Although treatment of colorectal cancer with 5-florouracil and oxaliplatin is widely used, it is frequently followed by a relapse. Therefore, there is an urgent need for profound understanding of chemotherapy resistance mechanisms as well as the profiling of predictive markers for individualized treatment. In this study, we identified the changes in 14 miRNAs in 5-fluouracil and 40 miRNAs in oxaliplatin-resistant cell lines by miRNA sequencing. The decrease in miR-224-5p expression in the 5-fluorouracil-resistant cells correlated with drug insensitivity due to its overexpression-induced drug-dependent apoptosis. On the other hand, the miR-23b/27b/24-1 cluster was overexpressed in oxaliplatin-resistant cells. The knockout of miR-23b led to the partial restoration of oxaliplatin susceptibility, showing the essential role of miR-23b in the development of drug resistance by this cluster. Proteomic analysis identified target genes of miR-23b and showed that endothelial–mesenchymal transition (EMT) was implicated in oxaliplatin insensibility. Data revealed that EMT markers, such as vimentin and SNAI2, were expressed moderately higher in the oxaliplatin-resistant cells and their expression increased further in the less drug-resistant cells, which had miR-23b knockout. This establishes that the balance of EMT contributes to the drug resistance, showing the importance of the miR-23b-mediated fine-tuning of EMT in oxaliplatin-resistant cancer cells.

MicroRNAs (miRNAs) are critical regulators of the functional pathways involved in the pathogenesis of cardiovascular diseases. Understanding of the disease-associated alterations in tissue and plasma will elucidate the roles of miRNA in modulation of gene expression throughout development of sporadic non-syndromic ascending thoracic aortic aneurysm (TAA). This will allow one to propose relevant biomarkers for diagnosis or new therapeutic targets for the treatment. The high-throughput sequencing revealed 20 and 17 TAA-specific miRNAs in tissue and plasma samples, respectively. qRT-PCR analysis in extended cohort revealed sex-related differences in miR-10a-5p, miR-126-3p, miR-155-5p and miR-148a-3p expression, which were the most significantly dysregulated in TAA tissues of male patients. Unexpectedly, the set of aneurysm-related miRNAs in TAA plasma did not resemble the tissue signature suggesting more complex organism response to the disease. Three of TAA-specific plasma miRNAs were found to be restored to normal level after aortic surgery, further signifying their relationship to the pathology. The panel of two plasma miRNAs, miR-122-3p, and miR-483-3p, could serve as a potential biomarker set (AUC = 0.84) for the ascending TAA. The miRNA-target enrichment analysis exposed TGF-β signaling pathway as sturdily affected by abnormally expressed miRNAs in the TAA tissue. Nearly half of TAA-specific miRNAs potentially regulate a key component in TGF-β signaling: TGF-β receptors, SMADs and KLF4. Indeed, using immunohistochemistry analysis we detected increased KLF4 expression in 27% of TAA cells compared to 10% of non-TAA cells. In addition, qRT-PCR demonstrated a significant upregulation of ALK1 mRNA expression in TAA tissues. Overall, these observations indicate that the alterations in miRNA expression are sex-dependent and play an essential role in TAA via TGF-β signaling.

Altered expression of miRNAs in tumor tissue encourages the translation of this specific molecular pattern into clinical practice. However, the establishment of a selective biomarker signature for many tumor types remains an inextricable challenge. For this purpose, a preclinical experimental design, which could maintain a fast and sensitive discovery of potential biomarkers, is in demand. The present study suggests that the approach of 3D cell cultures as a preclinical cancer model that is characterized to mimic a natural tumor environment maintained in solid tumors could successfully be employed for the biomarker discovery and validation. Subsequently, in this study, we investigated an environment-dependent miRNA expression changes in colorectal adenocarcinoma DLD1 and HT29 cell lines using next-generation sequencing (NGS) technology. We detected a subset of 16 miRNAs differentially expressed in both cell lines cultivated in multicellular spheroids compared to expression levels in cells grown in 2D. Furthermore, results of in silico miRNA target analysis showed that miRNAs, which were differentially expressed in both cell lines grown in MCS, are involved in the regulation of molecular mechanisms implicated in cell adhesion, cell-ECM interaction, and gap junction pathways. In addition, integrins and platelet-derived growth factor receptors were determined to be the most significant target genes of deregulated miRNAs, which was concordant with the environment-dependent gene expression changes validated by RT-qPCR. Our results revealed that 3D microenvironment-dependent deregulation of miRNA expression in CRC cells potentially triggers essential molecular mechanisms predominantly including the regulation of cell adhesion, cell–cell, and cell–ECM interactions important in CRC initiation and development. Finally, we demonstrated increased levels of selected miR-142-5p in rectum tumor tissue samples after neoadjuvant long course treatment compared to miR-142-5p expression levels in tumor biopsy samples collected before the therapy. Remarkably, the elevation of miR-142-5p expression remained in tumor samples compared to adjacent normal rectum tissue as well. Therefore, the current study provides valuable insights into the molecular miRNA machinery of CRC and proposes a potential miRNA signature for the assessment of CRC in further clinical research.

Background: Colorectal cancer (CRC) is the third most prevalent cancer worldwide. Despite substantial advancements in CRC therapy in recent years, ionizing radiation (IR) continues to be the predominant treatment for colon malignances. However, it still lacks the precision required for excellent therapeutic outcomes, ultimately resulting in tumor radioresistance. This study seeks to explore the potential of atypical PARPs including PARP9, PARP12, PARP13 and PARP14 as innovative radiosensitizing targets for CRC. Methods: We utilized CRISPR/Cas9-mediated gene editing to knockout the PARP9, PARP12, PARP13 and PARP14 in HT29 and DLD1 cells. The cells were exposed to either a single dose of 6–10 Gy or to fractionated dose of 5 × 2 Gy X-ray radiation cultivating cells in 2D, laminin-rich ECM 3D and multicellular spheroid models. The transcriptomes of nonirradiated and irradiated cells were analyzed using microarrays. Gene set enrichment analysis was conducted to determine the pathways in which PARP13 is engaged. Cell viability was assessed using a clonogenic assay. Gene expression levels in cells and patient samples were quantified using RT-qPCR. Results: The expression of PARP9, PARP12, PARP13 and PARP14 was particularly elevated in irradiated colorectal cancer HT29 cells in a microenvironment-dependent manner. PARP13 deficiency significantly enhanced the sensitivity of HT29 cells to both single-dose and multifractionated irradiation regimens, resulting in reduced colony formation and spheroidal integrity. Microarray analysis indicated that PARP13 may modulate the expression genes associated with immune response signaling pathways, including members of PARP family. Furthermore, PARP13 loss in HT29 cells markedly impaired the expression of immune response related genes following multifractionated ionizing irradiation. Finally, chemoradiotherapy significantly elevated the expression of PARP9, PARP12, PARP13 and PARP14 in rectal tumors, while having no effect on adjacent normal colon tissues. Elevated pre-treatment PARP9 expression levels and a blunted post-treatment increase in PARP9 and PARP14 expression predicted poor overall survival in rectal cancer patients, while PARP13 emerged as the most significant discriminator between tumor and healthy tissue. Conclusions: Collectively, the PARP9/13/14 axis is implicated in the response of CRC to radiation treatment in both preclinical and clinical settings, demonstrating the atypical members of the PARP family as attractive targets for neoadjuvant radiotherapy.