Preliminary Study On The Biological Roles Of Cellular Inhibitor Of Apoptosis Protein-1 In Gallbladder Carcinoma

?Background?Gallbladder cancer is a highly malignant tumor in digestive system tumors,and currently there is no effective treatment for patients who cannot be operated on.Cellular Inhibitor of Apoptosis Inhibitor-1(cIAP1)is an important member of the family of Inhibitors of Apoptosis Protein and was originally named for its role in inhibiting apoptosis.It was laterly discovered that cIAP1 can be ubiquitinate multiple substrates by its intramolecular RING domain and regulates the activities including proliferation,migration,autophagy and immunity.To date,various literatures have reported that cIAP1 is highly expressed in a variety of tumors and has been shown to be involved in the development,progression,and indication of prognosis of corresponding tumors.This article mainly illustrated the role of cIAP1 in proliferation,migration and anti-apoptosis in gallbladder cancer cells and preliminary research on its anti-apoptotic mechanism,aiming to provide new therapeutic ideas for the treatment of gallbladder cancer.?Method? 1.Immunohistochemistry was used to detect the expression of cIAP1 in gallbladder carcinoma and adjacent tissues.2.The expression of cIAP1 in gallbladder cancer cell lines NOZ and SGC-996 was ilenced by lentivirus techonology,which were then screened by puromycin and estified as knockdown of cIAP1 were built as LV-shcIAP1.The cell line transfected with the random control sequence lentivirus was marked as the control group was marked as NC.CCK-8 reagent assay and clone formation assay were used to detect the effect of cIAP1 on the proliferation of gallbladder cancer cells.Transwell chamber migration assay was used to detect the effect of cIAP1 on the migration of gallbladder cancer cells.3.Cycloheximide,TNF-? and Caspase inhibitor z-VAD were applied,respectively r in combination to both gallbladder cancer NC group and LV-shcIAP1 group nd then CCK-8 reagent was added to detect cell viability.Flow cytometry was mployed to detect the rate of apoptotic cell.Western-blot to detect the expression f apoptosis-associated proteins including cleaved Caspase-8,cleaved Caspase-3 nd cleaved PARP in each group 4.Immunoprecipitation was used to detect the measure the interaction between aspase-8 and RIP1 in NC group and LV-shcIAP1 group stimulated by TNF-? ombined with cycloheximide.Western-blot was used to evaluate the nuclear ransfer of NF-?B subunit P65 protein in both NC and LV-shcIAP1 group timulated by TNF-?.The corresponding c-FLIP transcription and translation evels were detected by q-RTPCR and Western-blot,respectively.?Result? 1.Expression of cIAP1 in gallbladder carcinoma tissuesThe expression of cIAP1 protein in 32 cases of gallbladder carcinoma tissues and corresponding adjacent tissues was detected by immunohistochemistry.The expression of cIAP1 was positive or negative in 27 cases of gallbladder carcinoma,and 5 cases were weakly positive or negative.The expression of cIAP1 in adjacent tissues showed moderate degree and above included 8 cases and weak positive or negative contained 24 cases.The expression of cIAP1 in gallbladder carcinoma tissues was significantly higher than that in adjacent tissues(X 2 = 22.76,P < 0.001).2.Effect of cIAP1 on proliferation and migration of gallbladder cancer cellsThe CCK-8 proliferation assay showed that the proliferation in LV-shcIAP1 group in NOZ was lower than that in NC group,and the OD value at 72 hours was significantly lower than that in the latter group(P<0.01).The proliferation in LV-shcIAP1 group in SGC-996 was lower than that in NC and the 72-hour OD value was lower than the latter(p<0.05).In colony formation assay,the number of visible cell clones in NOZ in NC group was significantly higher than that of LV-shcIAP1 group(411.2±24.2 VS 326.67±17.56,P<0.001);the number of cell clones formed by NC group of SGC-996 was significantly higher than that of LV-shcIAP1 group(481.0±31.44 VS 225.3±4.2,P<0.01).In Transwell migration assay,the number of transmembrane cells in the LV-shcIAP1 group of NOZ was significantly lower than that in NC group(34.0±3.3 VS 63.6±4.8,P<0.001);the number of transmembrane cells in the NC group of SGC-996 was significantly lower than that in the NC group(77.4 ± 7.22 VS 122.6 ± 8.24,P < 0.01).The proliferation and migration ability of the LV-shcIAP1 group were lower than those of the NC group.cIAP1 was proved to play roles in promoting proliferation and migration of gallbladder cancer cells.3.Effect of cIAP1 on TNF-?-mediated apoptosisCompared with the cycloheximide group,the cell viability of NOZ in the TNF-? plus cycloheximide group was significantly lower(P<0.001),and the cell viability in the LV-shcIAP1 group was significantly lower than that in the NC group(P<0.01).In 996,the cell viability of the TNF-? plus cycloheximide group was significantly lower than that of the cycloheximide group(P<0.001),and the cell viability in the LV-shcIAP1 group was significantly lower than that of the NC group(P<0.001).In both cells,the apoptotic protein inhibitor z-VAD restored the reduced cell viability of TNF-? plus cycloheximide(compared to the cycloheximide group,P=0.111 in NOZ;P=0.522 in SGC-996).Flow cytometry showed that the proportion of LV-shcIAP1 in NOZ and SGC-996 was significantly higher than that in NC group in the TNF-? plus cycloheximide group(P<0.05 in both NOZ and SGC-996).Consistent with the above results,Western-blot showed that in TNF-? plus cycloheximide groups,LV-shcIAP1 group was detected with more cleaved Caspase-8,cleaved Caspase-3,and cleaved PARP compared with the NC group(P<0.05 in both NOZ and SGC-996).In conclusion,with the assistance of cycloheximide,cIAP1 promoted Caspase-8-related apoptosis cascade induced by TNF-?.4.cIAP1 promotes the activation of NF-?B pathway with the transcription and translation of c-FLIPThe results of co-immunoprecipitation indicated that LV-shcIAP1 did not form more apoptotic complex II where Caspase-8 interacts with RIP1 under the stimulation of TNF-?/cycloheximide(P>0.05 in NOZ and SGC-996).Under the stimulation of TNF-?,the transcription level of CFLAR gene in LV-shcIAP1 group in NOZ was significantly lower than that in NC group(P<0.05).The transcription level of CFLAR in LV-shcIAP1 group in SGC-996 was significantly lower than that in NC group(P<0.01).The c-FLIP protein translation was significantly reduced in LV-shcIAP1 group compared with the NC group in the NC group with the stimulation of TNF-?(P<0.001);the c-FLIP protein translation in SGC-996 was significantly reduced compared with the NC group(P<0.05).Under the stimulation of TNF-?,the amount of NF-?B subunit P-65 cytoplasm-nuclear transfer in LV-shcIAP1 group was significantly lower than that in NC group(P<0.05);In SGC-996,the amount of P-65 cytoplasmic-nuclear transfer in the LV-shcIAP1 group was also significantly reduced compared to the NC group(P < 0.05).In conclusion,cIAP1 play an anti-apoptotic role possibly through the activation of the NF-?B pathway and the transcription and translation of the downstream anti-apoptotic protein c-FLIP under the stress of TNF-?.? ?Conclusion? 1.The expression of cIAP1 in gallbladder carcinoma tissues is significantly higher than that in adjacent tissues.2.cIAP1 promotes the proliferation and migration of gallbladder cancer cells.3.cIAP1 promotes TNF-?-induced Caspase-8-related apoptosis.4.The anti-apoptotic effect of cIAP1 in gallbladder cancer cells is associated with NF-?B pathway and c-FLIP expression.