NF-?B-mediated LPS Promotes Expression Of CXCR4 In Gallbladder Cancer Cells

Background: CXC chemokine receptor-4(CXCR4)is closely related to tumor proliferation,adhesion,invasion and metastasis.The preliminary study found that CXCR4 expression and gallbladder cancer proliferation,invasion is positively correlated,but the mechanism is unknown.Long-term chronic inflammation of the gallbladder can promote the development of gallbladder cancer.It is suggested that the expression of inflammatory mediator LPS(lipopolysaccharide)is related to the expression of CXCR4 in gallbladder carcinoma cells.Therefore,this subject intends to carry out research on two parts: 1,LPS on the expression of CXCR4 in gallbladder cancer cells;2,LPS induced gallbladder cancer cells to express CXCR4 molecular mechanism.Methods: 1.LPS enhances expression of CXCR4 by gallbladder cancer cells by NF-?B.1)The expression of CXCR4 in LPS group was significantly higher than that in control group(0.998 ± 0.10 vs 0.5977 ± 0.17),the difference was statistically significant(P <0.05).2)The expression of CXCR4 in si-NF-?B group was significantly lower than that in control group(0.3718 ± 0.10 vs 0.5898 ± 0.17),the difference was statistically significant(P <0.05).2.LPS to promote the expression of CXCR4 in gallbladder cancer cells 1)successfully constructed a series of recombinant plasmids containing CXCR4 promoter truncated fragments and sequenced.The results of double luciferase system showed that PGL4-319 was higher than PGL4-131(2.32 ± 0.04 vs 1.10 ± 0.04)(P <0.05),suggesting that there may be a regulatory site for regulating CXCR4 transcription activity in this interval from-319 to-131 nt.The 2)TFbind,Promoter Scan software showed that there were two potential NF-?B binding sites between the-319 and-131 nt regions of the ATCR upstream of the CXCR4 promoter transcription site: NF-?B binding site: CAGCAGGGTCC(-319 to-308nt)and CCTGGGCTTCCCAA(-307 to-293 nt);Were named NF-?B(1),NF-?B(2),respectively.3)Site-directed mutagenesis,electrophoretic mobility shift analysis(EMSA)and chromatin immunoprecipitation(Ch IP)experiments confirmed that the transcription factor NF-?B could bind directly to the NF-?B site of the CXCR4 promoter-319 to-131 nt,and LPS enhances the binding of NF-?B to this site.4)The effect of PGL4-NF-?B mut1(0.36 ± 0.06)on the phosphorylation activity of PGL4-319(0.61 ± 0.07)was significantly attenuated by the effect of NF-?B mutation on LPS-induced CXCR4 promoter activity.(P <0.05)."PGL4-NF-?B mut2"(0.61 ± 0.16)was not significantly attenuated compared with the control plasmid PGL4-319,and the difference was not statistically significant.The results showed that LPS could be detected by mutant 1 The promoter activity of CXCR4 was regulated by binding site,the difference was statistically significant(P <0.05).3.LPS enhances CXCR4 promoter activity by NF-?B.1)Effect of silencing NF-?B on LPS-induced CXCR4 promoter activity: The activity of PGL4-320 induced by LPS-induced si NF-?B NOZ cells was weaker than that of NOZ cells(0.44 ± 0.21 vs 0.57 ± 0.15)(P <0.05)..Indicating that LPS enhances CXCR4 promoter activity by NF-?B.Conclusions: 1,CXCR4 promoter-319 to-131 nt interval there is an active NF-?B binding site,and LPS can enhance the binding of NF-?B and this site.