The Roles And Mechanisms Of RIP1 Promote The Proliferation, Invasion And Matastasis Of Gallbladder Carcinoma

Background and aim TNF-α owns the ability of promoting the proliferation and invasion of gallbladder cancer cells. Receptor-interacting protein-1(RIP1) is one key protein in the TNF-α-TNFR1 signal pathway. But the roles and mechanisms of RIP1 are rarely reported in tumors. So, we conduct this study to test the expression of RIP1 in the gallbladder cancer specimens and to analyze the relationship between the expression of RIP1 and the clinicopathological parameters in gallbladder cancer. And to further explore the roles of RIP1 in growth and invasion of gallbladder carcinoma cells and further discussing the mechanisms RIP1 mediate the expression of VEGF-C.Methods Collected 60 formalin-fixed and paraffin-embedded gallbladder cancer samples and 17 samples of normal gallbladder tissues that were located away from the cancer were used as contorls. We using the method of immunohistochemistry assessed the expression of RIP1 gene in the gallbladder cancer samples and analyzed the correlation between the expression of RIP1 and clinicopathologic factors. Using the RT-PCR and Western-blot technology tested the expression situation of RIP1 in the all three gallbladder cell lines. Using RNAi technology knockdown the expression of RIP1 gene in the gallbladder cancer cells. Using the methods of MTT, Transwell assay, Flow cytometry tested the biology behavior changes of si RIP1 gene of gallbladder cancer cells. A series of luciferase reporter assay of VEGF-C promoters and were constructed, and the binding sites of NF-κB and AP-1 on the promoter were identified using site-directed mutagenesis technology,and the overexpression vectors of NF-κB, AP-1 and RIP1 were constructed, and using the methods of electrophoretic mobility shift assay(EMSA) and chromatin immunoprecipitation assay(Ch IP) to confirm that RIP1 mediated the activity VEGF-C promoter through the transcription factors NF-κB and AP-1.Results 1. The MOD of RIP1 in normal gallbladder tissues(0.1345±0.1220)was significantly lower than gallbladder cancer tissues(0.3134±0.0802;P<0.0001). RIP1 expression did not vary significantly with age, gender, tumor size, and histologic grade; however, there was a marked relationship between RIP1 overexpression and clinical stage(I-III vs. IV-V; P=0.0021<0.05), lymph node metastasis(negative vs. positive; P=0.047<0.05), and gallstones(negative vs. positive; P=0.0382<0.05).2. RIP1 m RNA and protein expressed in all three gallbladder cancer lines. The intensity of expression of RIP1 m RNA and protein in NOZcells was higher when compared to the SGC-996 and GBC-SD cell lines( P < 0.05). The P-1/si RNA vector resulted in a higher suppression of the levels of RIP1 m RNA and protein expression than the other vectors(P-2/si RNA、P-3/si RNA、P-4/si RNA),while non-transfected and NC/si RNA vectors had no effect on the levels of RIP1 m RNA and protein expression(P<0.05). Compared to the NC/si RNA cells, which was the same as the growth of non-transfected cells, cell proliferation in the P-1/si RNA cells was slower(P<0.05). The total number of cells in the P-1/si RNA group that invaded through Transwell polycarbonate filter was significantly lower than that of cells in the NC/si RNA group(P<0.05), which was similar to the number of cells in non-transfected group(P>0.05). The non-transfected cells, NC/si RNA cells and P-1/si RNA cells exhibited a similar rate of apoptosis. The western-blot analysis results indicated that the AKT、p-AKT、NF-κB(p65)、p-NF-κB(p-p65)、cjun(AP-1)、p-c-jun(p-AP-1) protein levels were significantly decreased in the P-1/si RNA NOZ cells group( P < 0.05) and the VEGF-C m RNA and protein expression levels were significantly decreased in the P-1/si RNA NOZ cells group(P<0.05).3.-332bp--190 bp was the critical area and kept improtant activity of VEGF-C promotor. The luciferase of PGL3B-332 were activated by the expression of RIP1 with the increase of dose effect. And we found two overlapping AP-1 sites(-207 to-192 bp GGCGCGTCAGTCATG, GGCGCGTCAGT and CGTCAGTCATGrespectively)in the area of-332bp--190 bp, which could mediate the activity of VEGF-C promoter.4. To confirm that RIP1 could activate transcription factors NF-κB and AP-1 to combine with the-332bp--190 bp area and enhance the activity of VEGF-C promotor. Conclution 1. The expression of RIP1 in the gallbladder cancer tissue were significantly increased. And RIP1 expression was marked correlation with the lymph metastasis, clinical stages and gallstone of gallbladder cancer.2. RIP1 is very impotant in the aspect of promoting the proliferation and invasiveess of gallbladder cancer cells, but no influence on apoptosis. And the underlying mechanisms of RIP1 promoting the lymph metastasis of gallbldder cancer may be through RIP1-NF-κB/AP-1-VEGF-C signal pathway.3.-332bp--190 bp was the critical area and kept improtant activity of VEGF-C promotor. And two overlapping AP-1 sites(-207 to-192 bp GGCGCGTCAGTCATG)in the area of-332bp--190 bp mediated the activity of VEGF-C promotor.4. To confirm that RIP1 could activate transcription factors NF-κB and AP-1 to combine with the-332bp--190 bp area and enhance the activity of VEGF-C promotor.