Various strategies are underway to inhibit hmgb1 expression in cancer cells having considerable therapeutic value. OBJECTIVE: The present work involves selective transcriptional inhibition of the hmgb1 gene using selective DNA triplex structure-based gene technology. Here, the promoter region of the hmgb1 gene at position (-183 to -165) from the transcription start site as a target was selected using bioinformatic tools. METHODS: The DNA triplex formation by the DNA of the target gene and TFO was confirmed using UV absorption spectroscopy, Circular Dichroism, and Isothermal Calorimetry. RESULTS: Treatment of HepG2 cell with specific Triplex-forming Oligonucleotide significantly downregulated HMGB1 expression level at mRNA and protein levels by 50%, while the classical anticancer drugs, actinomycin/ adriamycin as positive controls showed 65% and the combination of TFO and drug decreased by 70%. The anti-proliferative effects of TFO correlated well with the fact of accumulation of cells in the Go phase and apoptotic cell death. Further, the binding of anti-cancer drugs to hmgb1 is stronger in DNA triplex state as compared to hmgb1 alone, suggesting the combination therapy as a better option. CONCLUSION: Therefore, the ability of hmgb1 targeted triplex-forming oligonucleotide in combination with triplex selective anticancer drug holds promise in the treatment of malignancies associated with hmgb1 overexpression. The result obtained may open up new vistas to provide a basis for the rational drug design and searching for high-affinity ligands with a high triplex selectivity.

Pharmacological and molecular approaches for the treatment of ß-hemoglobin disorders.

ß-hemoglobin disorders, such as ß-thalassemia and sickle cell anemia are among the most prevalent inherited genetic disorders worldwide. These disorders are caused by mutations in the gene encoding hemoglobin-ß (HBB), a vital protein found in red blood cells (RBCs) that carries oxygen from lungs to all parts of the human body. As a consequence, there has been an enduring interest in this field in formulating therapeutic strategies for the treatment of these diseases. Currently, there is no cure available for hemoglobin disorders, although, some patients have been treated with bone marrow transplantation, whose scope is limited because of the difficulty in finding a histocompatible donor and also due to transplant-associated clinical complications that can arise during the treatment. On account of these constraints, reactivation of fetal hemoglobin (HbF) synthesis holds immense promise and is a viable strategy to alleviate the symptoms of ß-hemoglobin disorders. Development of new genomic tools has led to the identification of important natural genetic modifiers of hemoglobin switching which include BCL11A, KLF1, HBSIL-MYB, LRF, LSD1, LDB1, histone deacetylases 1 and 2 (HDAC1 and HDAC2). miRNAs are also promising therapeutic targets for development of more effective strategies for the induction of HbF production. Many new small molecule pharmacological inducers of HbF production are already under pre-clinical and clinical development. Furthermore, recent advancements in gene and cell therapy includes targeted genome editing and iPS cell technologies, both of which utilizes a patient's own cells, are emerging as extremely promising approaches for significantly reducing the burden of ß-hemoglobin disorders.

Structural aspects of the interaction of anticancer drug Actinomycin-D to the GC rich region of hmgb1 gene.

The high mobility group box 1 protein has been identified as a key player in chromatin homeostasis including transcription regulation, recombination, repair, and chromatin remodeling. Emerging findings indicate HMGB1 protein over expression in nearly all types of human cancers and inflammatory disorders. Thus it is considered as a potential therapeutic target for treating various malignancies. We screened the promoter region of hmgb1 gene and selected a positive regulatory element of 25 base pair duplex (25RY) (-165 to -183) as a potential target for chemotherapeutic intervention. The molecular interaction of actinomycin (ACT) with the regulatory region of hmgb1 gene was characterized by spectroscopic, calorimetric and molecular docking studies. The hypochromic and bathochromic shift in the absorption spectrum, stabilization of 25RY duplex against thermal denaturation, perturbation of CD spectrum of duplex and enhancement of fluorescence intensity of actinomycin indicate strong binding of actinomycin to the hmgb1 promoter region (25RY).The energetics was characterized to be endothermic and entropy driven. All these results are in good agreement with in silico investigation that suggest minor groove binding with effective intercalation at GC bases of actinomycin to 25RY. This study identifies hmgb1 gene promoter region a potential target for the anticancer therapautiucs.

Dichotomous Life of DNA Binding High Mobility Group Box1 Protein in Human Health and Disease.

The High mobility group box 1 (HMGB1) protein is an extremely versatile, highly conserved nuclear protein, with its unique intracellular and extracellular functions mediated by its relatively simple domain structure. Within the nucleus, HMGB1 binds to DNA minor groove in a nonspecific manner and causes bends in the double helix thus helps in recruiting a number of DNA binding protein and transcription factors, to facilitate transcription of various genes. HMGB1 also helps in DNA repair, chromatin remodeling, V (D) J recombination, and assembly of nucleosome on the chromatin. On contrary, under pathological conditions HMGB1 displays inflammatory response by interaction with specific cell surface receptors like RAGE, TLR-4, TLR9, and TLR2 and activates NF-kB downstream signaling pathways. The upregulation of HMGB1 is directly associated with the pathogenesis of cancer, sepsis, ischemia, hemorrhagic shock, anorexia, rheumatic disease, periodontal disease etc. Therefore, HMGB1 has been considered as a promising target in the treatment of various human diseases. The interest in HMGB1 is evident and reflected in the exponential increase in the recent publications, and therefore there is a need for an update on the understanding of the role of HMGB1 in pathogenesis and its potential application of HMGB1 as a therapeutic target in a number of human diseases.

