Chromosome Kinesin KIF4A Affects Gastric Cancer Cell Migration And Invasion By Interacting With The Membrane Protein Supervillin

Gastric cancer is one of the highest mortality rates of malignant disease all over the world. Gastric cancer metastasis is the main factor that lead to its high fatality rate. At present, there has been widely studied for the pathogenesis of gastric cancer and its treatment, but still no good treatment for gastric cancer, the gastric cancer still is one of the major diseases threatening human life and health. In order to investigate the molecular mechanism of clinical gastric cancer metastasis, find effective potential molecular targets for the treatment of gastric cancer, on the basis of previous work, we study the chromokinesin KIF4A regulating of gastric cancer cell migration.Studies have suggested that the chromokinesin KIF4A is the guarantee of normal cell division, a key protein KIF4A abnormal expression will lead to occurrence and development of tumor. However, KIF4A involved in regulating tumor metastasis process are reported rarely, we further study the KIF4A protein molecule’s regulation function on gastric cancer cell migration, thus illustrating KIF4A in regulating the molecular mechanism in the process of tumor metastasis.Supervillin (SVIL) is a membrane protein binding with F-actin, expressed in the human body as a outer membrane protein commonly, studies have shown that SVIL played a role in the process of cancer cell invasion, and through the yeast two hybrid experiment proved that the combined with KIF4A in the cell, because of these we guess that whether KIF4A regulating the molecular mechanism of tumor cell metastasis process due to SVIL, and on the assumption that we design the related experimental to verify.This research can be carried out according to the following aspects:1. We use Lipofectamine 2000 transfecting pGPU6-GFP-KIF4A and pGPU6-GFP-NC plasmid and pEGFP-C1, pEGFP-KIF4A and pEGFP-SVIL plasmid into SGC-7901 cells respectively, we construct the KIF4A lower expression SGC cell lines, KIF4A high expression SGC cell line, SVIL high expression SGC cell line and two comparing SGC cell lines using G418 screening, and then the cell lines were identified using immunofluorescence staining and Western Blot assays.2.We proceed wound healing and Transwell experiments using KIF4A lower expression gastric cancer cell lines and KIF4A high expression gastric cancer cell lines, observed KIF4A’s effecting on SGC-7901 gastric cancer cell migration.3. We conducted immunofluorescence assay with staining vinculin in the KIF4A lower expression gastric cancer cell lines and KIF4A high expression gastric cancer cell lines.and then counted the number of vinculin, applied Origin8.0 mapping software in making a bar chart, observed KIF4A’s affection on gastric cancer cell SGC-7901 invasion.4. Using IP technology and immunofluorescence assay to prove KIF4A combine with supervillin in molecular and cellular level respectively, laid a foundation for further study of KIF4A interaction between SVIL.5. Analyze the supervillin molecular function structure domain, using the molecular cloning technology to build Flag-SVIL truncated mutants, which, in turn, can study KIF4A with supervillin functional domains.