Abstract
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.
Highlights
Colorectal cancer (CRC) is the most common malignancy of the gastrointestinal tract
The cell growth in multicellular spheroid (MCS) is followed by the formation of the heterogeneous cellular zones within spheroid which was highly anticipated in the present study
To observe fully developed characteristic proliferation zones in MCS, CRC cells were cultivated for 6 days
Summary
Colorectal cancer (CRC) is the most common malignancy of the gastrointestinal tract. Approximately 1.23 million new cases are estimated each year worldwide, which defines CRC as the third leading oncologic disease after lung and breast cancer [1]. Detected CRC is effectively cured by surgical tumor resection, cancer diagnosed at advanced stages leads to poor 12.5% five-year survival, regardless of anti-cancer treatment applied [2]. The progression of colorectal cancer is an asymptomatic process and its clinical manifestations are observed only when the adjacent tissues are invaded. Another challenging feature of CRC is its exclusively rapid development, which exacerbates full recovery even when the early stage is detected. The early diagnosis, prognosis, and evaluation of the current status of disease development of CRC could be greatly facilitated by cancer-specific molecular biomarkers [3]
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