Epigenetic approaches in direct correlation with assessment of critical genetic mutations in non-small cell lung cancer (NSCLC) are currently very intensive, as the epigenetic components underlying NSCLC development and progression have attained high recognition. In this level of research, established human NSCLC cell lines as well as experimental animals are widely used to detect novel biomarkers and pharmacological targets to treat NSCLC. The epigenetic background holds a great potential for the identification of epi-biomarkers for treatment response however, it is highly complex and requires precise definition as these phenomena are variable between NSCLC subtypes and systems origin.

The intracellular topography of RNA molecules, encompassing ribonucleotides with biochemical modifications, such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), adenosine to inosine (A → I) editing, and isomerization of uridine to pseudouridine (Ψ), as well as of non-coding RNA molecules, is currently studied within the frame of the epigenome. Circulating RNA molecules in the intracellular space that have incorporated information by carrying specific modifications depend on the balanced activity and correct subcellular installation of their modifying enzymes, the “writers”, “readers” and “erasers”. Modifications are critical for RNA translocation from the nucleus to the cytoplasm, for stability and translation efficiency, and for other, still-uncovered functions. Moreover, trafficking of non-coding RNA molecules depends on membrane transporters capable of recognizing signal sequences and RNA recognition-binding proteins that can facilitate their transport to different intracellular locations, guiding the establishment of interconnection possibilities with different macromolecular networks. The potential of long non-coding RNAs to form multilayer molecular connections, as well as the differential topology of micro-RNAs in cell nuclei, compared to cytoplasm, has been recognized by several studies. The study of the intercellular compartmentalization of these molecules has recently become feasible thanks to technological progress; however, a wealth of information has not yet been produced that would lead to safe conclusions regarding non-coding RNA’s contributions to the early steps of pathogenesis and disease progression in hematological malignancies. Both, the bone marrow, as the main hematopoietic tissue, and the lymphoid tissues are composed of cells with highly reactive potential to signals affecting the epigenome and initiating cascade pathways in response. Independently or in combination with coexistent driver genetic mutations, especially mutations of enzymes involved in epigenomic surveillance, intracellular microenvironmental alterations within the cell nuclear, cytoplasmic, and mitochondrial compartments can lead to disorganization of hematopoietic stem cells’ epigenomes, promoting the generation of hematological malignancies. In this review, we discuss the various intracellular processes that, when disrupted, may result in the ectopic placement of RNA molecules, either inducing specific modifications or non-coding molecules or promoting hematological malignant phenotypes. The crosstalk between mitochondrial and nuclear genomes and the complex regulatory effects of mis-localized RNA molecules are highlighted. This research approach may constitute a field for new, more specifically targeted therapies in hematology based on RNA technology.

The intracellular topography of RNA molecules, encompassing ribonucleotides with biochemical modifications, such as N6-methyladenosine(m6A), 5-methylcytosine (m5C) and isomerization of uridine to pseudouridine (Ψ), as well as of the non-coding RNA molecules, are currently studied within the frame of the epigenome. Circulating RNA molecules in the intracellular space, that have incorporated information by carrying specific modifications, depend on the balanced activity and correct subcellular installation of their modifying enzymes, the “writers”, “readers” and “erasers”. Modifications are critical for RNA translocation from nucleus to cytoplasm, stability and translation efficiency and other, still uncovered functions. Moreover, intracellular movement of non-coding RNA molecules depends on membrane transporters, capable of recognizing signal sequences and RNA recognition-binding proteins, that can facilitate their transport to different intracellular locations, guiding the establishment of interconnection possibilities with different macromolecular networks. The potential of long non-coding RNAs to form multilayer molecular connections, as well as the differential topology of micro-RNAs in cell nucleus over cytoplasm have been recognized by several studies. The study of the intracellular compartmentalization of these molecules has recently become feasible thanks to technological progress, however, a wealth of information has not yet been produced that would lead to safe conclusions, regarding non-coding RNA contribution to the early steps of pathogenesis and disease progression of hematological malignancies. Both the bone marrow, as the main hematopoietic tissue, and the lymphoid tissues are composed of cells with high potential of reaction to signals affecting the epigenome and initiating cascade pathways in response. Independently or in combination with the coexistent driver genetic mutations, especially of enzymes involved in epigenomic surveillance, intracellular microenvironmental alterations within the cell nuclear, cytoplasmic and mitochondrial compartments, can lead to disorganization of hematopoietic stem cells’ epigenome, promoting the generation of hematological malignancies. In this review, we discuss the various intracellular processes that, when disrupted, may result in the ectopic placement of RNA molecules, either inducing specific modifications or non-coding molecules, promoting hematological malignant phenotypes. The crosstalk between mitochondrial and nuclear genomes and the complex regulatory effects of mis-localized RNA molecules is highlighted. This research approach may constitute a field for new, more specifically targeted therapies in hematology, based on the RNA technology.

Established epigenetic mechanisms, including nuclear (n)DNA methylation, are also known to regulate the mitochondrial (mt) genome despite its lack of histones. However, studies referring to evaluations of mtDNA methylation are often influenced by technical and biological confounders. We present an optimized protocol for extracting mtDNA from peripheral blood and bone marrow mononuclear cells, followed by steps for qualitative assessment to exclude nDNA contaminations and a downstream quantitative analysis of the mtDNA global methylation profile using liquid chromatography combined with mass spectrometry.

Epigenetic approaches in direct correlation with assessment of critical genetic mutations in non-small cell lung cancer (NSCLC) are currently very intensive, as the epigenetic components in NSCLC development and progression have attained high recognition. In this level of research, established human NSCLC cell lines as well as experimental animals are widely used to detect novel biomarkers and pharmacological targets to treat NSCLC. The epigenetic background of NSCLC is highly complex and requires precise definition as these phenomena are variable between NSCLC subtypes and systems origin. We engaged an in-depth characterization of non-coding (nc)RNAs prevalent in human KRAS-mutant NSCLC cell lines A549 and H460 and mouse KRAS-mutant NSCLC tissue by Next Generation Sequencing (NGS) and quantitative Real Time PCRs (qPCRs). Also, the effect of transcription factor (TF) LRF, a known epigenetic silencer, was examined on the epigenome modulation. Finally, interacting networks underlying epigenetic variations in NSCLC subtypes were created. Data derived from our study highlights the divergent epigenetic profiles of NSCLC of human and mouse origin, as well as the significant contribution of 12qf1: 109,709,060-109,747,960 mouse chromosomal region to micro-RNA upregulated species. Fur-thermore, the novel epigenetic miR-148b-3p/PVT1/ZBTB7A axis was identified, which differentiates lung adenocarcinoma from large cell lung carcinoma, two characteristic NSCLC subtypes. The detailed recording of epigenetic events in NSCLC and combinational studies including networking between ncRNAs and TFs validate the identification of significant epigenetic features, prevailing in NSCLC subtypes and among experimental models. Our results enrich knowledge in the field and empower research on the epigenetic prognostic biomarkers of the disease and patient-tailored treatments.