Establishment of YAP1 knockdown system induced by tetracycline and its effect on the function of gastric cancer cells
- Author:
He WANG
1
Author Information
- Publication Type:Journal Article
- Keywords: Gastric cancer; Migration; Proliferation; Small harp RNA; Tetracycline-induced expression system; YAP1 gene
- From: Journal of Xi'an Jiaotong University(Medical Sciences) 2020;41(5):690-695
- CountryChina
- Language:Chinese
- Abstract: Objective: To establish a tetracycline-induced gene knockdown system and study the effect of YAP1 on the function of gastric cancer cells. Methods: We constructed pLKO.1-tetON-YAP1 knock-down lentivirus and detected the vector by enzyme digestion and sequencing. Gastric cancer cell lines SGC-7901 and MKN-28 were infected with lentivirus, and YAP1 knocked-down gastric cancer cell lines induced by DOX were established. The mRNA level of YAP1 was detected by RT-qPCR, and the protein level of YAP1 was detected by Western blotting. Cell proliferation was detected by plate cloning experiment, and cell migration was detected by scratch-healing assay and Transwell assay. Results: The results of double enzyme digestion showed two bands at 6 000 bp and 3 000 bp, and that the sequencing results were consistent with the designed shRNA sequence. In the DOX-induced group, the mRNA and protein levels of YAP1 in gastric cancer cells infected with pLKO.1-tetON-YAP1 lentivirus significantly decreased compared with those in non-induced group. In the plate cloning experiment, the number of clones in shYAP1 groups decreased significantly after DOX induction, but there was no significant change in the non-induced group. Scratch-healing assay and Transwell assay showed that after DOX induction, the cell migration ability of shYAP1 groups was inhibited, but without significant change in the non-induced group. Results: We have successfully established a tetracycline-induced lentivirus system, and knocked down YAP1 gene of gastric cancer cells with this system. The proliferation and migration of gastric cancer cells are inhibited by YAP1 in this tetracycline induced lentivirus system.
