Construction and Identification of a Yeast Two-Hybrid Bait Vector and Its Effect on the Growth of Yeast Cells and the Self-Activating Function of Reporter Genes for Screening of HPV18 E6-Interacting Protein

By using a yeast two-hybrid system,a yeast two-hybrid bait vector was constructed and identified for screening of the HPV18 E6-interacting proteins,and its effects on the growth of yeast cells and the activation of reporter genes were investigated.Total mRNA extracted from Hela cells was reversely transcribed into cDNA.Fragment of HPV18 E6 cDNA was amplified using RT-PCR and directly ligated to the pGBKT7 vector.The recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing.Th...

Mechanism of Chinese herbal formula QHF against breast cancer MCF-7 cells invasion and migration

Objective： To investigate the effects of Chinese herbal formula Qinghuofu（QHF） on the migration and invasion of breast cancer MCF-7 cells and its possible molecular mechanisms, thereby providing a theoretical basis to find effective anti-cancer medicine and therapeutic targets for the treatment of anti-migration and anti-invasion of breast cancer.Methods： Breast cancer MCF-7 cells were treated with different QHF and other different reagents, CCK8 assay was used to detect the influence of the reagents on the proliferation of MCF-7 cells; Scrape migration and Transwell assay were used to quantitatively determine the migration and invasion effects of QHF and hepatocyte growth factor（HGF） on the MCF-7 cells. Subsequently, the c-Met inhibitor and its downstream ERK and PI3 K inhibitors were used to investigate the relationship between the migration and invasion of MCF-7 cells, as well as its downstream MAPK/ERK and PI3 K/Akt signaling pathways. The expression levels of HGF, c-Met, ERK, p-Akt, p-c-Met, p-ERK, p-Akt, MMP2, MMP9, and VEGF in breast cancer MCF-7 cells treated with QHF and other reagents were also examined.Results： The result indicated that formula QHF not only significantly inhibited the proliferation of MCF-7 cells, but also significantly suppressed the effects of HGF（40 ng/m L） on the proliferation and movement of MCF-7 cells, reducing the ability of the cells to invade and migrate. Western blot analysis indicated that QHF and c-Met inhibitor significantly decreased the expression of p-c-Met, p-ERK1, p-ERK2, p-Akt, MMP-2, MMP-9, and VEGF, while HGF significantly increased the expression of p-c-Met in MCF-7 cells; c-Met downstream ERK and PI3 K inhibitors also significantly decreased the expression of MMP-2, MMP-9, and VEGF in MCF-7 cells; But the difference among c-Met, PI3 K, ERK, and QHF group were not statistically significant.Conclusion： QHF can prevent the proliferation, migration, and invasion of MCF-7 cells by inhibiting the HGF/c-Met and its downstream PI3 K/Akt and MAPK/ERK signaling pathways; Thereby down-regulating the expression of HGF, p-Met, p-ERK1, p-ERK2, p-Akt, MMP-2, MMP-9, and VEGF.