Structural, conformational and thermodynamic aspects of groove-directed-intercalation of flavopiridol into DNA.

Certain plant-derived alkaloids and flavonoids have shown propitious cytotoxic acitvity against different types of cancer, having deoxyribose nucleic acid (DNA) as their main cellular target. Flavopiridol, a semi-synthetic derivative of rohitukine (a natural compound isolated from Dysoxylum binectariferum plant), has attained much attention owing to its anticancer potential against various haematological malignancies and solid tumours. This work focuses on investigating interaction between flavopiridol and DNA at molecular level in order to decipher its underlying mechanism of action, which is not well understood. To define direct influence of flavopiridol on the structural, conformational and thermodynamic aspects of DNA, various spectroscopic and calorimetric techniques have been used. ATR-FTIR and SERS spectral outcomes indicate a novel insight into groove-directed-intercalation of flavopiridol into DNA via direct binding with nitrogenous bases guanine (C6=O6) and thymine (C2=O2) in DNA groove together with slight external binding to its sugar-phosphate backbone. Circular dichroism spectral analysis of flavopiridol-DNA complexes suggests perturbation in native B-conformation of DNA and its transition into C-form, which may be localized up to a few base pairs of DNA. UV-visible spectroscopic results illustrate dual binding mode of flavopiridol when interacts with DNA having association constant, Ka = 1.18 × 10(4) M(-1). This suggests moderate type of interaction between flavopiridol and DNA. Further, UV melting analysis also supports spectroscopic outcomes. Thermodynamically, flavopiridol-DNA complexation is an enthalpy-driven exothermic process. These conclusions drawn from this study could be helpful in unveiling mechanism of cytoxicity induced by flavopiridol that can be further applied in the development of flavonoid-based new chemotherapeutics with more specificity and better efficacy.

Interaction of adriamycin with a regulatory element of hmgb1: spectroscopic and calorimetric approach.

HMGB1 is a non-histone nuclear protein which plays important role in transcription, variable, diverse and joining (VDJ) recombination, chromatin remodeling, and DNA repair, etc. and its over expression is directly correlated with various human malignancies and inflammatory diseases. Because of the clear association between HMGB1 and cancer, we studied the binding of adriamycin (ADM), a well-known anticancer drug with the promoter region (-165 to -183) of hmgb1 by using a variety of spectroscopic, calorimetric techniques, and in-silico molecular modeling. Changes in UV and CD spectral characteristics (intensity and wavelength) of ADM and DNA associated with an induced peak (300 nm) in CD spectrum of DNA and a high binding constant of 2.0 × 10(5) M(-1) suggest a strong and stable complex formation between DNA and ADM. Scatchard analysis of spectroscopic data indicate that ADM binds to DNA in a non-cooperative nature. Further the quenching of fluorescence emission of ADM and isothermal titration calorimetry of ADM in presence of DNA points out to the intercalative mode of ADM binding to DNA which is enthalpically driven with additional small entropic contribution. Results from molecular modeling, Isothermal titration calorimetry, and Fourier transform infrared spectroscopy reveal that ADM has no marked preference between AT vs. GC base pair in binding to DNA. Therefore, hmgb1 can be considered as a novel potential chemotherapeutic target in treating cancers associated with HMGB1 upregulation.

A structural insight into major groove directed binding of nitrosourea derivative nimustine with DNA: a spectroscopic study.

Nitrosourea therapeutics occupies a definite place in cancer therapy but its exact mechanism of action has yet to be established. Nimustine, a chloroethyl nitrosourea derivative, is used to treat various types of malignancy including gliomas. The present work focuses on the understanding of nimustine interaction with DNA to delineate its mechanism at molecular level. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) has been used to determine the binding sites of nimustine on DNA. Circular dichroism (CD) spectroscopy has been used to confirm conformational variations in DNA molecule upon nimustine-DNA interaction. Thermodynamic parameters of nimustine-DNA reaction have been calculated by isothermal titration calorimetry. Results of the present study demonstrate that nimustine is not a simple alkylating agent rather it causes major grove-directed-alkylation. Spectroscopic data suggest binding of nimustine with nitrogenous bases guanine (C6 = O6) and thymine (C4 = O4) in DNA major groove. CD spectra of nimustine-DNA complexes point toward the perturbation of native B-conformation of DNA and its partial transition into C-form. Thermodynamically, nimustine-DNA interaction is an entropy driven endothermic reaction, which suggests hydrophobic interaction of nimustine in DNA-major groove pocket. Spectral results suggest base binding and local conformational changes in DNA upon nimustine interaction. Investigation of drug-DNA interaction is an essential part of rational drug designing that also provides information about the drug's action at molecular level. Results, demonstrated here, may contribute in the development of new nitrosourea therapeutics with better efficacy and fewer side effects.