Study On The Mechanism Of How Fusobacterium Nucleatum Promote Incidence And Development Of CRC

objective: To study the influence of Fusobacterium nucleatum(Fn)LPS on PAK1?PAK1(Thr423)?beta-catenin?beta-catenin(S675)?Cyclin-D1?C-myc's expression when Fn LPS upregulates the expression of TLR4,which can help us to explore whether Fn plays a role in colorectal cancer process.Methods : Testing the expression of PAK1?TLR4 by immunohistochemical staining in colorectal cancers (CRCs) ?hyperplastic polyps (HPs)?adenomas (ADs) tissue; Detecting Fn by 16S rRNA fluorescence in situ hybridization (FISH) oligonucleotide probes to determine the presence and distribution of Fn in proximal CRC?distal CRC?SSA?proximal HP?distal HP?proximal TA?distal TA?CRC and matched lymph nodes with or without metastases tissues; SW480 cells incubated with LPS extracted from Fn which isolated and cultured from clinical CRC specimens, at the same time using commercial LPS incubate with SW480 cells the same condition as Fn LPS,and then extracting cell protein, testing it by western-blot to analyze the influence of Fn on TLR4?PAK1?PAK1(Thr423)?beta-catenin?beta-catenin(S675)? Cyclin-D1?C-myc's expression. To further investigate the interaction effect between intracellular proteins, we use TLR4 specific antagonist TAK-242 and PAK1 specific antagonist IPA-3 to incubate with SW480 cells, and then extract the cell proteins ,testing by western-blot to see what kind of expression changes the intracellular downstream proteins will be with TLR4?PAK1 inhibiton by specific antagonists. Moreover, we use Cell immunofluorescence to investigate expression and cellular location changes of beta-catenin(S675) after the use of Fn LPS?TAK-242?IPA-3 on SW480 cells. Results: (1)Immunohistochemical staining: the expression of PAK1?TLR4?beta-catenin in CRC?TA?HP tissues is high(P>0.05). What's more, there's a obvious nuclei gathered phenomenon of beta-catenin in CRC;(2)FISH: Fn were detected in 89.6% of proximal CRCs?65.7% of proximal HPs, 78.8% of SSAs, 28.9% of proximal TAs,42.2% of distal CRCs,47.5% of distal HPs,24.4% of distal TAs; Fn was present in 100% CRC matched lymph nodes with metastases and 40% of matched lymph nodes without metastases. Fn was prevalent in proximal SPs but rare in TAs(P<0.05), and Fn was more frequent in proximal CRCs and matched metastatic lymph nodes than that in distal CRCs and matched nonmetastatic lymph nodes(P<0.05).(3)Western-blot:Fn LPS and commercial LPS both have the same effection on Cyclin-D1?C-myc?PAK1?PAK1(Thr423)?beta-catenin ? beta-catenin(S675) ? TLR4 expression in SW480 cells. The rising expression tendency of TLR4?PAK1(Thr423)?beta-catenin(S675)?Cyclin-D1?C-myc shows in SW480 cells when it incubated with those two kind of LPS along with the time extending (0,2,6,12,24h)(P<0.05),and this rising expression tendency is more obvious when cell incubated with Fn LPS. When we incubate SW480 cells with TAK-242, the expression of PAK1(Thr423)?beta-catenin(S675)?Cyclin-D1 shows a decline tendency with time extending (0, 2, 6, 12, 24h) (P<0.05); we also incubate SW480 cells with IPA-3 ,and the expression of PAK1(Thr423)?beta-catenin(S675)?Cyclin-D1 shows a decline tendency(P<0.05) but TLR4 shows a rising tendency with time extending (0, 2, 6, 12, 24h) .Put these results together,we can conclude that Fn upregulates the expression of TLR4 by its LPS, with upregulation of TLR4 the phosphorylation of PAK1 S423? beta-catenin S675 is enhanced to stimulate beta-catenin signaling pathway, maybe this is how Fn promoting the incidence and development of CRC.(4)Using Fn LPS?TAK-242? IPA-3 incubated with SW480 cells, respectively, and then by Cell immunofluorescence we can observe that cells incubated with TAK-242 ?IPA-3 prior to LPS, the expression of beta-catenin(S675) is lower than that in cells only cultivated with LPS. Also beta-catenin(S675) in cells only cultivated with LPS is obviously gathering in nuclei. Conclusion: by stimulating TLR4/ PAK1(Thr423)/ beta-catenin(S675) signaling pathway, Fn LPS can promote the incidence and development of CRC.