ARTICLE | doi:10.20944/preprints202106.0102.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: colorectal cancer; alternative splicing; mucins; biomarkers; precision medicine
Online: 3 June 2021 (11:30:37 CEST)
Colorectal cancer prognosis get worse with advancement of disease into metastatic stage. There is a pertinent need to develop prognostic biomarkers that can be used for personalized and precision medicine. Alternative splicing provides an insight into understanding of changes at isoform expression level which may not be evident at gene level. In this direction, we utilized our prior knowledge about significant alternatively spliced genes and chose ADAM12 and MUC4 for further characterization in a metastatic cell line model. These genes were found to be good prognostic indicators in The Cancer Genome Atlas database. We studied the gene organization and designed primers to specifically amplify a group of isoforms. Differential expression of these group of isoforms was observed in normal, primary and metastatic colorectal cancer cell lines. We further validated the results using sanger sequencing. Isoform expression was found to respond to the 5-fluorouracil treatment. RNAseq analysis of the cell lines further validated the differential expression of gene isoforms. Successful detection of ADAM12 and MUC4 in cell lysates varied according to the antibody used which may reflect differential expression of isoforms. This comprehensive study underscores the importance of studying alternatively spliced isoforms and their probable used as prognostic or predictive biomarkers.
ARTICLE | doi:10.20944/preprints202204.0082.v1
Subject: Biology And Life Sciences, Agricultural Science And Agronomy Keywords: Caco2/HT-29 cells; lipopolysaccharide(LPS); mucins; focal adhesion pathway; ECM receptor in-teraction pathway
Online: 8 April 2022 (14:04:17 CEST)
Endotoxins are toxic substances that widely exist in the environment and can enter the intestine with food and other substances. Intestinal epithelial cells are protected by a mucus layer that contains MUC2 as its main structural component. However, a detailed understanding of the mechanisms involved in the function of the mucus barrier in endotoxin penetration is lacking. Here, we established the most suitable proportion of Caco-2/HT-29 co-culture cells as a powerful tool to evaluate the intestinal mucus layer. Our findings significantly advance current knowledge as focal adhesion and ECM-receptor interaction were identified as the two most significantly implicated pathways in MUC2 small interfering RNA (siRNA)-transfected Caco-2/HT-29 co-culture cells after 24 h of LPS stimulation. When the mucus layer was not intact, LPS was found to damage the tight junctions of Caco-2/HT29 co-cultured cells. Furthermore, LPS was demonstrated to inhibit the integrin-mediated focal adhesion structure and damage the matrix network structure of the extracellular and actin microfilament skeletons. Ultimately, LPS inhibited the interactive communication between the extracellular matrix and the cytoskeleton for 24 h in the siMUC2 group compared with the LPS(+) and LPS(-) groups. Overall, we recognized the potential of MUC2 as a tool for barrier function in several intestinal bacterial diseases.
ARTICLE | doi:10.20944/preprints202301.0150.v2
Subject: Biology And Life Sciences, Cell And Developmental Biology Keywords: oral; mucosa; mucins; tight junctions; wound-healing; E-liquids; cytotoxicity; viability; confluency.
Online: 3 April 2023 (03:33:37 CEST)
Background: Expansion of OKF6/TERT-2 oral epithelial cells in vitro is important for studying the molecular biology of disease and pathology affecting the oral cavity. Keratinocyte Serum-Free Medium (KSFM) is the medium of choice for this cell line. This study compares three media for OKF6/TERT-2 cultures: KSFM, Dulbecco’s Modified Eagle Medium/Nutrient Mixture of Hams F-12 (DMEM/F12) and a composite medium comprised of DMEM/F-12 and KSFM (1:1 v/v), referred as DFK. The toxicological effects of electronic cigarette liquids (E-liquids) on OKF6/TERT-2 cells cultured in these media were also compared. Methods: Cells were cultured in KSFM, DMEM/F12 or DFK and cellular morphology, growth, wound healing and gene expression of mucins and tight junctions were evaluated. Additionally, cytotoxicity was determined after E-liquid exposures. Results: Switching from KSFM to DMEM/F12 or DFK 24-hours post-seeding leads to typical cellular morphologies, and these cultures reach confluency faster than those in KSFM. Wound-healing recovery occurred fastest in DFK. Except for claudin-1, there is no difference in expression of the other genes tested. Additionally, E-liquid cytotoxicity appears to be amplified in DFK cultures. Conclusions: DMEM/F12 and DFK are alternative media for OKF6/TERT-2 cell culture to study molecular biology of disease and pathology, provided cells are initially seeded in KSFM.