LPS Activates CUL4A/NF-κB Signaling To Promote Gastric Cancer Cells Invasion

Objective:To evaluate the impacts of CUL4 A on gastric cancer cells invasion promoted by LPS and clarify the mechanisms of how CUL4 A regulate the NF-κB signaling to take part in it. Methods:1. Human gastric cancer HGC27 cells transfected with CUL4 A si RNA or NC. Then, transwell assays and wound healing assays were used to evaluate the cell migration and invasion.2. Western blot was used to investigate the expression of CUL4 A and NF-κB induced by LPS,and found the best time and concentration of LPS stimulation.3. To test the efficiency of the si RNA construct to knockdown CUL4 A expression by real-time PCR and western blot. In the best time and concentration of LPS stimulation, western blot was used to detected the expression of NF-κB protein, and real-time PCR was used to detected the m RNA of NF- k B downstream genes following CUL4 A knockdown.4. Two step immunohistochemistry(IHC) and western blot was used in this study to detect the expression of CUL4 A and NF-κB in gastric cancer and para-carcinoma tissues at the same time, then analysis the relation of CUL4 A and NF-κB. Result:1. The result of Transwell assays and wound healing assays were indicated that LPS could enhance the migratory and invasive abilities of gastric cancer cells, but knockdown of CUL4 A would suppressed it.2. CUL4 A and NF-κB expression were upregulated by LPS in a concentrationdependent manner up to 100 ng/m L at 2 hour.3. The date of real-time PCR and Western blot show that CUL4 A expression was significantly reduced at both the protein and m RNA levels in comparison control si RNA transfeced cells. At the same time, CUL4 A downregulation by si RNA resulted in a significant reduction of NF-κB response to LPS stimulation. Moreover, real-time PCR showed the m RNA level of NF-κB downstream genes were decreased in gastric cancer cells transfected with CUL4 A si RNA compared with those transfected with scrambled si RNA.4. The result of immunohistochemistry found that the positive expression of CUL4 A protein as a medium brown or brownish yellow stain in the cytoplasm and/or nucleus of tumor tissues, and NF-κB was mainly localized in the nucleus. Moreover, Correlation analysis revealed that CUL4 A was positively related to nuclear expression of NF-κB expression. And Western blot assay demonstrated that CUL4 A and NF-κB were overexpression in the GC tissues. Conclusion:CUL4A could regulate the the NF-κB signaling to promote gastric cancer cells invasion in LPS stimulation.