Establishment Of MARVELD1-Tet-on Cell Line And Analysis Of Mechanical Property

MARVELD1is a tumor suppressor which was firstly found in our research group.Its accession number of GenBank is AF325259. Our research group clarifiedMARVELD1has an influence on biological process of cell adhesion and cell migrationwhen paid close attention to its function study earlier. The reduction of homogeneityadhesion and the increase of heterotypic adhesion between tumor cell and surroundingtissue, is a prerequisite of metastasis and invasion of malignant tumor, and thebiological process such as proliferation, differentiation and migration have a closerelationship with mechanical property in tumor cells. It suggested that MARVELD1andthe change of adhesion of cell surface and mechanical properties of the cell itself mayhave a close relationship.To establish controllability gene expression system is extremely important for theresearch of gene function. So this project chose a widely recognized gene expressionregulation system, Tet system, was trying to build a cell line which could regulateexpression of MARVELD1. We selected HepG2cell line which has no MARVELD1expression to construct the MARVELD1-Tet-on cell line. Depending on Tet systemManual, we transfected pTet-on plasmid and pTRE2hygMARVELD1recombinant thento screen cells with stability. We extracted protein of dox induced/non-dox inducedMARVELD1-Tet-on cell clone, through western blot test, got a cell line with highinduction efficiency and low background.We also observed the cell surface morphology of dox induced/non-dox inducedMARVELD1-Tet-on cell line separately by using optical microscopy at high resolutionand SEM to taked a look at how MARVELD1effects on cell morphology and adhesion.It showed that dox induced MARVELD1-Tet-on cell line has a better spreadability andmore cell junctions. It demonstrated that MARVELD1has an influence on cellularmorphology and cell adhesion. At last, we observed dox induced/non-dox inducedMARVELD1-Tet-on cell line separately under the liquid of atomic force microscopyobservation of living, access to the cell surface topography and the several groups offorce-distance curve. As the process of atomic force microscope in contact with thebiological samples satisfied approximately Hertz contact theory, so has taken thestatistical analysis of the force-distance curve to Hertz model, will get the young’smodulus of samples. To campare Young’s modulus between dox induced/non-doxinduced MARVELD1-Tet-on cell line, it demonstrated that dox inducedMARVELD1-Tet-on cell has a firmer surface.